METHODS OF INHIBITING LIVER-TYPE GLUTAMINASE, GLS2
20250275964 ยท 2025-09-04
Inventors
- Richard Cerione (Ithaca, NY)
- William P. Katt (Brooktondale, NY, US)
- Michael J. LUKEY (Ithaca, NY, US)
- Sekar Ramachandran (Ithaca, NY, US)
Cpc classification
A61K31/501
HUMAN NECESSITIES
International classification
Abstract
The present application relates to a method of reducing the production of glutamate from glutamine by GLS and by GLS2 in a cancerous cell or cancerous tissue. This method includes inhibiting glutaminase activity of GLS and GLS2 in the cancerous cell or cancerous tissue by a method involving selecting a cancerous cell or cancerous tissue; and contacting GLS and GLS2 in the cell or tissue with a dual GLS/GLS2 inhibitor, where the contacting reduces the production of glutamate from glutamine by GLS and by GLS2 in the cell or tissue.
Claims
1. A method of reducing the production of glutamate from glutamine by GLS and by GLS2 in a cancerous cell or cancerous tissue, said method comprising: inhibiting glutaminase activity of GLS and GLS2 in the cancerous cell or cancerous tissue by a method comprising: selecting a cancerous cell or cancerous tissue; and contacting GLS and GLS2 in the cell or tissue with a dual GLS/GLS2 inhibitor; wherein said contacting reduces the production of glutamate from glutamine by GLS and by GLS2 in the cell or tissue.
2. A method of treating a subject with a condition mediated by production of glutamate from glutamine by GLS and by GLS2, said method comprising: selecting a subject with a condition mediated by production of glutamate from glutamine by GLS and by GLS2 and administering to said selected subject a dual GLS/GLS2 inhibitor under conditions effective to treat the condition mediated by production of glutamate from glutamine.
3. A method of reducing the production of glutamate from glutamine by GLS2 in a cancerous cell or cancerous tissue, said method comprising: inhibiting glutaminase activity of GLS2 in the cancerous cell or cancerous tissue by a method comprising: selecting a cancerous cell or cancerous tissue characterized by GLS2 overexpression and/or GLS2 hyperactivity; and contacting GLS2 in the cell or tissue with an inhibitor of GLS2 glutaminase activity; wherein said contacting reduces the production of glutamate from glutamine by GLS2 in the cell or tissue.
4. A method of treating a subject with a condition mediated by production of glutamate from glutamine by GLS2, said method comprising: selecting a subject with a condition mediated by production of glutamate from glutamine by GLS2 and administering to said selected subject an inhibitor of GLS2 glutaminase activity under conditions effective to treat the condition mediated by production of glutamate from glutamine.
5. The method according to claim 3, wherein the inhibitor is a dual GLS/GLS2 inhibitor.
6. The method according to claim 1, wherein the inhibitor inhibits GLS2 and GAC, and/or inhibits GLS2 and KGA, and/or inhibits LGA, and/or inhibits GAB.
7-9. (canceled)
10. The method according to claim 1, wherein the inhibitor is a compound, or a pharmaceutically acceptable salt, ester, enol ether, enol ester, solvate, hydrate, or prodrug thereof, selected from the group consisting of: I) compounds of Formula IA: ##STR00016## wherein: the dotted circle identifies an active moiety; X is independently CR.sub.14a or N; R.sub.1a is independently H, OH, OR.sub.14a, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aS(O), or R.sub.14aS(O).sub.2; R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are each independently a photoreactive moiety, H, halogen, NO.sub.2, OH, OR.sub.14a, SR.sub.14a, NH.sub.2, NHR.sub.14a, NR.sub.14aR.sub.15a, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aC(O)O, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; or R.sub.2a and R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and R.sub.5a, or R.sub.5a and R.sub.6a are combined to form a heterocyclic ring optionally substituted with a photoreactive moiety; R.sub.7a, R.sub.8a, R.sub.9a, and R.sub.10a are each independently a photoreactive moiety, H, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the aryl, heteroaryl, and aryl C.sub.1-C.sub.6 alkyl are optionally substituted from 1 to 3 times with substituents selected from the group consisting of, halogen, OH, NH.sub.2, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkoxy, SH, and C.sub.1-C.sub.6 thioalkyl, and wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; and R.sub.11a, R.sub.12a, R.sub.13a, R.sub.14a, R.sub.15a, R.sub.16a, and R.sub.17a are each independently a photoreactive moiety, H, halogen, OH, OC.sub.1-C.sub.6 alkyl, OC.sub.2-C.sub.6 alkenyl, OC.sub.2-C.sub.6 alkynyl, NO.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, and mono or polycyclic aryl are optionally substituted with a photoreactive moiety and each one of R.sub.11a-R.sub.17a is optionally substituted with NH.sub.2, OH, halogen, COOH, NO.sub.2, or CN; II) compounds of Formula IB: ##STR00017## wherein: the dotted circle identifies an active moiety; R.sub.1a is H, OH, OR.sub.14a, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aS(O), or R.sub.14aS(O).sub.2; R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are each independently a photoreactive moiety, H, halogen, NO.sub.2, OH, OR.sub.14a, SR.sub.14a, NH.sub.2, NHR.sub.14a, NR.sub.14aR.sub.15a, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aC(O)O, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; or R.sub.2a, and R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and R.sub.5a, or R.sub.5a and R.sub.6a are combined to form a heterocyclic ring optionally substituted with a photoreactive moiety; wherein at least two of R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are not hydrogen; R.sub.7a, R.sub.8a, R.sub.9a, and R.sub.10a are each independently a photoreactive moiety, H, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the aryl, heteroaryl, and aryl C.sub.1-C.sub.6 alkyl are optionally substituted from 1 to 3 times with substituents selected from the group consisting of, halogen, OH, NH.sub.2, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkoxy, SH, and C.sub.1-C.sub.6 thioalkyl, and wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; and R.sub.11a, R.sub.12a, R.sub.13a, R.sub.14a, R.sub.15a, R.sub.16a, R.sub.17a, and R.sub.18a are each independently a photoreactive moiety, H, halogen, OH, OC.sub.1-C.sub.6 alkyl, OC.sub.2-C.sub.6 alkenyl, OC.sub.2-C.sub.6 alkynyl, NO.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, and mono or polycyclic aryl are optionally substituted with a photoreactive moiety and each one of R.sub.11a-R.sub.18a is optionally substituted with NH.sub.2, OH, halogen, COOH, NO.sub.2, or CN; III) compounds of Formula IC: ##STR00018## wherein: the dotted circle identifies an active moiety; R.sub.1a is H, OH, OR.sub.14a, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aS(O), or R.sub.14aS(O).sub.2; R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are each independently a photoreactive moiety, H, halogen, NO.sub.2, OH, OR.sub.14a, SR.sub.14a, NH.sub.2, NHR.sub.14a, NR.sub.14aR.sub.15a, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aC(O)O, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; or R.sub.2a and R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and R.sub.5a, or R.sub.5a and R.sub.6a are combined to form a heterocyclic ring optionally substituted with a photoreactive moiety; wherein at least two of R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are not hydrogen; R.sub.7a, R.sub.8a, R.sub.9a, and R.sub.10a are each independently a photoreactive moiety, H, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the aryl, heteroaryl, and aryl C.sub.1-C.sub.6 alkyl are optionally substituted from 1 to 3 times with substituents selected from the group consisting of, halogen, OH, NH.sub.2, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkoxy, SH, and C.sub.1-C.sub.6 thioalkyl, and wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; and R.sub.11a, R.sub.12a, R.sub.13a, R.sub.14a, R.sub.15a, and R.sub.16a, are each independently a photoreactive moiety, H, halogen, OH, OC.sub.1-C.sub.6 alkyl, OC.sub.2-C.sub.6 alkenyl, OC.sub.2-C.sub.6 alkynyl, NO.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, and mono or polycyclic aryl are optionally substituted with a photoreactive moiety and each one of Ruia-R.sub.16, is optionally substituted with NH.sub.2, OH, halogen, COOH, NO.sub.2, or CN; IV) compounds of Formula ID: ##STR00019## wherein: the dotted circle identifies an active moiety; R.sub.1a is H, OH, OR.sub.14a, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aS(O), or R.sub.14aS(O).sub.2; R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are each independently a photoreactive moiety, H, halogen, NO.sub.2, OH, OR.sub.14a, SR.sub.14a, NH.sub.2, NHR.sub.14a, NR.sub.14aR.sub.15a, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aC(O)O, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; or R.sub.2a, and R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and R.sub.5a, or R.sub.5a and R.sub.6a are combined to form a heterocyclic ring optionally substituted with a photoreactive moiety; wherein at least two of R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are not hydrogen; R.sub.7a, R.sub.8a, R.sub.9a, and R.sub.10a are each independently a photoreactive moiety, H, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the aryl, heteroaryl, and aryl C.sub.1-C.sub.6 alkyl are optionally substituted from 1 to 3 times with substituents selected from the group consisting of, halogen, OH, NH.sub.2, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkoxy, SH, and C.sub.1-C.sub.6 thioalkyl, and wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; and R.sub.11a, R.sub.12a, R.sub.13a, R.sub.14a, R.sub.15a, R.sub.16a, R.sub.17a, R.sub.18a, R.sub.19a and R.sub.20a are each independently a photoreactive moiety, H, halogen, OH, OC.sub.1-C.sub.6 alkyl, OC.sub.2-C.sub.6 alkenyl, OC.sub.2-C.sub.6 alkynyl, NO.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, and mono or polycyclic aryl are optionally substituted with a photoreactive moiety and each one of R.sub.11a-R.sub.20a is optionally substituted with NH.sub.2, OH, halogen, COOH, NO.sub.2, or CN; V) compounds of Formula II: ##STR00020## wherein: the dotted circle identifies an active moiety; n is an integer from 1 to 4; R.sub.1b is independently at each occurrence H, OH, OR.sub.5b, halogen, CN, NO.sub.2, NH.sub.2, NHR.sub.5b, NR.sub.5bR.sub.6b, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen; R.sub.2b is independently H, halogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, or mono or polycyclic aryl; R.sub.3b and R.sub.4b are independently H, OR.sub.5b, SR.sub.5b, R.sub.5bS(O), R.sub.5bS(O).sub.2, COOR.sub.5b, C(O)NR.sub.5bR.sub.6b, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen; or R.sub.3b and R.sub.4b can combine together to form a mono or polycyclic heterocyclyl or heteroaryl containing from 1-5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, each formed heteroaryl or heterocyclyl optionally substituted with substituents selected from the group consisting of oxo, thio, amino, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl; and R.sub.5b and R.sub.6b are independently H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, each one of R.sub.5b or R.sub.6b optionally substituted from 1 to 3 times with substituents selected from the group consisting of H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, and C.sub.4-C.sub.7 cycloalkylalkyl; VI) compounds of Formula III: ##STR00021## wherein: the dotted circle identifies an active moiety; m and n are integers from 1 to 4; B is a substituted or unsubstituted mono or polycyclic aryl or mono or polycyclic heterocyclyl or heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen; R.sub.1c and R.sub.2c are independently H, OH, OR.sub.3c, halogen, CO, CN, NO.sub.2, COOH, NH.sub.2, NHR.sub.3c, NR.sub.3cR.sub.4c, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen; and R.sub.3c and R.sub.4c are independently H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen; and VII) compounds of Formula IV ##STR00022## wherein: R is selected from the group consisting of monocyclic or bicyclic aryl, monocyclic or bicyclic heteroaryl, and monocyclic or bicyclic heterocyclyl, wherein each monocyclic or bicyclic aryl, monocyclic or bicyclic heteroaryl, and monocyclic or bicyclic heterocyclyl can be optionally substituted from 1 to 4 times with substituents independently selected at each occurrence thereof from the group consisting of H, halogen, C.sub.1-6 alkyl, aryl, OR.sup.8, CF.sub.3, and CHF.sub.2; R.sup.1 and R.sup.2 are each independently selected from the group consisting of a photoreactive moiety, H, halogen, and C.sub.1-6 alkyl; or R.sup.1 and R.sup.2 are combined to form O; R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each independently selected from the group consisting of a photoreactive moiety, H, halogen, NO.sub.2, NR.sup.8R.sup.9, SO.sub.2NR.sup.8R.sup.9, N.sub.3, C(O)R.sup.8, aryl, heteroaryl, heterocyclyl, and ##STR00023## and R.sup.8 and R.sup.9 are each independently selected from the group consisting of H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, and aryl; or R.sup.8 and R.sup.9 are combined with the nitrogen to which they are attached to form a heterocyclyl, wherein the heterocyclyl can be optionally substituted with COOH or COOMe.
