LACTATE DEHYDROGENASE INHIBITOR POLYPEPTIDES FOR USE IN THE TREATMENT OF CANCER
20220289791 · 2022-09-15
Assignee
Inventors
- Pierre Sonveaux (Sterrebeek, BE)
- Raphaël Frédérick (Godinne, BE)
- Léopold THABAULT (Ixelles, BE)
- Lucie BRISSON (Tours, FR)
- Tamara COPETTI (Trieste, IT)
Cpc classification
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
A polypeptide that modulates the activity of at least one isoform of the native tetrameric lactate dehydrogenase, and the use thereof as a medicament for the treatment of a cancer. More particularly, linear and cyclic polypeptides that inhibit the tetramerization of the lactate dehydrogenase subunits, and compositions and kits including the polypeptides.
Claims
1-15. (canceled)
16. A polypeptide that inhibits the tetramerization of the lactate dehydrogenase subunits, said polypeptide comprising the amino acid sequence of formula (I)
X1-X2-X3-X4-X5-X6-X7-X8 (I) (SEQ ID NO: 5), wherein: X1 represents any amino acid residue, preferentially selected from the group consisting of amino acid residues A, G, K and C; X2 represents C, T or S; X3 represents C, L, A, T, cpA (cyclopropyl-L-alanine), chG (L-cyclohexylglycine), chA (cyclohexyl-L-alanine) or mlL (γ-Methyl-L-leucine); X4 represents any amino acid residue, preferentially a positively charged or neutral amino acid residue, preferentially selected from the group consisting of amino acid residues K, C, A and Aib (2-aminoisobutyric acid), and more preferentially amino acid K; X5 represents any amino acid residue, preferentially a negatively or positively charged or neutral amino acid residue, preferentially selected from the group consisting of amino acid residues E, D, K, A and C, and more preferentially amino acid E; X6 represents any amino acid residue, preferentially a negatively or positively charged or neutral amino acid residue, preferentially selected from the group consisting of amino acid residues E, K, Q, A, Aib (2-aminoisobutyric acid) and C, and more preferentially amino acid K; X7 represents C, L, I, cpA (cyclopropyl-L-alanine), chG (L-cyclohexylglycine), chA (cyclohexyl-L-alanine) or mlL (γ-methyl-L-leucine); and X8 represents C, I or G.
17. The polypeptide according to claim 16, wherein said polypeptide is a linear polypeptide.
18. The polypeptide according to claim 16, wherein said polypeptide is a linear polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 22.
19. The polypeptide according to claim 16, wherein said polypeptide is a cyclic polypeptide.
20. The polypeptide according to claim 16, wherein said polypeptide is a cyclic polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 30 to SEQ ID NO: 35, SEQ ID NO: 55 to SEQ ID NO: 58, SEQ ID NO: 61 to SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 68.
21. The polypeptide according to claim 16, wherein said polypeptide is a cyclic polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, SEQ ID NO: 67 and SEQ ID NO: 68.
22. The polypeptide according to claim 16, wherein said polypeptide is a cyclic polypeptide comprising an amino acid sequence represented by SEQ ID NO: 61, SEQ ID NO: 67 or SEQ ID NO: 68.
23. The polypeptide according to claim 16, wherein said lactate dehydrogenase subunit is lactate dehydrogenase B (LDHB) subunit.
24. The polypeptide according to claim 16, wherein the —OH group of the free —COOH group of the last amino acid residue at the C-terminus of the polypeptide is replaced by a group selected from an —O-alkyl group, an —O-aryl group, a —NH.sub.2 group, a —N-alkyl amine group, a —N-aryl amine group or a —N-alkyl/aryl group.
25. A polynucleotide encoding a polypeptide according to claim 16.
26. A pharmaceutical composition comprising at least one polypeptide according to claim 16, and at least one pharmaceutically acceptable vehicle.
27. A kit for preventing and/or treating a cancer comprising at least one polypeptide according to claim 16, a polynucleotide encoding said polypeptide, or a pharmaceutical composition comprising said polypeptide with at least one pharmaceutically acceptable vehicle, and optionally an anticancer agent.
28. A medicament comprising a polypeptide according to claim 16, a polynucleotide encoding said polypeptide, or a pharmaceutical composition comprising said polypeptide with at least one pharmaceutically acceptable vehicle.
29. A method for preventing and/or treating a cancer in a subject in need thereof comprising the step of administering to the subject an effective amount of a polypeptide according to claim 16, a polynucleotide encoding said polypeptide, or a pharmaceutical composition comprising said polypeptide with at least one pharmaceutically acceptable vehicle.
30. A method for screening a compound affecting the tetramerization of the lactate dehydrogenase subunits comprising the steps of: a. providing a system comprising truncated lactate dehydrogenase (LDHtr) subunit; b. providing the system with a candidate compound modulating the activity of a native tetrameric LDH; and c. measuring a level of binding of the candidate compound to a dimer of LDHtr subunits in the presence or in the absence of a polypeptide according to claim 16; wherein the observation of a competition between the polypeptide and the candidate compound for the binding to the dimer of LDHtr subunits is indicative of the candidate compound being an inhibitor of the tetramerization of the lactate dehydrogenase subunits.
31. The method according to claim 30, wherein the observation of a competition between the polypeptide and the candidate compound for the binding to the LDHtr subunit is indicative of the specificity of the binding of the candidate compound towards the tetramerization site onto the lactate dehydrogenase subunits.
32. A method for screening a compound affecting the tetramerization of the lactate dehydrogenase subunits comprising the steps of: a) providing a system (1) comprising truncated lactate dehydrogenase (LDHtr) subunits and a system (2) comprising native tetrameric LDH; b) providing the systems (1) and (2) with a candidate compound modulating the activity of a native tetrameric LDH; and c) measuring a level of binding (Kd) of the candidate compound to a dimer of LDHtr subunits in system (1) and to a native tetrameric LDH in system (2); wherein the observation of a binding of the candidate compound to the dimer of LDHtr subunits in system (1) and wherein the observation of an altered binding of the candidate compound to the native tetrameric LDH in system (2) are indicative of the candidate compound being an inhibitor of the tetramerization of the lactate dehydrogenase subunits, by interacting at the surface of the LDH subunits.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[0240] The present invention is further illustrated by the following examples.