11-16. (canceled)
17. The method according to claim 10, wherein ##STR00024## has the formula: ##STR00025## wherein X is carbon or nitrogen; R.sub.1a is H, OH, OR.sub.14a, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aS(O), or R.sub.14aS(O).sub.2; R.sub.14a is H, halogen, OH, NO.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, or mono or polycyclic aryl, with R.sub.1 is optionally substituted with NH.sub.2, OH, halogen, COOH, NO.sub.2, or CN; and the total number of R.sub.2c substituents is from 1 to 4.
18. The method according to claim 11, wherein R.sub.4a is not hydrogen.
19. The method according to claim 11, wherein at least two of R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are not hydrogen.
20-21. (canceled)
22. The method according to claim 11, wherein R.sup.5 is not hydrogen.
23. The method according to claim 11, wherein at least two of R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are not hydrogen.
24. (canceled)
25. The method according to claim 10, wherein the compound comprises an active moiety having a formula selected from the group consisting of: ##STR00026## ##STR00027##
26-28. (canceled)
29. The method according to claim 2, wherein the condition is a cancer.
30. The method according to claim 1, wherein the cancer exhibits active GLS2 glutaminase activity.
31. The method according to claim 1, wherein the cancer is selected from the group consisting of breast cancer, triple-negative breast cancer, receptor-positive breast cancer, acute myeloid leukemia, bladder cancer, bladder urothelial carcinoma, brain lower grade glioma, cervical cancer, cervical squamous cell carcinoma, radiation-resistant cervical cancer, colorectal cancer, colorectal tumor, colon adenocarcinoma, glioblastoma multiforme, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, kidney chromophobe, kidney renal papillary cell carcinoma, large B cell lymphoma, liver cancer, liver hepatocellular carcinoma, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, non-small cell lung cancer, neuroblastoma, ovarian cancer, ovarian serous cystadenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglial cancer, paraganglioma, prostate cancer, prostate adenocarcinoma, rectal cancer, rectal adenocarcinoma, testicular cancer, testicular germ cell tumors, thymal cancer, thymoma, thyroid cancer, thyroid carcinoma, and uterine corpus endometrial carcinoma.
32-33. (canceled)
34. The method according to claim 1, wherein the cancer is characterized by moderate-to-high GLS2 expression and low GLS expression.
35-43. (canceled)
44. The method according to claim 1, wherein the cancer is resistant to treatment with a GLS-specific inhibitor.
45. (canceled)
46. The method according to claim 1, wherein said contacting comprises inhibiting cell proliferation, tumorigenesis, tumor growth, tumor initiation, and/or metastasis.
47. The method according to claim 2, wherein said administering is performed parenterally, orally, subcutaneously, intravenously, intramuscularly, extraperitoneally, by intranasal instillation, or by application to mucous membranes.
48-55. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
DETAILED DESCRIPTION
[0033] One aspect of the present application relates to a method of reducing the production of glutamate from glutamine by GLS2 in a cancerous cell or cancerous tissue. This method includes inhibiting glutaminase activity of GLS2 in the cancerous cell or cancerous tissue by a method involving selecting a cancerous cell or cancerous tissue characterized by GLS2 overexpression and/or GLS2 hyperactivity; and contacting GLS2 in the cell or tissue with an inhibitor of GLS2 glutaminase activity; where the contacting reduces the production of glutamate from glutamine by GLS2 in the cell or tissue.
[0034] Another aspect of the present application relates to a method of reducing the production of glutamate from glutamine by GLS and by GLS2 in a cancerous cell or cancerous tissue. This method includes inhibiting glutaminase activity of GLS and GLS2 in the cancerous cell or cancerous tissue by a method involving selecting a cancerous cell or cancerous tissue; and contacting GLS and GLS2 in the cell or tissue with a dual GLS/GLS2 inhibitor; where the contacting reduces the production of glutamate from glutamine by GLS and by GLS2 in the cell or tissue.
[0035] Another aspect of the present application relates to a method of treating a subject with a condition mediated by production of glutamate from glutamine by GLS2. This method includes selecting a subject with a condition mediated by production of glutamate from glutamine by GLS2, and administering to said selected subject an inhibitor of GLS2 glutaminase activity under conditions effective to treat the condition mediated by production of glutamate from glutamine.
[0036] A second aspect of the present application relates to a method of treating a subject with a condition mediated by production of glutamate from glutamine by GLS and by GLS2. This method includes selecting a subject with a condition mediated by production of glutamate from glutamine by GLS and by GLS2 and administering to the selected subject a dual GLS/GLS2 inhibitor under conditions effective to treat the condition mediated by production of glutamate from glutamine.
[0037] The term reducing means to suppress, decrease, diminish, or lower the production of glutamate from glutamine. The term treatment or treating means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use for treating conditions mediated by the production of glutamate from glutamine by GLS2 alone or by both GLS and GLS2.
[0038] The term GLS refers to kidney-type glutaminases encoded by a GIS gene, including the isoforms GAC and KGA. The term GLS2 refers to liver-type glutaminases encoded by a GLS2 gene, including the isoforms LGA and GAB.
[0039] The methods of the present application involve the use of an inhibitor of GLS2 glutaminase activity. Such inhibitors include any inhibitor that is capable of inhibiting glutaminase activity of at least one GLS2 isoform (e.g., LGA and/or GAB). In at least some embodiments of all aspects of the present application, the inhibitor of GLS2 glutaminase activity is a dual GLS/GLS2 inhibitor. A dual GLS/GLS2 inhibitor is a compound that is capable of inhibiting glutaminase activity of at least one GLS isoform (e.g., GAC and/or KGA) and at least one GLS2 isoform (e.g., LGA and/or GAB), in any combination.
[0040] In at least one embodiment of all aspects of the present application, the inhibitor is a compound (or a pharmaceutically acceptable salt, ester, enol ether, enol ester, solvate, hydrate, or prodrug thereof) selected from the group consisting of: [0041] I) compounds of Formula IA:
##STR00001## [0042] wherein: [0043] the dotted circle identifies an active moiety; [0044] X is independently CR.sub.14a or N; [0045] R.sub.1a is independently H, OH, OR.sub.14a, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aS(O), or R.sub.14aS(O).sub.2; [0046] R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are each independently a photoreactive moiety, H, halogen, NO.sub.2, OH, OR.sub.14a, SR.sub.14a, NH.sub.2, NHR.sub.14a, NR.sub.14aR.sub.15a, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aC(O)O, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; or R.sub.2a and R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and R.sub.5a, or R.sub.5a and R.sub.6a are combined to form a heterocyclic ring optionally substituted with a photoreactive moiety; [0047] R.sub.7a, R.sub.8a, R.sub.9a, and R.sub.10a are each independently a photoreactive moiety, H, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the aryl, heteroaryl, and aryl C.sub.1-C.sub.6 alkyl are optionally substituted from 1 to 3 times with substituents selected from the group consisting of, halogen, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkoxy, SH, and C.sub.1-C.sub.6 thioalkyl, and wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; and [0048] R.sub.11a, R.sub.12a, R.sub.13a, R.sub.14a, R.sub.15a, R.sub.16a, and R.sub.17a are each independently a photoreactive moiety, H, halogen, OH, OC.sub.1-C.sub.6 alkyl, OC.sub.2-C.sub.6 alkenyl, OC.sub.2-C.sub.6 alkynyl, NO.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, and mono or polycyclic aryl are optionally substituted with a photoreactive moiety and each one of R.sub.11a-R.sub.17a is optionally substituted with NH.sub.2, OH, halogen, COOH, NO.sub.2, or CN; [0049] II) compounds of Formula IB:
##STR00002## [0050] wherein: [0051] the dotted circle identifies an active moiety; [0052] R.sub.1a is H, OH, OR.sub.14a, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aS(O), or R.sub.14aS(O).sub.2; [0053] R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are each independently a photoreactive moiety, H, halogen, NO.sub.2, OH, OR.sub.14a, SR.sub.14a, NH.sub.2, NHR.sub.14a, NR.sub.14aR.sub.15a, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aC(O)O, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; or R.sub.2a and R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and R.sub.5a, or R.sub.5a and R.sub.6a are combined to form a heterocyclic ring optionally substituted with a photoreactive moiety; wherein at least two of R.sub.2, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are not hydrogen; [0054] R.sub.7a, R.sub.8a, R.sub.9a, and R.sub.10a are each independently a photoreactive moiety, H, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the aryl, heteroaryl, and aryl C.sub.1-C.sub.6 alkyl are optionally substituted from 1 to 3 times with substituents selected from the group consisting of, halogen, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkoxy, SH, and C.sub.1-C.sub.6 thioalkyl, and wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; and [0055] R.sub.11a, R.sub.12a, R.sub.13a, R.sub.14a, R.sub.15a, R.sub.16a, R.sub.17a, and R.sub.18a are each independently a photoreactive moiety, H, halogen, OH, OC.sub.1-C.sub.6 alkyl, OC.sub.2-C.sub.6 alkenyl, OC.sub.2-C.sub.6 alkynyl, NO.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, and mono or polycyclic aryl are optionally substituted with a photoreactive moiety and each one of R.sub.11a-R.sub.18a is optionally substituted with NH.sub.2, OH, halogen, COOH, NO.sub.2, or CN; [0056] III) compounds of Formula IC:
##STR00003## [0057] wherein: [0058] the dotted circle identifies an active moiety; [0059] R.sub.1a is H, OH, OR.sub.14a, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aS(O), or R.sub.14aS(O).sub.2; [0060] R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are each independently a photoreactive moiety, H, halogen, NO.sub.2, OH, OR.sub.14a, SR.sub.14a, NH.sub.2, NHR.sub.14a, NR.sub.14aR.sub.15a, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aC(O)O, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; or R.sub.2a and R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and R.sub.5a, or R.sub.5a and R.sub.6a are combined to form a heterocyclic ring optionally substituted with a photoreactive moiety; wherein at least two of R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are not hydrogen; [0061] R.sub.7a, R.sub.8a, R.sub.9a, and R.sub.10a are each independently a photoreactive moiety, H, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the aryl, heteroaryl, and aryl C.sub.1-C.sub.6 alkyl are optionally substituted from 1 to 3 times with substituents selected from the group consisting of, halogen, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkoxy, SH, and C.sub.1-C.sub.6 thioalkyl, and wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; and [0062] R.sub.11a, R.sub.12a, R.sub.13a, R.sub.14a, R.sub.15a, and R.sub.16a are each independently a photoreactive moiety, H, halogen, OH, OC.sub.1-C.sub.6 alkyl, OC.sub.2-C.sub.6 alkenyl, OC.sub.2-C.sub.6 alkynyl, NO.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.7 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, and mono or polycyclic aryl are optionally substituted with a photoreactive moiety and each one of R.sub.11aR.sub.16a is optionally substituted with NH.sub.2, OH, halogen, COOH, NO.sub.2, or CN; [0063] IV) compounds of Formula ID:
##STR00004## [0064] wherein: [0065] the dotted circle identifies an active moiety; [0066] R.sub.1a is H, OH, OR.sub.14a, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aS(O), or R.sub.14aS(O).sub.2; [0067] R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are each independently a photoreactive moiety, H, halogen, NO.sub.2, OH, OR.sub.14a, SR.sub.14a, NH.sub.2, NHR.sub.14a, NR.sub.14aR.sub.15a, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aC(O)O, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; or R.sub.2a and R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and R.sub.5a, or R.sub.5a and R.sub.6a are combined to form a heterocyclic ring optionally substituted with a photoreactive moiety; wherein at least two of R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are not hydrogen; [0068] R.sub.7a, R.sub.8a, R.sub.9a, and R.sub.10a are each independently a photoreactive moiety, H, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, wherein the aryl, heteroaryl, and aryl C.sub.1-C.sub.6alkyl are optionally substituted from 1 to 3 times with substituents selected from the group consisting of, halogen, OH, NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkoxy, SH, and C.sub.1-C.sub.6 thioalkyl, and wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl, and mono or polycyclic heteroaryl are optionally substituted with a photoreactive moiety; and [0069] R.sub.11a, R.sub.12a, R.sub.13a, R.sub.14a, R.sub.15a, R.sub.16a, R.sub.17a, R.sub.18a, R.sub.19a and R.sub.20a are each independently a photoreactive moiety, H, halogen, OH, OC.sub.1-C.sub.6 alkyl, OC.sub.2-C.sub.6 alkenyl, OC.sub.2-C.sub.6 alkynyl, NO.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, and mono or polycyclic aryl are optionally substituted with a photoreactive moiety and each one of R.sub.11a-R.sub.20a is optionally substituted with NH.sub.2, OH, halogen, COOH, NO.sub.2, or CN; [0070] V) compounds of Formula II:
##STR00005## [0071] wherein: [0072] the dotted circle identifies an active moiety; [0073] n is an integer from 1 to 4; [0074] R.sub.1b is independently at each occurrence H, OH, OR.sub.5b, halogen, CN, NO.sub.2, NH.sub.2, NHR.sub.5b, NR.sub.5bR.sub.6b, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen; [0075] R.sub.2b is independently H, halogen, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, or mono or polycyclic aryl; [0076] R.sub.3b and R.sub.4b are independently H, OR.sub.5b, SR.sub.5b, R.sub.5bS(O), R.sub.5bS(O).sub.2, COOR.sub.5b, C(O)NR.sub.5bR.sub.6b, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen; or [0077] R.sub.3b and R.sub.4b can combine together to form a mono or polycyclic heterocyclyl or heteroaryl containing from 1-5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, each formed heteroaryl or heterocyclyl optionally substituted with substituents selected from the group consisting of oxo, thio, amino, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl; and [0078] R.sub.5b and R.sub.6b are independently H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, each one of R.sub.5b or R.sub.6b optionally substituted from 1 to 3 times with substituents selected from the group consisting of H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, and C.sub.4-C.sub.7 cycloalkylalkyl; [0079] VI) compounds of Formula III:
##STR00006## [0080] wherein: [0081] the dotted circle identifies an active moiety; [0082] m and n are integers from 1 to 4; [0083] B is a substituted or unsubstituted mono or polycyclic aryl or mono or polycyclic heterocyclyl or heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen; [0084] R.sub.1c and R.sub.2c are independently H, OH, OR.sub.3c, halogen, CO, CN, NO.sub.2, COOH, NH.sub.2, NHR.sub.3c, NR.sub.3cR.sub.4c, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl with each cyclic unit containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen; and [0085] R.sub.3c and R.sub.4c are independently H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl containing from 1 to 5 heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen; and [0086] VII) compounds of Formula IV
##STR00007## [0087] wherein: [0088] R is selected from the group consisting of monocyclic or bicyclic aryl, monocyclic or bicyclic heteroaryl, and monocyclic or bicyclic heterocyclyl, wherein each monocyclic or bicyclic aryl, monocyclic or bicyclic heteroaryl, and monocyclic or bicyclic heterocyclyl can be optionally substituted from 1 to 4 times with substituents independently selected at each occurrence thereof from the group consisting of H, halogen, C.sub.1-6 alkyl, aryl, OR.sup.8, CF.sub.3, and CHF.sub.2; [0089] R.sup.1 and R.sup.2 are each independently selected from the group consisting of a photoreactive moiety, H, halogen, and C.sub.1-6 alkyl; or R.sup.1 and R.sup.2 are combined to form O; [0090] R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each independently selected from the group consisting of a photoreactive moiety, H, halogen, NO.sub.2, NR.sup.8R.sup.9, SO.sub.2NR.sup.8R.sup.9, N.sub.3, C(O)R.sup.8, aryl, heteroaryl, heterocyclyl, and
##STR00008## and [0091] R.sup.8 and R.sup.9 are each independently selected from the group consisting of H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, and aryl; or R.sup.8 and R.sup.9 are combined with the nitrogen to which they are attached to form a heterocyclyl, wherein the heterocyclyl can be optionally substituted with COOH or COOMe.
[0092] The term halo or halogen means fluoro, chloro, bromo, or iodo.
[0093] The term optionally substituted indicates that a group may have a substituent at each substitutable atom of the group (including more than one substituent on a single atom), and the identity of each substituent is independent of the others.
[0094] The term substituted or substitution of an atom means that one or more hydrogen on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded. Unsubstituted atoms bear all of the hydrogen atoms dictated by their valency. When a substituent is oxo (i.e., O), then 2 hydrogens on the atom are replaced. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds; by stable compound or stable structure is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Exemplary substituents include, without limitation, oxo, thio (i.e. S), nitro, cyano, halo, OH, NH2, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C4-C7 cycloalkylalkyl, monocyclic aryl, monocyclic heteroaryl, polycyclic aryl, and polycyclic heteroaryl.
[0095] The term monocyclic indicates a molecular structure having one ring.
[0096] The term polycyclic indicates a molecular structure having two (bicyclic) or more rings, including, but not limited to, fused, bridged, or spiro rings.
[0097] The term alkyl means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and 3-pentyl.
[0098] The term thioalkyl means an alkyl group as described above bonded through a sulfur linkage.
[0099] The term alkenyl means an aliphatic hydrocarbon group containing a carbon-carbon double bond and which may be straight or branched having about 2 to about 6 carbon atoms in the chain. Preferred alkenyl groups have 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to a linear alkenyl chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, and i-butenyl.
[0100] The term alkynyl means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched having about 2 to about 6 carbon atoms in the chain. Preferred alkynyl groups have 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to a linear alkynyl chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, and n-pentynyl.
[0101] The term alkoxy means an alkyl-O, alkenyl-O, or alkynyl-O group wherein the alkyl, alkenyl, or alkynyl group is described above. Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, pentoxy, and hexoxy.
[0102] The term cycloalkyl refers to a non-aromatic saturated or unsaturated mono- or polycyclic ring system which may contain 3 to 6 carbon atoms; and which may include at least one double bond. Exemplary cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, anti-bicyclopropane, and syn-bicyclopropane.
[0103] The term cycloalkylalkyl refers to a radical of the formula RaRb where Ra is an alkyl radical as defined above and Rb is a cycloalkyl radical as defined above. The alkyl radical and the cycloalkyl radical may be optionally substituted as defined above.
[0104] The term aryl refers to aromatic monocyclic or polycyclic ring system containing from 6 to 19 carbon atoms, where the ring system may be optionally substituted. Aryl groups of the present application include, but are not limited to, groups such as phenyl, naphthyl, azulenyl, phenanthrenyl, anthracenyl, fluorenyl, pyrenyl, triphenylenyl, chrysenyl, and naphthacenyl.
[0105] The term arylalkyl refers to a radical of the formula RaRb where Ra is an alkyl radical as defined above and Rb is an aryl radical as defined above. The alkyl radical and the cycloalkyl radical may be optionally substituted as defined above.
[0106] The term arylarylalkyl refers to a radical of the formula RaRbRc where Ra is an alkyl as defined above, Rb is an aryl radical as defined above, and Rc is an aryl radical as defined above. The alkyl radical and both aryl radicals may be optionally substituted as defined above.
[0107] The term heterocyclyl refers to a stable 3- to 18-membered ring radical which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For purposes of this application, the heterocyclyl radical may be a monocyclic, or a polycyclic ring system, which may include fused, bridged, or spiro ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the ring radical may be partially or fully saturated. Examples of such heterocyclyl radicals include, without limitation, azepinyl, azocanyl, pyranyl dioxanyl, dithianyl, 1,3-dioxolanyl, tetrahydrofuryl, dihydropyrrolidinyl, decahydroisoquinolyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, oxazolidinyl, oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydropyranyl, thiamorpholinyl, thiamorpholinyl sulfoxide, and thiamorpholinyl sulfone.
[0108] The term heteroaryl refers to an aromatic ring radical which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. For purposes of this application the heteroaryl may be a monocyclic or polycyclic ring system; and the nitrogen, carbon, and sulfur atoms in the heteroaryl ring may be optionally oxidized; the nitrogen may optionally be quaternized. Examples of heteroaryl groups include, without limitation, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, furyl, thiophenyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thienopyrrolyl, furopyrrolyl, indolyl, azaindolyl, isoindolyl, indolinyl, indolizinyl, indazolyl, benzimidazolyl, imidazopyridinyl, benzotriazolyl, benzoxazolyl, benzoxadiazolyl, benzothiazolyl, pyrazolopyridinyl, triazolopyridinyl, thienopyridinyl, benzothiadiazolyl, benzofuyl, benzothiophenyl, quinolinyl, isoquinolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, cinnolinyl, quinazolinyl, quinolizilinyl, phthalazinyl, benzotriazinyl, chromenyl, naphthyridinyl, acrydinyl, phenanzinyl, phenothiazinyl, phenoxazinyl, pteridinyl, and purinyl.
[0109] Further heterocycles and heteroaryls are described in COMPREHENSIVE HETEROCYCLIC CHEMISTRY: THE STRUCTURE, REACTIONS, SYNTHESIS AND USE OF HETEROCYCLIC COMPOUNDS Vol. 1-8 (Alan R. Katritzky et al. eds., 1st ed. 1984), which is hereby incorporated by reference in its entirety.
[0110] A photoreactive moiety as used herein is a moiety that becomes reactive when exposed to ultraviolet or visible light. Suitable photoreactive moieties include, for example, aryl azides, diazirines, and benzophenone; when the photoreactive moiety is an aryl azide or benzophenone, it is attached to an aromatic ring. Suitable examples include, without limitation, NN.sup.+N.sup.:
##STR00009##
[0111] Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to, N, N-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine, and tris(hydroxymethyl) aminomethane; alkali metal salts, such as but not limited to, lithium, potassium, and sodium; alkali earth metal salts, such as but not limited to, barium, calcium, and magnesium; transition metal salts, such as but not limited to, zinc; and other metal salts, such as but not limited to, sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to, hydrochlorides and sulfates; and salts of organic acids, such as but not limited to, acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids, and boronic acids. Pharmaceutical acceptable enol ethers include, but are not limited to, derivatives of formula CC (OR) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of formula CC (OC(O) R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Pharmaceutical acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.
[0112] In accordance with some embodiments of the inhibitor compound of Formula III,
##STR00010##
has the formula:
##STR00011##
where [0113] X is carbon or nitrogen; [0114] R.sub.1a is H, OH, OR.sub.14a, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, R.sub.14aC(O), R.sub.14aOC(O), R.sub.14aS(O), or R.sub.14aS(O).sub.2; [0115] R.sub.14a is H, halogen, OH, NO.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, or mono or polycyclic aryl, with R.sub.14a is optionally substituted with NH.sub.2, OH, halogen, COOH, NO.sub.2, or CN; and [0116] the total number of R.sub.2c substituents is from 1 to 4.
[0117] In accordance with some embodiments of the compounds of Formula IA, compounds of Formula IB, compounds of Formula IC, compounds of Formula ID, compounds of Formula II, and compounds of Formula III, R.sub.4a is not hydrogen. In a further embodiment, at least two of R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and R.sub.6a are not hydrogen. In another embodiment, at least two of R.sub.3a, R.sub.4a, and R.sub.5a are not hydrogen.
[0118] In at least one embodiment of the compounds of Formula IV, R.sup.5 is not hydrogen. In a further embodiment, at least two of R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are not hydrogen. In another embodiment, at least two of R.sup.4, R.sup.5, and R.sup.6 are not hydrogen.