Example 1
1—Experimental Procedures
1.1—Peptide Synthesis
[0241] All polypeptides employed herein were purchased from GeneCust® (www.genecust.com). The level of purity of peptides was >95%. Structure conformity and purity grade was checked by analytical HPLC analyses and mass spectrometry. All peptides were amidated at their C-terminal unless stated otherwise.
1.2—Nuclear Magnetic Resonance (NMR)
[0242] Full length human LDHB (LDHB; SEQ ID NO: 2) and truncated LDHB, i.e. a LDHB subunit lacking the first N-terminal 19 amino acid residues (LDHBtr; SEQ ID NO: 4) tagged with a 6His Tag were expressed and purified from E. coli cells as described previously. All experiments were acquired on a Bruker Ascend Avance III 600 MHz equipped with a broadband cryoprobe (Bruker® GmBH, Germany)
1.3—NMR WaterLOGSY Experiments
[0243] NMR WaterLOGSY was performed on samples prepared in 10% D.sub.2O buffer containing 50 mM sodium phosphate buffer, pH 7.6 and 100 mM NaCl. The concentration of LDH subunits was 15-20 μM. Ligand binding was detected using a NMR WaterLOGSY ephogsygpno.2 advance-version sequence with a is mixing time. Water signal suppression was achieved using excitation sculpting scheme and a 50 ms spinlock was used to suppress protein background signal. For each experiment, 512 scans were collected to yield a 16K points FID. NMR WaterLOGSY intensity was corrected by plotting the intensity difference of the ligand NMR WaterLOGSY spectra recorded in the presence and absence of protein.
[0244] For NMR WaterLOGSY screening experiments a correction factor was applied to account for slight concentration variation between samples. To do so, 8 scans 1H NMR spectra with 50 ms spinlock were recorded before NMR WaterLOGSY experiments. The intensity ratio of the aliphatic region (0.700 ppm to 0.955 ppm) with and without protein was used as a correction factor to compare the NMR WaterLOGSY intensity of the polypeptides of interest with and without LDH subunits. An arbitrary threshold of 0.1, corresponding to a 10% decrease in the NMR WaterLOGSY signal intensity between the spectra with and without protein, was set to discriminate between binders and non-binders.
1.4-2D Experiments
[0245] Polypeptides were dissolved in a 50 mM phosphate buffer pH 7.0 containing 100 mM NaCl, 1 mM TSP and 10% D20. For all experiments, water suppression was achieved using an excitation sculpting scheme. 4 Ktime domain points and 256 increments were applied for all the 2D spectra.
[0246] TOCSY experiments were performed using the homonuclear Hartman-Hahn transfer with the dipsi2 sequence with an 80 ms mixing time. 8 scans per spectra, 4 Ktime domain points and 256 increments were recorded.
[0247] ROESY experiments were performed using a 2D ROESY sequence with cw spinlock for mixing. 400 ms mixing times were used, and the number of scans taken for was 32.
1.5—Size Exclusion Experiments
[0248] Size exclusion chromatography was performed using a ÄKTA explorer (GE Healthcare®) equipped with a Superdex 200 Increase 10/300 GL equilibrated with 50 mM sodium phosphate pH 7.6, 100 mM NaCl at 0.7 ml/min. LDHBfl (SEQ ID NO: 2) and LDHBtr (SEQ ID NO: 4) were diluted to 3 μM in assay buffer. The final Injection volume was 100 μl. Prior to experiment, the column was equilibrated for 2× column volume with distillated and filtrated H.sub.2O followed by 3× column volume filtrated buffer. Molecular weight was determined using the Biorad gel filtration standard in the same assay buffer following the manufacturer instructions.
1.6—Fluorescence-Based Thermal Shift
[0249] Thermal shift assays were performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific®) in 96-well white plates (Roche®). Each well contained 20 μl of 5 μM protein and 5×SYPRO Orange in 50 mM sodium phosphate pH 7.6, 100 mM NaCl. Each plate was sealed with an optically clear foil and centrifuged for 1 min at 1000 rpm before performing the assay. The plates were heated from 20-99° C. at approximately 4° C./min.sup.−1. The fluorescence intensity was measured with λex=480 nm and λem=580 nm. The melting temperature (Tm) was obtained by determining the minimum of the first derivative curve of the melt curve.
1.7—Microscale Thermophoresis (MST)
[0250] MST measurements were performed on a Nanotemper Monolith NT.115 instrument (Nanotemper Technologies®, GmbH) using Red-dye-NHS fluorescent labeling. Each LDH (WT or truncated) sample, purified to homogeneity, was labeled with the Monolith RED-NHS 2.sup.nd generation labeling dye according to the supplied protocol (Nanotemper Technologies®, GmbH). Measurements were performed in 50 mM Na-Phosphate pH 7.6 and 100 mM NaCl containing 0.05% Tween-20 in premium treated capillaries (Nanotemper Technologies®, GmbH). The final concentrations of either labeled protein in the assay were 100 nM. The ligands (NADH and peptides) were titrated in 1:1 dilutions following manufacturer's recommendations. All binding reactions were incubated 5′ at room temperature after loading into capillaries. Experiments were performed in triplicates using 40% LED power and medium MST power, LaserOn time was 20 sec, Laser Off time 3 sec. Linear octapeptides were evaluated for their thermophoretic pattern. Longer and cyclic peptide were found to interact with the labelling dye, hence raw fluorescence instead of the thermophoretic pattern was used to extract dissociation constant according to the manufacturer instructions.