[0119] With respect to compounds of Formula IA, Formula IB, Formula IC, Formula ID, Formula II, Formula III, and Formula IV, suitable active moieties include, but are not limited to:
##STR00012## ##STR00013## ##STR00014##
In a preferred embodiment, the active moiety is
##STR00015##
[0120] In at least one embodiment of the methods of the present application, the inhibitor is a compound selected from the group consisting of glutaminase inhibitors identified in International Application No. PCT/US2010/028688 to Cerione et al. (filed Mar. 25, 2010) (which is hereby incorporated by reference in its entirety), optionally modified to include a photoreactive moiety, if not already present; glutaminase inhibitors identified in Katt et al., Mol. Cancer Ther. 11:1269-78 (2012) (Katt 2012, which is hereby incorporated by reference in its entirety), optionally modified to include a photoreactive moiety, if not already present; and glutaminase inhibitors identified in International Application No. PCT/US2015/064152 to Cerione et al. (filed Dec. 5, 2015) (which is hereby incorporated by reference in its entirety), optionally modified to include a photoreactive moiety, if not already present, and optionally modified to exclude a photoreactive moiety, if present. In at least one embodiment of all aspects of the present application, the inhibitor is selected from the group consisting of Compound 968, Compound 27 of Katt 2012, Compound 17 of Katt 2012, Compound 23 of Katt 2012, Compound SU-1 of PCT/US2015/064152, Compound SU-6 of PCT/US2015/064152, Compound SU-12 of PCT/US2015/064152, Compound SU-14 of PCT/US2015/064152, Compound SU-21 of PCT/US2015/064152, and Compound SU-29 of PCT/US2015/064152.
[0121] Some aspects of the present application involve selecting a cancerous cell or cancerous tissue. In some aspects/embodiments, the cancerous cell or cancerous tissue is characterized by GLS2 overexpression and/or GLS2 hyperactivity. In some embodiments, the cancerous cell/tissue is also characterized by GLS overexpression and/or hyperactivity. Suitable cells/tissue include cells/tissue of the cancer types set forth infra. Suitable cells/tissue include cells/tissue taken or derived from the subjects set forth infra.
[0122] Some aspects of the present application involve treating a subject with a condition mediated by production of glutamate from glutamine by GLS2. In some embodiments, the condition is also mediated by production of glutamate from glutamine by GLS. As will be apparent to the skilled artisan, such conditions include those in which GLS and GLS2 are simultaneously active in the absence of a GLS2 inhibitor or dual GLS/GLS2 inhibitor, as well as conditions in which, in the absence of a GLS2 inhibitor or dual GLS/GLS2 inhibitor, GLS and GLS2 are active at different stages of the condition (for example, when a reduction in GLS activity is followed by subsequent activation of (or increase in) GLS2 activity).
[0123] In at least one embodiment of the methods of the present application, the condition mediated by production of glutamate from glutamine is a cancer. The cancer may exhibit active GLS2 glutaminase activity. Exemplary cancers include, but are not limited to, breast cancer, triple-negative breast cancer, receptor-positive breast cancer, acute myeloid leukemia, bladder cancer, bladder urothelial carcinoma, brain lower grade glioma, cervical cancer, cervical squamous cell carcinoma, radiation-resistant cervical cancer, colorectal cancer, colorectal tumor, colon adenocarcinoma, glioblastoma multiforme, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, kidney chromophobe, kidney renal papillary cell carcinoma, large B cell lymphoma, liver cancer, liver hepatocellular carcinoma, lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, non-small cell lung cancer, neuroblastoma, ovarian cancer, ovarian serous cystadenocarcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglial cancer, paraganglioma, prostate cancer, prostate adenocarcinoma, rectal cancer, rectal adenocarcinoma, testicular cancer, testicular germ cell tumors, thymal cancer, thymoma, thyroid cancer, thyroid carcinoma, and uterine corpus endometrial carcinoma.
[0124] In some embodiments of the methods of the present application, the cancer is characterized by moderate-to-high GLS2 expression. In some embodiments of the methods of the present application, the cancer is characterized by low GLS expression. See Table 2 for the expression patterns of GLS and GLS2 in various cancer types. In Table 2 below, low is defined as the majority of samples having gene expression less than the average expression level across all tissues examined. Moderate is defined as the majority of samples having gene expression close to the average expression level across all tissues examined. High is defined as the majority of samples having gene expression levels above the average expression level across all tissues examined.
TABLE-US-00002 TABLE 2 Expression patterns of GLS and GLS2 in various cancer types 5 year Cancer GLS GLS2 survival (%) Acute Myeloid Leukemia Moderate High 65 Bladder Urothelial Carcinoma Low High 78 Brain Lower Grade Glioma Low Moderate 35 Cervical Squamous cell carcinoma Low High 69 Colon Adenocarcinoma High High 65 Gliobolastoma Multiforme Low Low 35 Head and Neck Squamous Cell High Moderate 21 Carcinoma Kidney Chromophobe High High 75 Kidney Renal Clear Cell High Low 75 Kidney Renal Papillary Cell High High 75 Carcinoma Large B Cell Lymphoma High Low 74 Liver Hepatocellular Carcinoma Low High 19 Lung Adenocarcinoma High High 20 Lung Squamous Cell Carcinoma Low High 20 Melanoma High Low 94 Mesothelioma High Low Ocular Melanoma High Low Ovarian Serous Low High 48 Cystadenocarcinoma Pancreatic Adenocarcinoma High Moderate 9 Paraganglioma Low High Prostate Adenocarcinoma Low High 99 Receptor-positive Breast Low High 91 Rectal Adenocarcinoma High High 69 Sarcoma High Low Testicular Germ Cell Tumors Low High 97 Thymoma Low High Thyroid Carcinoma High High 98 Triple-negative Breast High Low 91 Uterine Carcinoma Low Low 69 Uterine Corpus Endometrial Low Moderate 83 Carcinoma
[0125] In at least some preferred embodiments of the methods of the present application, the cancer is selected from the group consisting of receptor-positive breast cancer, bladder urothelial carcinoma, brain lower grade glioma, cervical squamous cell carcinoma, liver hepatocellular carcinoma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, paraganglioma, prostate adenocarcinoma, testicular germ cell tumors, thymoma, and uterine corpus endometrial carcinoma). In at least one embodiment of the methods of the present application, the cancer is a breast cancer. In at least one embodiment of the methods of the present application, the cancer can is a receptor-positive breast cancer or a luminal type breast cancer (e.g., a luminal type A breast cancer or a luminal type B breast cancer).
[0126] In some embodiments of the methods of the present application, the cancer is characterized by GLS2 hyperactivity. Furthermore, the cancer can be characterized by GLS2 overexpression. Additionally, the cancer may be characterized by only one of GLS2 overexpression and GLS2 hyperactivity.
[0127] In some embodiments of the methods of the present application, the cancer is characterized by GLS overexpression and/or GLS hyperactivity and characterized by GLS2 overexpression and/or GLS2 hyperactivity.
[0128] In some embodiments of the methods of the present application, the cancer is resistant to treatment with a GLS-specific inhibitor (i.e., glutaminase inhibitor that inhibits at least one GLS isoform but does not inhibit an GLS2 isoform). Such GLS-specific inhibitors include CB-839, BPTES, and BPTES-like compounds.
[0129] The methods of the present application involve contacting a cell/tissue with an inhibitor or administering an inhibitor to a subject. In at least one embodiment of the methods of the present application, the contacting or administering includes inhibiting cell proliferation, tumorigenesis, tumor growth, tumor initiation, and/or metastasis.
[0130] Administration may be performed parenterally, orally, subcutaneously, intravenously, intramuscularly, extraperitoneally, by intranasal instillation, or by application to mucous membranes.
[0131] Numerous standard references are available that describe procedures for preparing various formulations suitable for administering the compounds according to the application. Examples of potential formulations and preparations are contained, for example, in the H
[0132] Any pharmaceutically acceptable liquid carrier suitable for preparing solutions, suspensions, emulsions, syrups and elixirs may be employed in the composition of the application. Compounds for administration may be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, or a pharmaceutically acceptable oil or fat, or a mixture thereof. The liquid composition may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, coloring agents, viscosity regulators, stabilizers, osmo-regulators, or the like. Examples of liquid carriers suitable for oral and parenteral administration include water (particularly containing additives as above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) or their derivatives, or oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration the carrier may also be an oily ester such as ethyl oleate or isopropyl myristate.
[0133] It will be understood that the specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
[0134] In all aspects of the present application directed to methods involving contacting a sample with an inhibitor, contacting can be carried out using methods that will be apparent to the skilled artisan, and can be done in vitro, ex vivo, or in vivo.
[0135] Compounds may be delivered directly to a targeted cell/tissue/organ. Additionally and/or alternatively, the compounds may be administered to a non-targeted area along with one or more agents that facilitate migration of the compounds to (and/or uptake by) a targeted tissue, organ, or cell. As will be apparent to one of ordinary skill in the art, the compound itself can be modified to facilitate its transport to a target tissue, organ, or cell, including its transport across the blood-brain barrier; and/or to facilitate its uptake by a target cell (e.g., its transport across cell membranes).
[0136] In vivo administration can be accomplished either via systemic administration to the subject or via targeted administration to affected tissues, organs, and/or cells, as described above. Typically, the therapeutic agent (i.e., a GLS2 inhibitor or dual GLS/GLS2 inhibitor) will be administered to a patient in a vehicle that delivers the therapeutic agent(s) to the target cell, tissue, or organ. Typically, the therapeutic agent will be administered as a pharmaceutical formulation, such as those described above.
[0137] The compounds can be administered, e.g., by intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, topical application, sublingual, intraarticular (in the joints), intradermal, buccal, ophthalmic (including intraocular), intranasally (including using a cannula), or by other routes. The compounds can be administered orally, e.g., as a tablet or cachet containing a predetermined amount of the active ingredient, gel, pellet, paste, syrup, bolus, electuary, slurry, capsule, powder, granules, as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, via a micellar formulation (see, e.g. WO 97/11682, which is hereby incorporated by reference in its entirety) via a liposomal formulation (see, e.g., European Patent No. 736299, WO 99/59550, and WO 97/13500, which are hereby incorporated by reference in their entirety), via formulations described in WO 03/094886, which is hereby incorporated by reference in its entirety, or in some other form. The compounds can also be administered transdermally (i.e. via reservoir-type or matrix-type patches, microneedles, thermal portion, hypodermic needles, iontophoresis, electroporation, ultrasound or other forms of sonophoresis, jet injection, or a combination of any of the preceding methods (Prausnitz et al., Nature Reviews Drug Discovery 3:115 (2004), which is hereby incorporated by reference in its entirety). The compounds can be administered locally, for example, at the site of injury to an injured blood vessel. The compounds can be coated on a stent. The compounds can be administered using high-velocity transdermal particle injection techniques using the hydrogel particle formulation described in U.S. Patent Publication No. 20020061336, which is hereby incorporated by reference in its entirety. Additional particle formulations are described in WO 00/45792, WO 00/53160, and WO 02/19989, which are hereby incorporated by reference in their entirety. An example of a transdermal formulation containing plaster and the absorption promoter dimethylisosorbide can be found in WO 89/04179, which is hereby incorporated by reference in its entirety. WO 96/11705, which is hereby incorporated by reference in its entirety, provides formulations suitable for transdermal administration.
[0138] For use as aerosols, a compound in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The compounds also may be administered in a non-pressurized form.
[0139] Exemplary delivery devices include, without limitation, nebulizers, atomizers, liposomes (including both active and passive drug delivery techniques) (Wang & Huang, pH-Sensitive Immunoliposomes Mediate Target-Cell-Specific Delivery and Controlled Expression of a Foreign Gene in Mouse, Proc. Nat'l Acad. Sci. USA 84:7851-55 (1987); Bangham et al., Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids, J. Mol. Biol. 13:238-52 (1965); U.S. Pat. No. 5,653,996 to Hsu; U.S. Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613 to Holland et al.; U.S. Pat. No. 5,631,237 to Dzau & Kaneda; U.S. Pat. No. 5,059,421 to Loughrey et al.; Wolff et al., The Use of Monoclonal Anti-Thy1 IgG1 for the Targeting of Liposomes to AKR-A Cells in Vitro and in Vivo, Biochim. Biophys. Acta 802:259-73 (1984), each of which is hereby incorporated by reference in its entirety), transdermal patches, implants, implantable or injectable protein depot compositions, and syringes. Other delivery systems which are known to those of skill in the art can also be employed to achieve the desired delivery of the compound to the desired organ, tissue, or cells in vivo to effect this aspect of the present application.
[0140] Contacting (including in vivo administration) can be carried out as frequently as required and for a duration that is suitable to provide the desired effect. For example, contacting can be carried out once or multiple times, and in vivo administration can be carried out with a single sustained-release dosage formulation or with multiple (e.g., daily) doses.
[0141] The amount to be administered will, of course, vary depending upon the particular conditions and treatment regimen. The amount/dose required to obtain the desired effect may vary depending on the agent, formulation, cell type, culture conditions (for ex vivo embodiments), the duration for which treatment is desired, and, for in vivo embodiments, the individual to whom the agent is administered.
[0142] Effective amounts can be determined empirically by those of skill in the art. For example, this may involve assays in which varying amounts of the compound of the application are administered to cells in culture and the concentration effective for obtaining the desired result is calculated. Determination of effective amounts for in vivo administration may also involve in vitro assays in which varying doses of agent are administered to cells in culture and the concentration of agent effective for achieving the desired result is determined in order to calculate the concentration required in vivo. Effective amounts may also be based on in vivo animal studies.
[0143] The compounds can be administered alone or as an active ingredient of a pharmaceutical formulation, such as those described above. The compounds can be administered in a form where the active ingredient is substantially pure.
[0144] In the methods of the present application involving selecting a subject, the subject is preferably a human subject, but can be any animal, including a laboratory animal in the context of a clinical trial or screening or activity experiment. Thus, as can be readily appreciated by one of ordinary skill in the art, the methods of the present application are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, humans, domestic animals, such as feline (e.g., cats) or canine (e.g., dogs) subjects, farm animals, such as but not limited to bovine (e.g., cows), equine (e.g., horses), caprine (e.g., goats), ovine (e.g., sheep), and porcine (e.g., pigs) subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, guinea pigs, goats, sheep, pigs, dogs, cats, horses, cows, camels, llamas, monkeys, zebrafish etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.
[0145] Preferences and options for a given aspect, feature, embodiment, or parameter of the technology described herein should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features, embodiments, and parameters of the technology.
[0146] The present technology may be further illustrated by reference to the following examples.
EXAMPLES
[0147] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.
Materials and Methods
Human Cell Lines
[0148] Human breast cancer cell lines MCF7, BT-474, T-47D, MDA-MB-453, MDA-MB-231, Hs 578T, HCC38, SK-BR-3, and DU4475 were purchased from the American Type Culture Collection (ATCC) and no additional cell authentication was performed. The TSE human breast cancer cell line was supplied by Dr. Steven Abcouwer (University of Michigan). All breast cancer cells were cultured at 37 C. under a 5% CO.sub.2 atmosphere in RPMI 1640 medium containing 2 mM glutamine (Gibco) and supplemented with 10% fetal bovine serum (FBS) (Gibco). The 293T cell line (ATCC) used for generating lentivirus particles was cultured as above but using DMEM, high glucose (Gibco).
Animals
[0149] All experiments involving mice were carried out according to protocols approved by the Institutional Animal Care and Use Committee at Cornell University. In all cases, 6-8 week old female NOD.Cg-Prkdc.sub.scid Il2rg.sup.tm1Wjl/SzJ (NSG) mice (The Jackson Laboratory) were used. For xenograft experiments, a suspension of MDA-MB-453 breast cancer cells (or derivative cell lines stably expressing shRNAs) was mixed 1:1 with Matrigel Matrix (BD Biosciences) to give a final concentration of 310.sup.6 cells per 100 l, and 310.sup.6 cells were immediately injected into each of the two flanks of 6-8 week old female NOD.Cg-Prkdc.sup.scid Il2rg.sup.tm1Wjl/SzJ (NSG) mice (n=3 mice per condition). Tumor sizes were measured using calipers, and estimated volumes were calculated using the formula V=(/6)lengthwidth.sup.2, as described previously (Tomayko et al., Determination of Subcutaneous Tumor Size in Athymic (Nude) Mice, Cancer Chemother Pharmacol. 24:148-154 (1989), which is hereby incorporated by reference in its entirety). For experiments using MDA-MB-453 cells stably expressing shRNAs, mice were sacrificed after 6 weeks. For 968 treatment studies, when tumors of 1-2 mm diameter were detected, the mice were randomly divided into two groups and intraperitoneal (IP) injections of 968 (10 mg/kg) solution or carrier solution were initiated and carried out three times weekly. The formulation was prepared immediately prior to injection and consisted of 70% PBS, 20% Cremophor EL, 10% DMSO, and 968 diluted from a 21 mM DMSO stock. Mice were sacrificed after 3 weeks of treatment.
Cell Lysis and Western Blot Analysis
[0150] Cells, or fractionated mitochondria and nuclei, were lysed with ice-cold lysis buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 25 mM NaF, 1% (v/v) Triton X-100, 1 mM MgCl.sub.2, 50 mM -glycerophosphate, 30 g/ml leupeptin, 5 g/ml aprotinin), and insoluble debris cleared by centrifugation at 4 C. Protein concentrations were determined by Bradford assay (Bio-Rad), and lysate was then boiled for 10 min in reducing SDS-sample buffer. Lysate proteins (20 g total protein/lane, except for the cell fractionation experiment in which 5 g total protein/lane was used) were then resolved on Novex 12% Tris-Glycine Mini Gels (Thermo Fisher Scientific) and transferred to PVDF membrane (PerkinElmer). Membranes were blocked in 7% BSA in Tris-Buffered Saline plus 0.05% TWEEN 20 (TBST) for 1 hour at room temperature and probed overnight at 4 C. in primary antibody solution in TBST (see Table 3 for antibody dilutions). They were then washed with TBST and incubated in TBST containing 25% (v/v) non-fat dry milk powder and anti-Rabbit or anti-Mouse secondary antibody (1:2500) for 1 hour. Finally, membranes were washed in TBST, and imaged using Western Lightning Plus-ECL (PerkinElmer) and HyBlot ES autoradiography film (Denville Scientific Inc.).
TABLE-US-00003 TABLE 3 Antibody dilutions used for western blot analysis related to STAR Methods (see FIG. 2 for Star Method Resource Table). Antibody Dilution ASCT2 Antibody 1:2000 /-Tubulin Antibody 1:5000 GLS antibody (C-term) 1:10000 Anti-GLS2 antibody (C-term) 1:8000 4F2hc/CD98 (SLC3A2) antibody 1:2000 xCT/SLC7A11 antibody 1:2000 VDAC Rabbit mAb 1:4000 ASNS Antibody 1:4000 Lamin A/C Mouse mAb 1:5000 p53 Mouse mAb 1:4000 p63 Rabbit mAb 1:5000 p73 Rabbit mAb 1:5000 N-Myc Rabbit mAb 1:5000 c-Myc Rabbit mAb 1:2000 GATA-3 Rabbit mAb 1:32000 HER2/ErbB2 Rabbit mAb 1:4000 Progesterone Receptor A/B Rabbit mAb 1:4000 Estrogen Receptor Rabbit mAb 1:4000 V5 Tag Monoclonal Antibody 1:8000
Breast Tumor Tissue Microarray
[0151] Tissue microarray BRC961 (US Biomax) was probed as follows, using the anti-GLS2 antibody (Abgent, AP6650D). Reagents were from Vector Laboratories VECTASTAIN Elite ABC HRP kit (Cat #PK-6200), Avidin/Biotin blocking kit (Cat #SP-2001), and ImmPACT DAB Peroxidase (HRP) Substrate (Cat #SK-4105). Deparaffinization was carried out by heating the slide to 60 C. for 20 min and then immersing the slide in mixed xylenes (210 min), 100% ethanol, 95% ethanol, 70% ethanol (5 min each) and finally H.sub.2O (25 min). The antigen retrieval step involved immersing the slide in 10 mM sodium citrate buffer, pH 6.0 at 95 C. for 15 min and then cooling at room temperature for 20 min. To remove endogenous peroxidase the slide was washed with H.sub.2O (25 min) and then incubated in 3% H.sub.2O.sub.2 in H.sub.2O for 10 min. The slide was then washed in H.sub.2O for 5 min followed by PBS for 5 min. Blocking was carried out at room temperature using horse serum (20 min), PBS rinse, avidin solution (15 min), PBS rinse, biotin solution (15 min), PBS rinse, followed by PBS washes (25 min). The slide was then incubated overnight at 4 C. with the anti-GLS2 primary antibody (1:100 in horse serum). Next, the slide was washed with PBS (45 min), incubated for 30 min at room temperature with biotinylated universal secondary antibody, and washed again with PBS (45 min). Then, the slide was incubated for 30 min at room temperature with VECTASTAIN ABC reagent and washed again with PBS (45 min). To develop, the slide was incubated with diluted ImmPACT DAB chromogen for 2 min and then washed in H.sub.2O (25 min). Finally, the stained slide was dehydrated by immersing in 70% ethanol, 95% ethanol, 100% ethanol (5 min each) and mixed xylenes (25 min), mounted using Permount mounting medium (Fisher) and sealed. Signal intensity was quantified using ImageJ. Receptor staining intensity data were from US Biomax.
RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)
[0152] Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) and QIAshredder (Qiagen), and cDNAs were prepared using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). RT-PCR was carried out using the 7500 fast real-time PCR system (Applied Biosystems), using appropriate primers (Table 4) with cDNA as the template. In all cases, 18S rRNA served as the endogenous control. All primer sequences were obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/), and primers were synthesized by Integrated DNA Technologies. Reactions were carried out using Power SYBR Green PCR Mix (Thermo Fisher Scientific).
TABLE-US-00004 TABLE4 ListofprimersrelatedtoSTARMethods Name Sequence SequenceID 18SrRNAFWD CGGCGACGACCCATTCGAAC SEQIDNO:4 18SrRNAREV GAATCGAACCCTGATTCCCCGTC SEQIDNO:5 GLSFWD TCTACAGGATTGCGAACGTCT SEQIDNO:6 GLSREV CTTTGTCTAGCATGACACCATCT SEQIDNO:7 GLS2FWD GCCTGGGTGATTTGCTCTTTT SEQIDNO:8 GLS2REV CCTTTAGTGCAGTGGTGAACTT SEQIDNO:9 SLC7A11FWD GGTCCATTACCAGCTTTTGTACG SEQIDNO:10 SLC7A11REV AATGTAGCGTCCAAATGCCAG SEQIDNO:11 GLS2_000_10F AGGAAGAGAGGGAGGTAGGAGGTTGTTTTATAT SEQIDNO:12 TT GLS2_000_T7R CAGTAATACGACTCACTATAGGGAGAAGGCTCT SEQIDNO:13 TACCCCCACTCCCACTATAATTC GLS2_001_10F AGGAAGAGAGATTATAGTGGGAGTGGGGGTAA SEQIDNO:14 GT GLS2_001_T7R CAGTAATACGACTCACTATAGGGAGAAGGCTTC SEQIDNO:15 CTAAAAATACCCCTAACCCTAAA GLS2_002_10F AGGAAGAGAGGGGTTTGTTTTTTTAAAGTTGGTT SEQIDNO:16 T GLS2_002_T7R CAGTAATACGACTCACTATAGGGAGAAGGCTAC SEQIDNO:17 TCTCATTAAACAACCTTACCCTTT GLS2_003_10F AGGAAGAGAGGAGTTGGAATGGATAATGTTTAG SEQIDNO:18 GTT GLS2_003_T7R CAGTAATACGACTCACTATAGGGAGAAGGCTCC SEQIDNO:19 CAATACCTCCCTAAAATACTAATC GLS2_004_10F AGGAAGAGAGAAAGGGTAAGGTTGTTTAATGA SEQIDNO:20 GAGT GLS2_004_T7R CAGTAATACGACTCACTATAGGGAGAAGGCTCA SEQIDNO:21 CAAACTAAAACCCAAACTTCCTA GLS2_005_10F AGGAAGAGAGTAAGGTTTTTGGGATAGGGTAGG SEQIDNO:22 GLS2_005_T7R CAGTAATACGACTCACTATAGGGAGAAGGCTAC SEQIDNO:23 ACAAACCCCAATACACCTAAAC GLS2_006_10F AGGAAGAGAGTGAAGGTTTTGTAGAAGGTTTTG SEQIDNO:24 AG GLS2_006_T7R CAGTAATACGACTCACTATAGGGAGAAGGCTCC SEQIDNO:25 ACAATAAAAATAAAAACAAACCC GLS2_007_10F AGGAAGAGAGTGATTGGATTTGGGTATTGTTTA SEQIDNO:26 TT GLS2_007_T7R CAGTAATACGACTCACTATAGGGAGAAGGCTCT SEQIDNO:27 CAAAACCTTCTACAAAACCTTCA GLS2_008_10F AGGAAGAGAGGTTTAATGTTTTTTTGAGGTGTTG SEQIDNO:28 G GLS2_008_T7R CAGTAATACGACTCACTATAGGGAGAAGGCTAA SEQIDNO:29 AATAAACAATACCCAAATCCAATC GLS2_009_10F AGGAAGAGAGTAAATAAATGGTAGAGGGATAT SEQIDNO:30 AAATTGG GLS2_009_T7R CAGTAATACGACTCACTATAGGGAGAAGGCTTT SEQIDNO:31 CAAAAAATCACTCAAAAAACCTC
Genomic DNA Isolation and DNA Methylation Analysis
[0153] High molecular weight genomic DNA was isolated from breast cancer cells using QIAamp DNA Mini Kit (Qiagen), and contaminating RNA was digested using RNase One Ribonuclease (Promega) followed by re-purification of DNA, elution with water, and adjustment of DNA concentration to 50 ng/l. To assess DNA purity, UV/visible absorption spectra were measured, and for all samples the A260/280 ratio was >1.7 and the A260/230 ratio was >2.0. Samples were then submitted to the Epigenomics Core at Weill Cornell Medical College, where bisulfite conversion was carried out followed by DNA methylation analysis using Mass ARRAY EpiTYPER1.2 Suite (Agena Bioscience). The sequence of the CpG island in the GLS2 gene promoter was obtained from human reference genome GRCh38/hg38 using the UCSC Genome Browser (https://genome.ucsc.edu). Primers for DNA methylation analysis were designed using EpiDesigner (Agena Bioscience) and synthesized by Integrated DNA Technologies (Table 4). Relative methylation ratios at CpG sites are presented as a heatmap, generated using MORPHEUS (https://software.broadinstitute.org/morpheus).