1.8—Purification of 6His Tagged Human LDH Polypeptides
[0251] hLDH (wt and truncated) sequences cloned into pET-28a expression vector was ordered from Genecust®, Luxembourg. The recombinant plasmids were then transformed into host bacterium Escherichia coli Rosetta strain (DE3). The transformants were cultured in LB medium with 50 μg/ml kanamycin and 34 μg/ml chloramphenicol at 37° C. until an optical density of 0.6 was reached. LDHs expression were induced by 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 20° C. for 20 h. Then, cells were collected by centrifugation at 5,000 rpm, 4° C. for 25 min. Pellets were suspended into a lysis buffer and then disrupted by sonication, followed by centrifugation at 4° C., 10,000 rpm for 30 min. Insoluble fraction was discarded and 1 μl of β-Mercaptoethanol was added per milliliters of soluble fraction. The purification of recombinant polypeptides was performed using 1 ml His-trap FF-crude columns (GE Healthcare®) according to the instruction of the manufacturer. Finally, concentration was measured using the Bradford method with the Biorad Protein Assay Kit and sample homogeneity was assessed using sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue as staining agent.
1.9—Spectrophotometric Experiments
[0252] All spectrophotometric experiments were performed with transparent or opaque 96 wells using a spectramax m2e spectrophotometer.
1.10—Enzymatic Assay
[0253] The dehydrogenase reaction was run in the LDH1 (tetramer of LDHB) physiologically relevant lactate to pyruvate direction following the NADH fluorescence generated during lactate oxidation. The progression of the reaction was monitored as the increase of fluorescence at 340/460 nm.
[0254] The Michaelis-Menten constant determination was performed using the GraphPad prism software. Enzymatic reactions were performed in a solution containing phosphate buffer 100 mM at pH 8.3 to enhance lactate to pyruvate oxidation, EDTA 1 mM and DTT 1 mM. Final protein concentration was 7.7 nM for full length LDHB subunit and 13.5 nM for the truncated LDHB subunit (LDHBtr). For NAD.sup.+Km determination, concentrations of lactate were set to 20 mM while co factor concentrations ranged from 1 μM to 5 mM for full length enzyme and from 50 μM to 10 mM for the truncated form. For lactate Km determination, concentrations of NAD.sup.+ were set to 1 mM while substrate concentrations ranged from 1 μM to 40 mM for full length enzyme and from 1 mM to 40 mM for the truncated form.
1.11—Intrinsic Fluorescence Assay
[0255] Following described procedure; rabbit LDHA commercial solution in an ammonium sulfate suspension in (pH-7.0, 3.2 M) was first diluted to 1 mg/ml in a solution of NaCl 200 mM and then dialyzed at 4° C. 2×2 h against 200 mM NaCl. The stock solution of 1 mg/ml was then diluted to 30 μg/ml in NaCl 200 mM to give the assay solution.
[0256] Assay solution was mixed 1:1 with either an acetate-chloride buffer (20 mM Acetic acid/Acetate, 180 mM NaCl, pH 5.0) or phosphate buffer (250 mM phosphate pH 7.6). After storage at 4° C. for 30′, samples were removed and diluted 1:1,250 mM phosphate buffer (pH 7.6) to yield a 7.5 μg/ml final concentration of re-associating LDH-M.
[0257] Samples of the resulting solutions were then subjected to kinetic experiments of the recovery of intrinsic fluorescence (Exc=286 nm, Em=350 nm, 10′, rt). Full tryptophan fluorescence spectra were afterward recorded (Exc=286 nm, Em=320-400 nm, rt).
1.12—Polypeptide Cyclization
[0258] The lyophilized crude peptide solution (˜3 mg/mL, —1.5 mM) in NH.sub.4HCO.sub.3 buffer (100 mM, pH=8.0) was treated with TCEP (1.5 equiv) (2.25 μl from 1 M solution in the same NH.sub.4HCO.sub.3 buffer) and stirred for 1 h (700 rpm). The alkylating agent in DMF (˜3 equiv) (100 μl from a 50 mM solution) was added to the solution and shaken for the 2 h (700 rpm). The reaction was quenched by adjusting the pH of the mixture to slightly acidic conditions through the addition of 0.5 N HCl or TFA (150 μl/ml). The crude mixture was then centrifugated at 10,000 rpm 20 minutes. The Supernatant was then analyzed and purified by HPLC/MS.
1.13—in Silico Evaluation
[0259] Calculation of the free binding energy was performed using the MOE software with the available LDHB (SEQ ID NO: 2) crystallographic structure. No minimization was performed prior to calculation.
2—Results
2.1—LDH Tetramerization Site in Silico Study
[0260] As LDHA and LDHB subunits can hybridize in vitro and in cellulo to give the hetero-tetramers LDH2-3-4, LDHA and LDHB tetramerization site and N-terminal arm are structurally very close. Thus, no selectivity towards one sub-unit is expected to be reached using the considered approach. LDH1 (tetrameric LDHB) and its N-terminal arm were first studied. The analysis of the available LDHB crystallographic structure shows clearly the stabilization of the tetramer by interaction of the 19 N-terminal amino acid polypeptide of each subunit with two other subunits, like four arms embracing the tetramer (
[0261] Based on this structural analysis, the pharmacological properties of the LDHB 19 N-terminal amino acid peptide (ATLKEKLIAPVAEEEATVP, namely LB19; SEQ ID NO: 6) on the full length LDH1 enzyme was assessed. Unfortunately, biochemical as well as biophysical evaluation showed no interaction between LB19 and LDH1. It was hypothesized that this lack of effect was stemming from an “unfair” competition between the LDHB N-terminal 19-mer peptide arm and LB19 for the tetramerization site. This led to the design and evaluation of a new protein model allowing for the evaluation of this interaction.
2.2—Design and Evaluation of a Dimeric LDH
[0262] To address the challenge of evaluating tool compounds at the tetramerization site, a second LDHB truncated of its 19 N-terminal amino acid residues (LDHBtr) was produced. It was hypothesized that this truncated protein, lacking the tetramerization arm, would probably be in a native dimeric state and would therefore allow gaining accessibility to the LDHB tetramerization site.