Immunofluorescence
[0154] Cells were fixed with 3.7% formaldehyde in PBS for 20 min and then permeabilized with PBS containing 0.1% Triton X-100 (v/v) for 20 min. Samples were blocked with 10% BSA (w/v) in PBS for 1 hour, rinsed with PBS and then incubated with primary antibody in PBS containing 5% BSA (w/v) for 2 hours. Samples were washed 3 times with PBS, and then Texas Red- or Oregon Green 488-conjugated secondary antibody diluted 1:400 in PBS containing 5% BSA (w/v), along with DAPI counterstain, was added for 1 hour. Samples were washed 3 times with PBS, mounting medium was applied, and slides were sealed with a cover slip prior to imaging with a ZEISS Axioscope.
Cell Fractionation and Mitochondrial Isolation
[0155] Breast cancer cells were fractionated into cytosolic, mitochondrial, and nuclear components via partial lysis and centrifugation using the Qproteome Mitochondria Isolation Kit (Qiagen). For all samples, a portion of the starting material (i.e. whole cells) was retained for comparison with the isolated fractions. When cellular fractions were to be analyzed only by western blot, a single 10 cm dish of exponentially growing cells was used. When mitochondria were isolated for glutaminase assays, two 15 cm dishes of exponentially growing cells were used.
Mitochondrial Glutaminase Activity Assays
[0156] A two-reaction protocol was used to measure mitochondrial glutaminase activity. Mitochondria (5 pg total protein) were added to 105 l of Reaction Mix 1 (20 mM glutamine, 0.2 mM EDTA, 50 mM Tris-acetate pH 8.6), supplemented with 10 M BPTES when appropriate, and samples were incubated at 37 C. for 40 min. The reaction was then quenched by addition of 10 l of 2.4 M HCl, and samples placed on ice. Next, 20 l of quenched Reaction Mix 1 was added to 200 l of Reaction Mix 2 (1 unit bovine liver glutamate dehydrogenase (Sigma-Aldrich), 80 mM Tri-HCl pH 9.4, 200 mM hydrazine, 0.25 mM ADP, 2 mM NAD) and samples were incubated for 1 hour at room temperature. The absorbance at 340 nm was then measured against a matched sample in which heat-inactivated mitochondria (immersed in boiling water for 5 minutes) were used. A standard curve was prepared using given concentrations of glutamate in Reaction Mix 2, allowing the amount of glutamate produced in Reaction 1 to be determined.
Real-Time Recombinant Glutaminase Activity Assays
[0157] Real-time monitoring of glutaminase activity through NADH production was performed on a Cary Eclipse fluorescence spectrometer. The excitation and emission wavelengths were set at 340 nm and 460 nm, respectively. To a 1 ml cuvette, 900 l of assay buffer was added, followed by 10 l of GDH, 40 l of 50 mM NAD and 20 l of either DMSO or various dilutions of 968. Then, 100 l of either 100 nM GAC or 500 nM GLS2 was added to this mixture and the fluorescence emission was monitored in real time. After 30 s, 200 l of a mixture of glutamine and K.sub.2HPO.sub.4 was added such that the final concentrations of K.sub.2HPO.sub.4 and glutamine were 100 mM and 20 mM, respectively. The initial velocity of glutamine hydrolysis was obtained from the slopes of the linear portion of the kinetic curve.
Glutamine Consumption and Glutamate Release Assays
[0158] To 6-well plates containing 2 ml phenol red-free culture medium/well, 210.sup.5 cells/well were added and incubated overnight to attach. Wells were then rinsed twice with serum-free, phenol red-free culture medium, and 2 ml/well fresh serum-free/phenol red-free medium (containing 2 mM glutamine) was added, followed by incubation for 19 hours. As a negative control, wells containing culture medium only were used. Medium was then collected, cellular debris removed by centrifugation at 4 C., and the supernatant retained and stored on ice. Meanwhile, cells attached to the wells were lysed and total protein was quantified using the Bradford assay. Glutamine concentrations were determined using the L-Glutamine/Ammonia Assay Kit (Rapid) (Megazyme) following the manufacturer's instructions. Briefly, 50 l sample was mixed with 100 l Assay Buffer 1 and 10 l Glutaminase Suspension and incubated at room temperature for 5 min. For all reactions, a blank containing 50 l H.sub.2O was run in parallel. Then, 150 l. Assay Buffer 2, 100 l NADPH Solution, and H.sub.2O to bring the final volume to 1160 l was added, followed by incubation at room temperature for 4 min. Absorbance A.sub.1 was then measured at 340 nm. Next, 10 l. Glutamate Dehydrogenase Suspension was added, samples were mixed and incubated at room temperature for 5 min, and absorbance A.sub.2 was measured at 340 nm. Sample concentrations of glutamine were calculated using the extinction coefficient of NADPH at 340 nm. Changes in sample glutamine concentrations were measured relative to the culture medium samples which had been incubated in cell-free wells. To measure glutamate levels in culture medium, samples were analyzed by Reaction 2 described above for the mitochondrial glutaminase activity assays.
Cell Proliferation Assays
[0159] Culture medium was added to 12-well plates (1 ml/well) and wells were seeded with cells at Day 0 as follows. MCF7, T-47D, BT-474, HCC38 cells: 210W cells/well. MDA-MB-453 and MDA-MB-231 cells: 110.sup.4 cells/well. TSE and Hs 578T cells: 0.310.sup.4 cells/well. After 16 hours, culture medium was replaced with fresh medium supplemented with appropriate concentrations of inhibitors and was subsequently replaced every 48 hours. On Day 6 cells were trypsinized and suspended in an appropriate volume of culture medium, and the total number of cells per well was determined using a hemocytometer or a TC20 Automated Cell Counter (Bio-Rad).
DNA Constructs for Expressing GLS and GLS2
[0160] Vectors for expressing human GAC or GLS2 in breast cancer cell lines were based on pCDNA3.1/V5-His TOPO (Thermo Fisher Scientific), with the appropriate gene sub-cloned in and the tag switched to HA-tag or myc-tag for immunofluorescence experiments. Vectors pQE80-GAC-72-598 and pQE80-GLS2-38-602, for expressing the processed forms of human GAC (residues 72 to 598) or GLS2 (residues 38 to 602) in E. coli, were described in Huang et al., Characterization of the Interactions of Potent Allosteric Inhibitors with Glutaminase C, a Key Enzyme in Cancer Cell Glutamine Metabolism, J. Biol. Chem. 293:3535-3545 (2018), which is hereby incorporated by reference in its entirety.
Expression and Purification of Recombinant GAC and GLS2
[0161] Recombinant GAC and GLS2 were expressed in E. coli as described in Huang et al., Characterization of the Interactions of Potent Allosteric Inhibitors with Glutaminase C, a Key Enzyme in Cancer Cell Glutamine Metabolism, J. Biol. Chem. 293:3535-3545 (2018), which is hereby incorporated by reference in its entirety. Briefly, E. coli strain BL21(DE3) (Thermo Fisher Scientific) were transformed with vector pQE80-GAC-72-598 or with pQE80-GLS2-38-602 to express the processed forms of GAC and GLS2, respectively. For both constructs, expression was induced with 0.3 mM IPTG for 20 hours at 18 C., and cells were then harvested by centrifugation and lysed by sonication in binding buffer (500 mM NaCl, 50 mM Tris-HCl pH 8.5, 10 mM imidazole, 5 mM -mercaptoethanol, 1 mM benzamidine chloride). The lysate was centrifuged, and the supernatant applied to a Ni-NTA column which was then washed with 100 column volumes of binding buffer followed by 10 volumes of wash buffer (500 mM NaCl, 50 mM Tris-HCl pH 8.5, 40 mM imidazole, 5 mM -mercaptoethanol, 1 mM benzamidine chloride). Protein was eluted with 5 column volumes of elution buffer (500 mM NaCl, 300 mM imidazole-HCl pH 7.5, 5 mM -mercaptoethanol, 1 mM benzamidine chloride). The eluate was centrifugally concentrated and then further purified by FPLC using a HiLoad 16/600 Superdex 200 column (GE Healthcare) with 150 mM NaCl, 5 mM Tris-HCl pH 7.5.
Transfection of Breast Cancer Cells with DNA Constructs
[0162] For 60 mm dish format. 0.2 ml Opti-MEM (Gibco) containing 1.5 pg of the appropriate DNA construct, along with 0.2 ml Opti-MEM containing 12 l Lipofectamine 2000 (Invitrogen), were separately incubated at room temperature for 5 min. The two solutions were combined and incubated for an additional 20 min, mixed with 1.6 ml culture medium and added to cells. After 5 hours incubation at 37 C. the transfection mixture was replaced with fresh culture medium, and cells were then incubated for an additional 48 hours to allow for ectopic expression of GLS or GLS2. To select for cells stably expressing the DNA construct, culture medium supplemented with 500 g/ml G-418 disulfate (Research Products International) was added and replaced every 2 days for 2-3 weeks until isolated colonies 2 mm in diameter were present. Individual colonies were transferred to a 12-well plate (1 colony per well) using sterile blotting paper soaked in trypsin solution and were then cultured in medium supplemented with 250 g/ml G-418 disulfate. All colonies were screened by western blot for ectopic expression, and positive clones were maintained in medium supplemented with 250 g/ml G-418 disulfate.
Genetic Knockdowns Using siRNAs
[0163] Transient knockdowns of GATA3, GLS, and GLS2 were achieved using two rounds of transfection with Silencer Select pre-designed siRNAs (Invitrogen). For 60 mm dish format, 0.3 ml Opti-MEM (Gibco) containing 100 nM of the appropriate siRNA (to give a final siRNA concentration of 10 nM when diluted as below), along with 0.3 ml Opti-MEM containing 12 l Lipofectamine 2000 (Invitrogen), were incubated separately at room temperature for 5 min. The two solutions were then combined and incubated for an additional 20 min, mixed with 2.4 ml culture medium, and added to cells. After 5 hours incubation at 37 C. the transfection mixture was replaced with fresh culture medium. For all knockdowns, two independent siRNAs were used, along with a negative control siRNA.
Genetic Knockdowns Using shRNAs
[0164] The MISSION RNAi system (Sigma-Aldrich) was used for shRNA-mediated knockdown of GLS and GLS2. Lentivirus particles for each shRNA construct were generated using exponentially-growing 293T cells (ATCC) as follows. For 10 cm dish format, 570 l DMEM was mixed with 33 l FuGENE 6 (Promega) and incubated at room temperature for 5 min. Plasmids pLKO.1-shRNA (5 g), pCMV-dR8.2 (packaging vector) (5 g), and pMD2.G (envelope vector) (1 gg) were then added to the solution, incubated for an additional 15 min, mixed with 8 ml culture medium and added to cells. Cells were incubated at 37 C. overnight and the transfection medium was then replaced with fresh culture medium, followed by an additional 24 hours incubation to allow for production of virus particles. Virus-containing medium was then collected, and cellular debris removed by centrifugation. To transduce breast cancer cells, virus-containing supernatant was diluted 1:12 in fresh culture medium, and 6 g/ml polybrene was added before applying to cells. After 6 hours incubation at 37 C. the transduction medium was replaced with fresh culture medium, and cells were incubated for an additional 48 hours before knockdowns were validated. For both GLS and GLS2 knockdowns, two independent shRNA constructs were used (see Table 5), and for all experiments the effects of knockdown were compared with those of a control shRNA. To select for stable expression of the constructs, cells were cultured in medium containing 0.5 g/ml puromycin.
TABLE-US-00005 TABLE5 ListofshRNAConstructs Sigma-Aldrich Name ProductNo. TargetSequence SequenceID GLSshRNA1 TRCN0000051135 GCACAGACATGGTTGGTATAT SEQIDNO:32 GLSshRNA2 TRCN0000298987 GCACAGACATGGTTGGTATAT SEQIDNO:33 GLS2shRNA1 TRCN0000051324 GCCATGGATATGGAACAGAAA SEQIDNO:34 GLS2shRNA2 TRCN0000051326 GCCCTGTCCAAAGAGAACTTA SEQIDNO:35
Data from the Cancer Genome Atlas
[0165] Gene expression data (RNA-Seq V2, RSEM) from TCGA invasive breast cancer dataset (Koboldt et al., Comprehensive Molecular Portraits of Human Breast Tumours, Nature 490:61-70 (2012), which is hereby incorporated bt reference in its entirety) were accessed using UCSC Xena (https://xena.ucsc.edu) or cBioPortal (www.cbioportal.org). Breast tumor subtype calls made by UCSC Xena were based on RNA-Seq data. Outlier readings are not shown on box and whisker plots but are included in calculation of the mean. Copy-number analysis data were accessed using cBioPortal.
Metabolite Extraction
[0166] The procedures for metabolite extraction from cultured cells are described in previous studies (Cluntun et al., The Rate of Glycolysis Quantitatively Mediates Specific Histone Acetylation Sites, Cancer Metab. 3:10 (2015); Liu et al., A Strategy for Sensitive, Large Scale Quantitative Metabolomics, J. Vis. Exp. e51358-e51358 (2014), which are hereby incorporated by reference in their entirety). Briefly, adherent cells were grown in 6-well plates in biological triplicate to 80% confluence, medium was rapidly aspirated and cells were washed with cold PBS on ice. Then, 1 ml of extraction solvent (80% methanol/water) cooled to 80 C. was added to each well, and the dishes were transferred to 80 C. for 15 min. Cells were then scraped into the extraction solvent on dry ice. All metabolite extracts were centrifuged at 20,000g at 4 C. for 10 min. Finally, the solvent in each sample was evaporated in a Speed Vacuum. The cell extracts were dissolved in 15 l water and 15 l methanol/acetonitrile (1:1 v/v) (LC-MS optima grade, Thermo Fisher Scientific). Samples were centrifuged at 20,000g for 10 min at 4 C. and the supernatants were transferred to Liquid Chromatography (LC) vials. The injection volume for polar metabolite analysis was 8 l.
[0167] For metabolite abundances in
[U-.SUP.13.]-Glutamine Labeling
[0168] Cells were grown to 80% confluence in 6-well plates with standard culture medium and washed with sterile PBS. Then, culture medium in which glutamine was replaced by [.sup.13C.sub.5]-L-glutamine (Cambridge Isotope Laboratories), supplemented with dialyzed FBS (Gibco) and appropriate concentrations of inhibitors was added (1.5 ml/well). At the appropriate time-point, metabolites were extracted as described above.
Liquid Chromatography
[0169] A hydrophilic interaction liquid chromatography method (HILIC) with an Xbridge amide column (1002.1 mm, 3.5 m) (Waters) was employed on a Dionex (Ultimate 3000 UHPLC) for compound separation and detection at room temperature. The mobile phase A was 20 mM ammonium acetate and 15 mM ammonium hydroxide in water with 3% acetonitrile, pH 9.0, and the mobile phase B was acetonitrile. The linear gradient was as follows: 0 min, 85% B; 1.5 min, 85% B, 5.5 min, 35% B; 10 min, 35% B, 10.5 min, 35% B, 14.5 min, 35% B, 15 min, 85% B, and 20 min, 85% B. The flow rate was 0.15 ml/min from 0 to 10 min and 15 to 20 min, and 0.3 ml/min from 10.5 to 14.5 min. All solvents were LCMS grade and purchased from Thermo Fisher Scientific.
[0170] For metabolite abundances in
Mass Spectrometry
[0171] The Q Exactive MS (Thermo Scientific) is equipped with a heated electrospray ionization probe (HESI), and the relevant parameters are as listed: evaporation temperature, 120 C.; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.6 kV for positive mode and 2.5 kV for negative mode. Capillary temperature was set at 320 C., and S-lens was 55. A full scan range from 60 to 900 (m/z) was used. The resolution was set at 70,000. The maximum injection time was 200 ms. Automated gain control (AGC) was targeted at 3,000,000 ions.
[0172] For metabolite abundances in
Metabolomics and Data Analysis
[0173] Raw data collected from LC-Q Exactive MS were processed on Sieve 2.0 (Thermo Scientific) and ToxID 2.0 (Thermo Scientific). Peak alignment and detection were performed according to the protocol described by Thermo Scientific. For targeted metabolite analysis, the method peak alignment and frame extraction was applied. An input file of theoretical m/z and detected retention time of 204 known metabolites was used for targeted metabolites analysis with data collected in positive mode, while a separate input file of 278 metabolites was used for negative mode. M/Z width was set at 10 ppm. The output file including detected m/z and relative intensity in different samples was obtained after data processing. The quantity of the metabolite fraction analyzed was adjusted to the corresponding protein concentration and cell count upon processing a parallel 6-well plate. Quantitation and statistics were calculated and visualized with Microsoft Excel, MORPHEUS and MetaboAnalyst online software.
Example 1Luminal Breast Cancers Use Glutamine Anaplerosis but Resist GLS Inhibitors
[0174] The most extensively studied inhibitors of GLS are based on the BPTES molecular scaffold, with the potent analog CB-839 currently in clinical trials for a number of malignancies. CB-839 was originally reported to be effective against triple-negative breast cancer (TNBC) cells (Gross et al., Antitumor Activity of the Glutaminase Inhibitor CB-839 in Triple-negative Breast Cancer, Mol. Cancer Ther. 13:890-901 (2014), which is hereby incorporated by reference in its entirety), which are characterized by low expression of the receptors ER, PR, and HER2. Across a collection of breast cancer cell lines, it was observed that basal-subtype cells respond to BPTES or CB-839 treatment, whereas luminal-subtype cells resist these inhibitors, regardless of their specific receptor status (
TABLE-US-00006 TABLE 6 Description of breast cancer cell lines. Reported Reported receptor status Cell line subtype ER PR HER2 Notes MCF7 Luminal + + A BT-474 Luminal + + + ERBB2 gene amplified, but inverse B correlation to HER2+ molecular subtype. T-47D Luminal + + A MDA-MB- Luminal +/ ERBB2 gene amplified but not 453 (LAR) overexpressed. Luminal androgen receptor (LAR) triple-negative breast cancer. MDA-MB- Basal Basal B and Claudin-low subgroups. 231 TSE Basal Triple-negative with strong basal markers. Hs 578T Basal Basal B and Claudin-low subgroups. HCC38 Basal Basal B and Claudin-low subgroups. DU4475 Basal Derived from a metastatic lesion of the breast tumor located at the skin.
TABLE-US-00007 TABLE 7 EC.sub.50 values for BPTES and CB-839 derived from 6-day cell proliferation assay. BPTES CB-839 Cell line EC.sub.50/M EC.sub.50/nM MCF7 ~20 >1000 BT-474 ~20 >1000 T-47D ~20 >1000 MDA-MB-453 ~20 >1000 MDA-MB-231 2.1 19 TSE 3.1 41 Hs 578T 3.7 31 HCC38 4.7 49
[0175] However, the sensitivity of breast cancer cells to GLS inhibitors does not correspond to their rate of glutamine consumption or to their expression of SLCIA5 (
Example 2Expression of GLS2 Is Elevated in Luminal-Subtype Breast Cancers
[0176] Previously, the glutaminase isozyme GLS2 has been described as a tumor suppressor in some contexts, with downregulated expression in liver and brain cancers (Mats et al., Glutaminase Isoenzymes in the Metabolic Therapy of Cancer, Biochim. Biophys. ActaRev. Cancer 1870:158-164 (2018), which is hereby incorporated by reference in its entirety). However, since luminal-subtype breast cancer cells resist GLS inhibitors yet still exhibit glutamine-mediated anaplerosis, it was hypothesized that they might instead be dependent on GLS2. The Cancer Genome Atlas (TCGA) invasive breast cancer dataset (Koboldt et al., Comprehensive Molecular Portraits of Human Breast Tumours, Nature 490:61-70 (2012), which is hereby incorporated by reference in its entirety) was used to examine GLS2 transcript levels in the breast cancer molecular subtypes luminal A (LumA), luminal B (LumB), HER2+, and basal. Expression of GLS2 is indeed substantially higher in LumA and LumB tumors than in basal-subtype tumors, which instead have high levels of the GLS transcript (
[0177] To compare protein levels of GLS2 in breast tumors and normal mammary tissue, a tissue microarray was probed (
Example 3GLS2 Is Localized to Mitochondria in Breast Cancer Cells
[0178] Although GLS2 contains a predicted mitochondrial localization signal (Katt et al., A Tale of Two Glutaminases: Homologous Enzymes with Distinct Roles in Tumorigenesis, Future Med. Chem. 9:223-243 (2017), which is hereby incorporated by reference in its entirety), several subcellular localizations have been reported, including the nucleus in neurons and astrocytes and as a binding partner of the plasma membrane/cytosolic protein Rac1 in liver cancer cells (Cardona et al., Expression of GIs and Gls2 Glutaminase Isoforms in Astrocytes, Glia 63:365-382 (2015); Zhang et al., Glutaminase 2 is a Novel Negative Regulator of Small GTPase Rac1 and Mediates p53 Function in Suppressing Metastasis, Elife 5:e10727 (2016), which are hereby incorporated by reference in their entirety). To establish the localization of GLS2 in breast cancer cells, both MDA-MB-453 (high-GLS2) and MDA-MB-231 cells (high-GLS) were fractionated, and western blot analysis performed on the whole-cell lysates along with the cytosolic, mitochondrial, and nuclear fractions. As markers for these fractions, the samples were also probed for the cytosolic enzyme asparagine synthetase (ASNS), the mitochondrial ion channel VDAC, and the nuclear envelope protein lamin A. Both GLS2 and GLS, along with VDAC, were detected almost exclusively in the mitochondrial fractions of both cell lines (
Example 4GLS2 Expression Is Regulated by GATA3 and Promoter Methylation
[0179] To understand the upregulation of GLS2 in luminal-subtype breast cancers, the mechanisms influencing GLS2 gene expression was investigated. Data from TCGA show a high frequency of copy-number gains at the GLS2 gene locus in luminal-subtype and HER2+ breast tumors (as high as 37% copy-number gain, 2% gene amplification, in the case of LumB), but not in basal-subtype tumors (
[0180] Across the cell lines there is no clear association between GLS2 levels and any one of the receptors ER, PR, or HER2, with MDA-MB-453 cells, which have low expression of all three receptors, having the highest GLS2 levels (
[0181] Because GLS2 levels are so low in basal-subtype breast cancers, it was also tested if expression is epigenetically silenced in these cells via methylation of the GLS2 gene promoter. After isolating genomic DNA from the basal-subtype cell lines with the lowest levels of GLS2 transcript (TSE and Hs 578T) and the luminal-subtype cell lines with the highest levels (T-47D and MDA-MB-453), the MassARRAY system was used to quantify methylation levels at sites in the CpG island centered on the TSS of the GLS2 gene (
Example 5GLS2 Mediates Glutamine Anaplerosis in Luminal-Subtype Cells
[0182] To probe the role of GLS2 in luminal-subtype cells, it was examined whether GLS2 is involved in the observed supply of glutamine-derived carbon to the TCA cycle (