[0263] The recombinant LDHBtr (SEQ ID NO: 4) form was produced in E. coli and was shown to be in a native dimeric state by size exclusion chromatography (SEC), diffusion light scattering (DLS) and intrinsic fluorescence. Furthermore, the affinity of rLDHBtr for its cofactor was evaluated using microscale thermophoresis (MST) and a Kd value of 21 μM+/−5 μM was obtained, similarly to the one of the full length LDHB (Kd=24 μM+/−8 μM), thus indicating a correct fold of the protein “Rossman domain”. The catalytic properties of rLDHBtr were also evaluated using standard biochemical assay (
TABLE-US-00001 TABLE 1 Enzymatic characteristics of full length and truncated LDHB subunits LDHBtr LDHBfl Km NAD.sup.+ (mM) 0.578 0.153 Km Lactate (mM) 6.52 33.77 Vmax NAD.sup.+ (μM/min) 0.29 3.33 Vmax Lactate (μM/min) 1.11 11.41
[0264] Finally, LDHBtr stability was evaluated using thermal shift assay and TYCHO NT.6. The dimeric LDHB was found to be deeply destabilized when compared to the tetrameric LDHB with 18° C. and 24° C. shift in melting temperature, respectively (
2.3—Study and Optimization of the Interaction Between LB19 and LDHBtr Tetramerization Site
[0265] Biophysical evaluation of the interaction between LDHBtr (SEQ ID NO: 4) and LB19 (SEQ ID NO: 6) was performed using two biophysical orthogonal methods: NMR WaterLOGSY and Microscale thermophoresis (MST). According to MST analysis, LB19 interacts with LDHBtr with a Kd of 270 μM [+/−70 μM]. NMR WaterLOGSY analysis showed a positive signal stemming from an interaction between LB19 and LDHBtr. Analysis of NMR WaterLOGSY spectra allowed for an epitope mapping of the interaction between the two molecules. Interestingly, LB19 N-terminal residues underwent more saturation transfer than their C terminal counterpart, meaning that LB19 N-terminal residues could account for most of the binding strength. Accordingly, calculation of the overall binding energy of LDHB native arm showed similar results.
[0266] Following these observations, some C-terminal amino acids were removed from LB19 to keep only those from the N-terminal end accounting for the binding with LDHBtr. This led to evaluate the binding of the polypeptide LB13 (SEQ ID NO: 7) to LDHBtr (SEQ ID NO: 4). In accordance with the previous results, LB13 resumed all the interacting residues and thus presented the exact same NMR WaterLOGSY spectrum than LB19. Moreover, MST analysis confirmed that the interaction was only slightly weakened with a Kd=605 μM [+/−290 μM]. Further size reduction of LB13 led to the evaluation of LB8 (ATLKEKLI; SEQ ID NO: 8), which apart from a valine residue summarized the same interaction residues as in LB13. Again, apart from a small drop in the interaction strength (Kd=1.4 mM [+/−0.4 mM]), LB8 exactly matched with the N-terminal alpha-helix of LDHB N-terminal arm and therefore can be anticipated as a “Hot-spot” of the interaction between LDHB tetramerization site and its N-terminal arm. A LB19 central fragment (LIAPVAE, namely LBc; SEQ ID NO: 26) was also evaluated as a negative control and found not to demonstrate any appreciable saturation transfer under these conditions.
2.4—LB8 SAR
[0267] The evaluation of the structure-activity relationship between LB8 (SEQ ID NO: 8) and LDHB tetramerization site was further examined. As the active conformation of LB8 was expected to be an a-helix, a combination of in silico and experimental evaluation was used to unravel LB8 SARs. A set of 15 LB8 structural analogues was constructed and further analyzed by NMR WaterLOGSY experiments at a single concentration of 800 μM to identify structural modifications that would result in a loss of saturation transfer (Table 2 and
TABLE-US-00002 TABLE 2 Binding properties of linear polypeptides according to the invention Linear peptides Binding using WaterLOGS Kd using MST Name Sequence Y (800 μM) experiment LB19 ATLKEKLIAPVAEEEATVP + 270 μM +/− 70 μM (SEQ ID NO: 6) LB13 ATLKEKLIAPVAE + 605 μM +/− 290 μM (SEQ ID NO: 7) LA19 ATLKDQLIYNLLKEEQTPQ + N.D (SEQ ID NO: 21) LB8 ATLKEKLI + 1.44 mM +/− 0.4 mM (SEQ ID NO: 8) LA8 ATLKDQLI + 3.1 mM +/− 1.1 mM (SEQ ID NO: 22) LB8-A1 AALKEKLI − 8.2 mM +/− 5.1 mM (SEQ ID NO: 23) LB8-A2 ATAKEKLI + N.D (SEQ ID NO: 9) LB8-A3 ATLAEKLI + N.D (SEQ ID NO: 10) LB8-A4 ATLKAKLI + N.D (SEQ ID NO: 11) LB8-A5 ATLKEALI + N.D (SEQ ID NO: 12) LB8-A6 ATLKEKAI − >10 mM (SEQ ID NO: 24) LB8-A7 ATLKEKLA + N.D (SEQ ID NO: 13) LB8-AL7 ATLKEKL − N.D (SEQ ID NO: 25) Ac-LB8 Ac-ATLKEKLI + N.D (SEQ ID NO: 14) LBc Ac-LIAPVAE-NH2 − N.D (SEQ ID NO: 26) LB8-AcTI7 Ac-TLKEKLI − N.D (SEQ ID NO: 27) LB8-T17 TLKEKLI − N.D (SEQ ID NO: 28) LB8-A3-A5 ATLAEALI + N.D (SEQ ID NO: 15) LB8-G3 ATGKEKLI − >10 mM (SEQ ID NO: 29) LB8-G8 ATLKEKLG + N.D (SEQ ID NO: 17) LB8-G1 GTLKEKLI + N.D (SEQ ID NO: 16) LB8-Aib1 ATL(Aib)EKLI + N.D (SEQ ID NO: 18) LB8-Aib2 ATLKE(Aib)LI + N.D (SEQ ID NO: 19) LB8-Aib3 ATL(Aib)E(Aib)LI + N.D (SEQ ID NO: 20)
[0268] In agreement with the analysis of the crystallographic data, two L amino acid residues as well as the C-terminal isoleucine were found to be required for the binding. The in silico model extracted from the LDHB 3D structure indicated that these aliphatic side chains projected towards hydrophobic cavities at the tetramerization site. NMR WaterLOGSY mapping of the saturation transfer intensity confirmed that lipophilic residues undergo more saturation transfer that any other, thus interacting more closely at the tetramerization site.