Example 6GLS2 Is Essential in Luminal-Subtype Breast Cancer Cells
[0183] Because various functions have been reported for GLS2 in cancer, including tumor suppressive activity in liver cancer and glioblastoma (Mates et al., Glutaminase Isoenzymes in the Metabolic Therapy of Cancer, Biochim. Biophys. ActaRev. Cancer 1870:158-164 (2018), which is hereby incorporated by reference in its entirety), the importance of GLS2 for breast cancer cell proliferation and tumorigenesis was investigated. Luminal- and basal-subtype cell lines were transfected with either a control siRNA, or siRNAs selectively targeting GLS2, GLS, or both isozymes simultaneously. Western blot analysis 48 hours after transfection confirmed that potent and selective knockdowns had been achieved (
[0184] Next, the importance of GLS2 for luminal-subtype breast tumor growth in vivo was addressed. This experiment required stable rather than transient depletion of GLS2, but potent shRNA-mediated knockdowns severely impacted luminal-subtype cell viability after several days. Therefore, a low titer of virus was used for transducing MDA-MB-453 cells and generated stable GLS2 partial-knockdown cell lines (
Example 7GLS2 Mediates Resistance to GLS Inhibitors
[0185] The BPTES class of inhibitors have been extensively studied and are highly selective for GLS over GLS2 (
[0186] To gain further insight into the glutaminase expression profiles of breast cancer cells, western blot analyses were performed using known amounts of purified, recombinant GLS and GLS2 to estimate absolute levels of each glutaminase in cells (
[0187] Next cells were treated with 10 M BPTES in culture medium containing [U-.sup.13C]-glutamine and extracted and analyzed cellular metabolites. Consistent with the data above, BPTES treatment potently inhibited the supply of glutamine-derived carbon to glutamate and the TCA cycle in the basal-subtype cell lines Hs 578T and MDA-MB-231 (
[0188] Using MDA-MB-231 and TSE cells, which express almost exclusively GLS, derivative cell lines were generated that ectopically express GLS2 (
TABLE-US-00008 TABLE 8 EC50 values for BPTES and 968, derived from 6-day cell proliferation assay. Cell line BPTES EC.sub.50/M 968 EC.sub.50/M MDA-MB-231 (parental) 2 4 MDA-MB-231 vector only 1.3 2 MDA-MB-231 GLS2 clone 1 >20 4 MDA-MB-231 GLS2 clone 2 >20 5 TSE (parental) 3 4 TSE vector only 4 2 TSE GLS2 clone 1 >20 7 TSE GLS2 clone 2 >20 6 MCF7 ~20 4 BT-474 ~20 4 T-47D ~20 4 MDA-MB-453 ~20 3 MDA-MB-231 2.1 5 TSE 3.1 5 Hs 578T 3.7 4* HCC38 4.7 4** *Maximal inhibition ~75% **Maximal inhibition ~70%
[0189] Then a search was conducted for basal-subtype breast cancer cells that are intrinsically resistant to GLS-selective inhibitors, to test if resistance can be overcome by simultaneously targeting GLS2. The triple-negative, basal-subtype, breast cancer cell line DU4475, which was originally derived from a metastatic lesion, is highly resistant to BPTES treatment, with an EC.sub.50 value >20 M (
Example 8-968 Inhibits GLS2 and Suppresses BPTES-Resistant Breast Cancer Growth
[0190] Previously, it was reported that the small molecule 968 binds and inhibits the GLS splice variant GAC (Wang et al., Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation, Cancer Cell 18:207-219 (2010), which is hereby incorporated by reference in its entirety). This inhibitor has a much higher affinity for monomeric GAC than for the active tetramer, and is proposed to bind to newly-synthesized enzyme monomers and prevent the formation of activated tetramers (Stalnecker et al., Mechanism by which a Recently Discovered Allosteric Inhibitor Blocks Glutamine Metabolism in Transformed Cells, Proc. Natl. Acad. Sci. U.S.A. 112:394-399 (2014), which is hereby incorporated by reference in its entirety). However, the sensitivity of GLS2 to 968 has not previously been tested. Therefore, the effect of 968 on the activity of purified, recombinant, glutaminase was measured. In contrast to BPTES-class inhibitors, which are highly selective for GLS, 968 inhibited both GLS and GLS2, with a moderate (>3-fold) selectivity for GLS2 (
[0191] Treatment of breast cancer cells with 968 inhibited proliferation with EC.sub.50 values of 3-5 M (
[0192] Next basal- and luminal-subtype breast cancer cells were treated with 10 M 968 and extracted metabolites for analysis at different time points. Consistent with the mechanism of 968 binding to monomeric glutaminase and preventing the formation of active tetramers, a time-dependent inhibition of glutamine-mediated TCA cycle anaplerosis was observed (
[0193] Since 968 inhibits the proliferation of breast cancer cells ex vivo but does not affect the growth of primary human mammary epithelial cells or fibroblasts (Wang et al., Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation, Cancer Cell 18:207-219 (2010), which is hereby incorporated by reference in its entirety), it was tested if it can be used to treat luminal-subtype breast tumor growth in vivo. 310.sup.6 MDA-MB-453 cells in Matrigel suspension was injected into each flank of NSG mice (n=6 xenografts per condition) and waited until palpable tumors 1-2 mm in diameter were present (14 days). At this point, mice were separated into two groups, with one group receiving subcutaneous injections of 968 (10 mg/kg body weight) 3 times per week, and the other group receiving carrier solution only. The tumor growth was monitored for three weeks after initiating treatment and found that it was robustly inhibited in 968-treated animals (
Discussion of Examples 1-8
[0194] Efforts to target glutamine catabolism for cancer therapy have focused on inhibiting the glutaminase isozyme GLS, which is highly expressed and onco-supportive in diverse malignancies (Cluntun et al., Glutamine Metabolism in Cancer: Understanding the Heterogeneity, Trends in Cancer 3:169-180 (2017), which is hereby incorporated by reference in its entirety). The importance of the other mammalian glutaminase, GLS2, in tumorigenesis has remained less clear, and various subcellular localizations and functions have been described, including tumor suppressor activity (Mates et al., Glutaminase Isoenzymes in the Metabolic Therapy of Cancer, Biochim. Biophys. ActaRev. Cancer 1870:158-164 (2018), which is hereby incorporated by reference in its entirety). It is reported herein that GLS2 is upregulated in luminal-subtype/receptor-positive breast cancers, where it is essential for glutamine-mediated TCA cycle anaplerosis, cell proliferation, and tumorigenesis. These findings explain the identification of GLS2 as one of only 16 metabolic enzymes required for tumorigenesis in an earlier functional genomics screen (Possemato et al., Functional Genomics Reveal that the Serine Synthesis Pathway is Essential in Breast Cancer, Nature 476:346-350 (2011), which is hereby incorporated by reference in its entirety).
[0195] Previous studies found that triple-negative, but not receptor-positive, breast cancer cell lines often express high levels of the GLS splice variant GAC (Gross et al., Antitumor Activity of the Glutaminase Inhibitor CB-839 in Triple-negative Breast Cancer, Mol. Cancer Ther. 13:890-901 (2014), which is hereby incorporated by reference in its entirety). The results herein indicate that, rather than being directly dictated by the cellular receptor status, differences in glutaminase expression in breast cancer correspond to the intrinsic molecular subtype (
[0196] These findings highlight the importance of considering GLS2 when identifying target diseases for treatment with GLS-selective inhibitors. Gene expression data from TCGA show that GLS2 transcript levels are consistently upregulated relative to healthy tissue in colorectal tumors and also in a subset of lung tumors. With clinical trials underway to evaluate the efficacy of CB-839 against these cancers, it will be important to characterize further the function of GLS2 in these contexts. In pancreatic ductal adenocarcinoma (PDAC) cells, GLS inhibitors have only a temporary cytostatic effect which is followed by metabolic adaptation and recovery of proliferation (Biancur et al., Compensatory Metabolic Networks in Pancreatic Cancers Upon Perturbation of Glutamine Metabolism, Nat. Commun. 8:15965 (2017), which is hereby incorporated by reference in its entirety). Notably, GLS2 is present in both healthy pancreas and PDAC cells (Altman et al., From Krebs to Clinic: Glutamine Metabolism to Cancer Therapy, Nat. Rev. Cancer 16:619-634 (2016); Biancur et al., Compensatory Metabolic Networks in Pancreatic Cancers Upon Perturbation of Glutamine Metabolism, Nat. Commun. 8:15965 (2017), which are hereby incorporated by reference in their entirety), and thus might provide a critical supply of glutamate following GLS inhibition. The sensitivity of some cancer cells to GLS inhibitors requires high expression of the xCT antiporter, which exchanges intracellular glutamate for extracellular cystine and can therefore deplete intracellular glutamate reserves (Muir et al., Environmental Cystine Drives Glutamine Anaplerosis and Sensitizes Cancer Cells to Glutaminase Inhibition, Elife 6:e27713 (2017), which is hereby incorporated by reference in its entirety). Thus, a signature of concurrent high expression of GLS and xCT, along with low levels of GLS2, might identify tumors that are most likely to respond to GLS-targeted therapy.
GLS2 as a Potential Therapeutic Target
[0197] In healthy tissues GLS2 expression is highest in periportal regions of the liver, where it allows glutamine carbon to be directed via the TCA cycle into the gluconeogenic pathway in response to glucagon (Lacey et al., Increased Activity of Phosphate-Dependent Glutaminase in Liver Mitochondria as a Result of Glucagon Treatment of Rats, Biochem. J. 194:29-33 (1981); Watford and Smith, Distribution of Hepatic Glutaminase Activity and mRNA in Perivenous and Periportal Rat Hepatocytes, Biochem. J. 267:265-267 (1990), which are hereby incorporated by reference in their entirety). For GLS2 to be targeted for cancer therapy, any toxicity arising from inhibiting its normal physiological function must be within a tolerable range. It was observed that mice treated with 968 at 10 mg/kg body weight, three times weekly for three weeks, showed no gross evidence of toxicity. Moreover, it was recently reported that GLS2 knockout mice are viable, albeit with a decreased ability to maintain plasma glucose levels during fasting (Miller et al., Targeting Hepatic Glutaminase Activity to Ameliorate Hyperglycemia, Nat. Med. 24:518-524 (2018), which is hereby incorporated by reference in its entirety). These results indicate that GLS2 could be safely targeted as a strategy for treating luminal-subtype breast cancers, most likely as part of a combination therapy designed to maximize cancer cell dependence on the glutaminase reaction. To date, drug discovery efforts for blocking glutamine catabolism in cancer have focused almost exclusively on the GLS isozyme. However, a small number of molecular scaffolds have now been reported to inhibit GLS2 (Wu et al., Glutaminase Inhibitors: A Patent Review, Expert Opin. Ther. Pat. 28:823-835 (2018), which is hereby incorporated by reference in its entirety), including 968, which is shown to target both isozymes with a moderate (3-fold) selectivity for GLS2. The finding that GLS2 is upregulated and essential in the most prevalent subtypes of breast cancer support the notion of building on these scaffolds to develop more potent inhibitors for selective targeting of GLS2-high cancers.
[0198] Humans have two genes encoding glutaminase enzymes, GIS and GLS2. Efforts to target glutamine catabolism for cancer therapy have focused on GLS, an inhibitor of which (CB-839) is currently in clinical trials. The GLS2 isozyme has previously been described as a tumor suppressor, with downregulated expression in liver cancer. It is reported herein that GLS2 is overexpressed and essential for growth in the most prevalent subtypes of breast cancer, luminal A and B. Although GLS2 is insensitive to CB-839-class drugs, it is inhibited by the small molecule 968, which suppresses breast tumor growth in vivo. These findings establish a critical role for GLS2 in breast cancer and advance the understanding of how to target aberrant glutamine metabolism for cancer therapy generally.
[0199] Preferences and options for a given aspect, feature, embodiment, or parameter of the methods described herein should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features, embodiments, and parameters of the methods described herein.
[0200] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the application and these are therefore considered to be within the scope of the application as defined in the claims which follow.