[0269] In LB8 (SEQ ID NO: 8), amino acid residue switch T to A, as well as a removal of any of the terminal residues, also resulted in abrogation of the interaction (Table 2 and
2.5—Cyclization
[0270] It was expected that LB8 weak binding could be accounted for its poor helical propensity that would result in a huge entropy cost prior to binding. Indeed, 2D NOESY and ROESY analysis confirmed the absence of the alpha-helix characteristic cross coupling in the N-terminus region. Moreover, previous studies have shown entropy-mediated gain in potency by constraining the conformational freedom of peptides. LB 8 side chain to side chain cyclization was hence performed to promote its helicity. Many strategies are described for peptide macrocyclization (Hill et al. (2014)). Among them, cysteine alkylation with an alpha-helix promoting agent already demonstrated strong results in enhancing small peptides helicity, and hence affinity (
TABLE-US-00003 TABLE 3 Binding properties of cyclic peptides according to the invention Cyclic peptides Binding using WaterLOGSY Kd using MST Name Sequence (800 μM) experiment VS-142-BisAlk ACLKECLI + 233 μM +/− 113 μM (SEQ ID NO: 30) LT018 (SEQ ID NO: 31) CTLKCKLI + 66 μM +/− 32 μM LT020 (SEQ ID NO: 32) ATLKCKLIC N.D 477 μM +/− 116 μM LT021 (SEQ ID NO: 33) ACTLKCKLI + LT022 (SEQ ID NO: 34) CATLCEKLI CB-09 (SEQ ID NO: 35) ATCKEKCI
[0271] Among them, the LT018 polypeptide (SEQ ID NO: 31) revealed to be the most promising one with an apparent 30-fold increase in potency (Kd=66 μM+/−32 μM) compared to LB8 (SEQ ID NO: 8) and an intense saturation transfer. However, despite this increased affinity, LT018 polypeptide (SEQ ID NO: 31) was still not able to compete with LDHB native arm (
2.6—VS-142-BisAlk Polypeptide Inhibits LDH Tetramerization
[0272] Following the observation that LT018 polypeptide (SEQ ID NO: 31) was not able to compete with LDHB native arm and thus was not able to disrupt an already formed LDHB tetramer, it was reasoned that it could maybe bind to the tetramerization site in a pre-dissociation dependent manner. To confirm this hypothesis, experiments were therefore designed to follow the recovery of the LDH tetrameric form after a pre-dissociation initiated in slightly acidic conditions. Briefly, six tryptophan residues are found in the LDH structure, three of them being located at the dimer-dimer interface. As the tryptophan quantum yield decreases in polar environment, dimeric LDHs show very weak tryptophan fluorescence compared to tetrameric one. Accordingly, in acidic conditions (pH 5.0) LDH shows a decrease in tryptophan fluorescence that is correlated to the dissociation of the tetramer (Rudolph and Jaenicke (1976);
[0273] Strikingly, the LT018 polypeptide (SEQ ID NO: 31) nicely interfered with the fluorescence recovery at 50 μM (
Example 2
1—Materials and Methods
1.1—Chemicals and Peptides
[0274] All reagents were purchased from chemical suppliers and used without purification. Rabbit and recombinant human LDHA were purchased respectively from Sigma-Aldrich® and Abnova®. Linear peptides used directly in biophysical experiments were purchased from Genecust® and linear peptides used for cysteine stapling were synthetized by solid-phase peptide synthesis. Lactam cyclic peptides were purchased from Proteogenixa Structure conformity and purity grade (>95%) were assessed by analytical high-performance liquid chromatography (HPLC) analysis and mass spectrometry (MS) for both commercial and synthetized peptides. All peptides were amidated at their C-termini
1.2—Peptide Synthesis
[0275] All peptides used for cysteine cross-linking procedures were synthetized on a 0.05 or 0.1 mmol scale using a Rink amide AM resin (Bachem®) (substitution 0.5-1.2 mmol/g). Fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids (5-fold excess) were activated with 1 equivalent of hexafluorophosphate benzotriazole tetramethyl uronium (HBTU) and 2 equivalents of diisopropylethanolamine (DIPEA) (equivalent relative to the amino acid). Coupling was performed in N-methyl-2-pyrrolidone (NMP) for 60 min at room temperature. Fmoc deprotection was carried out using 20% piperidine in NMP for 10 min at room temperature. Side chain deprotection as well as simultaneous cleavage from the resin were achieved using a mixture of Trifluoroacetic acid (TFA)/Triisopropylsilane/water/thioanisole (90/2.5/2.5/5) at room temperature for 2 h. TFA was then evaporated under nitrogen flux, and the crude peptide was precipitated using ice cold diethyl ether. Crude peptides were then analyzed using an Agilent® (1100 series) HPLC single quadrupole (InfinityLab, ESI+) system equipped with a kinetex 5 μm EVO C18 (150×4.6 mm), and subsequently lyophilized for further use.
1.3—Synthesis of the Cross-Linked Peptides
[0276] Stapling using hexafluorobenzene was performed by following the procedure described by Spokoyny et al. (2013). To a lyophilized sample of peptide (˜7.5 μmoles) was added 1.9 mL of 100 mM solution (— 25 equiv.) of hexafluorobenzene in DMF and 1.5 mL of 50 mM solution of tris base in DMF. Solution was left under agitation at room temperature for 5 h. Resulting mixture was diluted with 2 times volume of 0.1% TFA solution in water and subjected to analysis and purification on HPLC as described above.
1.4—Microscale Thermophoresis (MST)
[0277] MST measurements were performed on a Nanotemper Monolith NT.115 instrument (NanoTemper Technologies®) using Red-dye-NHS fluorescent labeling. Each LDH sample, purified to homogeneity, was labeled with the Monolith Red-dye-NHS 2.sup.nd generation labeling dye (NanoTemper Technologies®), according to the manufacturer's instructions. Measurements were performed in 50 mM sodium phosphate, pH 7.6, and 100 mM NaCl containing 0.05% Tween-20 in premium-treated capillaries (NanoTemper Technologies®). The final concentrations of either labeled protein in the assay were 100 nM. The ligands (NADH and peptides) were titrated in 1:1 dilutions following manufacturer's recommendations. All binding reactions were incubated for 5 min at room temperature after loading into capillaries. Experiments were performed in triplicates using 40% LED power, medium MST power, Laser On time 20 s and Laser Off time 3 s. Peptides were evaluated for their thermophoretic pattern, and Kd's were extracted from raw data at a 10 to 20 s MST on time according to manufacturer's instructions. Regarding interaction of 7 with LDH5, Kd was extracted from the raw fluorescence. A denaturation test was performed accordingly to manufacturer recommendation and excluded any nonspecific spectral interaction between 7 and the red-dye. All Kd's of interacting macrocycles and peptides were obtained in triplicate and corrected by taking into account the molecular weight of the TFA counter ion. Peptide ATGKEKLI (LB8-G3; SEQ ID NO: 29) was used as a negative control, and displayed no appreciable binding when compared to LB8 (SEQ ID NO: 8).
1.5—Spectrophotometric Experiments
[0278] All spectrophotometric experiments were performed with opaque 96-well plates using a Spectramax m2e spectrophotometer (Molecular Devices).
a) Kinetic Assays
[0279] The dehydrogenase reaction was run in the LDH1 physiologically relevant lactate to pyruvate direction following the NADH fluorescence generated during lactate oxidation to pyruvate. The progression of the reaction was monitored as the increase of fluorescence at 340/460 nm. The Michaelis-Menten Km constant determination was performed using the GraphPad prism 7.0 software. Enzymatic reactions were performed in a solution containing phosphate buffer 100 mM at pH 8.0 to enhance lactate to pyruvate oxidation, EDTA 1 mM. Final protein concentration was 7.7 nM for LDHB and 13.5 nM for LDHBtr. For NAD+ Km determination, concentrations of lactate were set to 20 mM for LDHB and 150 mM for LDHBtr, while cofactor concentrations ranged from 1 μM to 5 mM for LDHB and from 50 μM to 10 mM for LDHBtr. For lactate Km determination, concentrations of NAD+ were set to 1 mM while substrate concentrations ranged from 1 μM to 30 mM for LDHB and from 1 mM to 40 mM for LDHBtr.
b) Intrinsic Fluorescence Assays
[0280] Full tryptophan fluorescence spectra were recorded using an excitation wavelength of 286 nm and recording the emission spectra from 320 nm to 400 nm at room temperature. Raw fluorescence of every experiments was further subtracted to a corresponding control experiment without the protein. Experiments were performed in a 50 mM sodium phosphate and 100 mM NaCl, pH 7.6, buffer. For LDH dissociation into subunits, increasing amounts of guanidinium/HCl were put in contact with the studied proteins (1.3 μM), and fluorescence spectra were recorded afterwards. Guanidinium/HCl concentrations ranged from 0.3 M to 2 M.
c) Denaturation Assays
[0281] Rabbit LDHA commercial solution (Sigma-Aldrich®) in an ammonium sulfate suspension (pH-7, 3.2 M) was first diluted to 1 mg/ml in a solution of NaCl 200 mM and then dialyzed at 4° C. 2×2 h against 200 mM NaCl. The stock solution of 1 mg/ml was then diluted to 30 μg/ml (800 nM) in NaCl 20 0 mM to give the assay solution. Assay solution was mixed 1:1 with acetate-chloride buffer (20 mM Acetic acid/Acetate, 180 mM NaCl, 1 mM DTT pH 5) and stored on ice for 30 minutes. Samples were then removed from ice and let warm up for 2 minutes. The acidic solution was then diluted 1:1 with a 250 mM phosphate buffer (pH 7.6) containing or not the inhibitory peptide to yield a 7.5 μg/ml (200 nM) final concentration of re-associating LDHA. Samples of the resulting solutions were then subjected to kinetic experiments of the recovery of intrinsic fluorescence (Exc=286 nm, Em=350 nm, 10′, rt).
1.6—Statistics
[0282] All quantitative data are expressed as means±SEM. Error bars are sometimes smaller than symbols. n refers to the total number of replicates per group. All experiments were repeated at least twice independently. Data were analyzed using the GraphPad Prism 7.0 software. Student's t test, one-way ANOVA and two-way ANOVA were used where appropriate. P<0.05 was considered to be statistically significant.
2—Results
2.1—Binding of Macrocyclic Peptides (MP) to Truncated LDHB (LDHBtr)
[0283] Following identification of the optimal i and i+4 position for LB8 polypeptide cyclization, macrocyclic peptides (MP) bearing other linkers (see Table 4) we investigated, including p-tetrafluorophenyl (MP7), o-benzyl (MP8), p-benzyl (MP9), as well as a lysine to aspartate lactam bridge between the side-chains of the K.sub.1 and D.sub.5 residues (MP10).
TABLE-US-00004 TABLE 4 Code, structure, dissociation constants (IQ) and 95% confidence interval of evaluated macrocycles against truncated LDHB Name Sequence Linker K.sub.d* CI.sub.95% LB8 (SEQ ID NO: 8) ATLKEKLI — 1.05 mM 0.55 to 2.01 mM MP1 (SEQ ID NO: CTLKCKLI m-benzyl 64 μM 55 to 75 μM 55) MP2 (SEQ ID NO: ACTLKCKLI m-benzyl 67 μM 55 to 82 μM 56) MP3 (SEQ ID NO: ACLKECLI m-benzyl 787 μM 529 to 1171 μM 57) MP4 (SEQ ID NO: ATLKCKLIC m-benzyl 398 μM 246 to 645 μM 58) MP5 (SEQ ID NO: ATLKECLIAC m-benzyl >>1 mM ND 59) MP6 (SEQ ID NO: CATLCEKLI m-benzyl >>1 mM ND 60) MP7 (SEQ ID NO: CTLKCKLI P- 113 μM 9 to 14 μM 61) tetrafluorophenyl MP8 (SEQ ID NO: CTLKCKLI o-benzyl 25 μM 21 to 29 μM 62) MP9 (SEQ ID NO: CTLKCKLI p-benzyl 11 μM 98 to 131 μM 63) MP10 Ac-KTLKDKLI Lactam bridge K.sub.1- 142 μM 117 to 174 μM (SEQ ID NO: 64) D.sub.5 MP11 ATLKEKLI Lactam bridge 465 μM 355 to 607 μM (SEQ ID NO: 65) Nter-E.sub.5 MP12 ATLKEKLI Lactam bridge K.sub.6- >>1 mM ND (SEQ ID NO: 66) Cter CT-44 CT(m1L)KCKLI.sup.1 p- 9.43 μM 7.30 to 12.17 μM (SEQ ID NO : 67) tetrafluorophenyl CT-45 CTLKCK(cpA)I.sup.2 p- 7.82 μM 6.13 to 9.96 μM (SEQ ID NO : 68) tetrafluorophenyl *K.sub.d were extracted from MST traces at 10 s to 20 s on time (n = 3 for macrocyclic peptides MP1-MP4 and MP7-MP11, n = 2 for macrocyclic peptides MPS-MP6 and MP12). ND, not determined. .sup.1mlL represents γ-methyl-L-leucine. .sup.2cpA represents cyclopropyl-L-alanine.
[0284] Strikingly, Kd evaluation of these macrocyclic peptides revealed an impact of the overall constrain imposed by the linker on the evaluated affinity. Indeed, p-tetrafluorophenyl (MP7; SEQ ID NO: 61) and o-benzyl (MP8; SEQ ID NO: 62) analogues yielded a supplementary 2-fold to 6-fold improvement in affinity when compared to macrocyclic peptide MP1 (SEQ ID NO: 55), with K.sub.d's of 11 μM and 25 μM, respectively. Comparatively, less constraining linkers, p-benzyl (MP9; SEQ ID NO: 63) and the Ki-D5 lactam bridge (MP10; SEQ ID NO: 64), resulted in weakly potent derivatives with K.sub.d's of 113 μM and 142 μM, respectively. As compared to macrocyclic peptide MP7 (SEQ ID NO: 61), substitution of leucine in amino acid position 3 with γ-methyl-L-leucine, as in macrocyclic peptide CT-44 (SEQ ID NO: 67) did not affect the binding properties. Similarly, substitution of leucine in amino acid position 7 with cyclopropyl-L-alanine, as in macrocyclic peptide CT-45 (SEQ ID NO: 45), resulted in an unaltered K.sub.d value, or even a slightly improved K.sub.d value.
[0285] The influence of a lactam bridge between the N-terminal amino group and the carboxylic acid on the side-chain of the E.sub.5 residue was further investigated, as these two moieties can be found close to each other in LB8 in silico model. The resulting macrocyclic peptide MP11 (SEQ ID NO: 65) was found to be slightly more potent than LB8, with a K.sub.d of 465 μM (see Table 4). For comparison, the impact of a lactam bridge between K.sub.6 side-chain NH.sub.2 and the C-terminal carboxylate was also evaluated. The resulting peptide MP12 (SEQ ID NO: 66) yielded no appreciable binding using either NMR or MST (see Table 4).
2.2—Destabilizing and Disrupting LDH Tetramerization with Designed Macrocyclic Peptides
[0286] It was further tested whether macrocyclic peptides MP1 and MP7 were able to compete with N-terminal domain of native LDHB. To this end, their ability to interact with tetrameric LDH1 and LDH5 using MST was first investigated. Interestingly, macrocyclic peptide MP7, the most potent analogue, displayed an interaction at high concentrations with LDH1 and LDH5 (
[0287] The ability of macrocyclic peptides MP1 and MP7 to destabilize tetrameric LDH1 and LDH5 was further investigated. Indeed, molecules interacting at oligomeric interfaces can reduce the melting temperature of the studied oligomers owing to a perturbation of the overall stability of the complex. The impact of macrocyclic peptides MP1 and MP7 on LDH1 and LDH5 thermal denaturation was therefore evaluated using nanoDSF. While MP1 had no effect up to 500 μM on both human LDH1 and LDH5 stabilities, macrocyclic peptide MP7 induced a destabilizing conformational change on both isoforms at 400 μM (
[0288] Next, it was evaluated whether these macrocyclic peptides could also bind to the tetramerization site during LDH tetramer formation. Such an approach was already reported, for instance, in the case of peptides interacting at the interface of human glutathione reductase.
[0289] An experiment was thus designed to follow the recovery of LDH tetramers after a dissociation step initiated by acidic conditions. These experiments were conducted on LDH5, as it is less stable and thus more prone to dissociation than LDH1. Because strong acidic conditions (pH 2.3) are necessary to disrupt the human LDH5 (hLDH5) homotetramer, which results in partial protein denaturation, the assay was performed on rabbit LDH5 (rLDH5) that dissociates at less acidic conditions (pH 5), does not denaturate and affords reproducible data. rLDH5 shares 94% sequence identity and 98% homology with the hLDH5, with similar nanoDSF denaturation patterns. Monitoring of the rLDH5 tetrameric state was performed by following its intrinsic tryptophan fluorescence: 6 tryptophan residues can be found in each rLDHA monomer, three of them being located at the dimer-dimer interface. As the tryptophan quantum yield decreases in a polar environment, dimeric LDHs show very weak tryptophan fluorescence compared to the tetrameric form. Accordingly, dimeric rLDHA showed very weak tryptophan fluorescence at pH 5 when compared to the high fluorescence of tetrameric rLDH5 at pH 7.6. Such decay can be compared to the difference in tryptophan fluorescence between tetrameric LDH1 and dimeric LDHBtr.
[0290] When restoring a neutral pH following acidification, macrocyclic peptides MP1 and MP7 significantly interfered with LDH5 fluorescence recovery (
[0291] Finally, the ability of macrocyclic peptide MP7 to disrupt LDH oligomerization state without prior dissociation was investigated. The impact of macrocyclic peptide MP7 on the protein native fluorescence was therefore directly evaluated. Strikingly, exposing LDH1 to macrocyclic peptide MP 7 resulted in a concentration dependent conversion of LDH1 fluorescence spectrum to the one of the dimeric model LDHBtr. Normalization of the fluorescence intensity allowed to approximate the disruption ratio of LDH1 upon exposure to increasing amount of macrocyclic peptide MP7 (
[0292] Together, these results demonstrate that the designed cyclic peptides can target the tetramerization sites of LDHs by competing with the N-terminal domain of LDHB and LDHA monomers, leading to a destabilization and disruption of the tetrameric complexes. Moreover, these macrocyclic peptides can also interfere with the formation of LDH tetramers. These data also confirm that targeting of LDH highly conserved tetramerization site can lead to molecules interacting on both isoforms of the protein.
Example 3
1—Materials and Methods
[0293] Macrocyclic peptide MP7 (SEQ ID NO: 61) was evaluated at 200 μM against Mia Paca-2 human pancreatic cancer cells (ATCC®).
[0294] The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured on a Seahorse XF96 analyzer (Agilent®) with a combination of XF cell mito stress kit (Agilent®) and 2-deoxy-D-glucose (2DG; Sigma Aldrich®). Seahorse experiments were performed using 10,000 cells/well in DMEM medium with 10 mmol/L of D-glucose and 1 mmol/L of L-Glutamine Cells were incubated for 1 h in a CO.sub.2-free incubator before analysis. In the Seahorse analyzer, oximetry was repeatedly performed in closed wells after the sequential addition of the components of the XF cell mito stress kit, namely, oligomycin to inhibit ATP-synthase, ionophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) to disrupt the mitochondrial potential, and rotenone together with antimycin A to simultaneously inhibit Complexes I and III of the mitochondrial electron transport chain (ETC). Oximetry before the addition of any agent provided the basal respiration rate of the cells; ATP-linked reparation was determined after the addition of 1 μM of oligomycin and the maximal respiration rate of the cells after the addition of 1 μM of FCCP. All data were normalized to cell numbers measured right before oximetry using a SpectraMax miniMax 300 imaging cytometer (Molecular Devices®). Macrocyclic peptide MP7 in PBS or PBS alone (control experiment) were added directly into the medium and incubated with Mia Paca-2 cells during 4 h before conducting Seahorse experiments on the medium composed of the Mia Paca-2 cells with or without macrocyclic peptide MP7.
2—Results
[0295] Seahorse evaluation revealed a strong decrease in mitochondrial oxygen consumption rate (OCR) as well as an increase in the glycolytic flux of Mia Paca-2 human pancreatic cancer cells (
TABLE-US-00005 TABLE 5 SEQUENCES Sequences used herein SEQ ID NO: Name Sequences 1 hLDHA subunit MATLKDQLIYNLLKEEQTPQNKITVVGVGAVGMACAISILMKDLA DELALVDVIEDKLKGEMMDLQHGSLFLRTPKIVSGKDYNVTANSK LVIITAGARQQEGESRLNLVQRNVNIFKFIIPNVVKYSPNCKLLIVSN PVDILTYVAWKISGFPKNRVIGSGCNLDSARFRYLMGERLGVHPLS CHGWVLGEHGDSSVPVWSGMNVAGVSLKTLHPDLGTDKDKEQW KEVHKQVVESAYEVIKLKGYTSWAIGLSVADLAESIMKNLRRVHP VSTMIKGLYGIKDDVFLSVPCILGQNGISDLVKVTLTSEEEARLKKS ADTLWGIQKELQF 2 hLDHB subunit MATLKEKLIAPVAEEEATVPNNKITVVGVGQVGMACAISILGKSLA DELALVDVLEDKLKGEMMDLQHGSLFLQTPKIVADKDYSVTANS KIVVVTAGVRQQEGESRLNLVQRNVNVFKFIIPQIVKYSPDCIIIVVS NPVDILTYVTWKLSGLPKHRVIGSGCNLDSARFRYLMAEKLGIHPS SCHGWILGEHGDSSVAVWSGVNVAGVSLQELNPEMGTDNDSEN WKEVHKMVVESAYEVIKLKGYTNWAIGLSVADLIESMLKNLSRIH PVSTMVKGMYGIENEVFLSLPCILNARGLTSVINQKLKDDEVAQLK KSADTLWDIQKDLKDL 3 hLDHAtr NKITVVGVGAVGMACAISILMKDLADELALVDVIEDKLKGEMMD LQHGSLFLRTPKIVSGKDYNVTANSKLVIITAGARQQEGESRLNLV QRNVNIFKFIIPNVVKYSPNCKLLIVSNPVDILTYVAWKISGFPKNR VIGSGCNLDSARFRYLMGERLGVHPLSCHGWVLGEHGDSSVPVW SGMNVAGVSLKTLHPDLGTDKDKEQWKEVHKQVVESAYEVIKLK GYTSWAIGLSVADLAESIMKNLRRVHPVSTMIKGLYGIKDDVFLSV PCILGQNGISDLVKVTLTSEEEARLKKSADTLWGIQKELQF 4 hLDHBtr NNKITVVGVGQVGMACAISILGKSLADELALVDVLEDKLKGEMM DLQHGSLFLQTPKIVADKDYSVTANSKIVVVTAGVRQQEGESRLN LVQRNVNVFKFIIPQIVKYSPDCIIIVVSNPVDILTYVTWKLSGLPKH RVIGSGCNLDSARFRYLMAEKLGIHPSSCHGWILGEHGDSSVAVW SGVNVAGVSLQELNPEMGTDNDSENWKEVHKMVVESAYEVIKLK GYTNWAIGLSVADLIESMLKNLSRIHPVSTMVKGMYGIENEVFLSL PCILNARGLTSVINQKLKDDEVAQLKKSADTLWDIQKDLKDL 5 LBX X1X2X3X4X5X6X7X8 6 LB19 ATLKEKLIAPVAEEEATVP 7 LB13 ATLKEKLIAPVAE 8 LB8 ATLKEKLI 9 LB8-A2 ATAKEKLI 10 LB8-A3 ATLAEKLI 11 LB8-A4 ATLKAKLI 12 LB8-A5 ATLKEALI 13 LB8-A7 ATLKEKLA 14 Ac-LB8 Ac-ATLKEKLI 15 LB8-A3-A5 ATLAEALI 16 LB8-G1 GTLKEKLI 17 LB8-G8 ATLKEKLG 18 LB8-Aib1 ATL(Aib)EKLI 19 LB8-Aib2 ATLKE(Aib)LI 20 LB8-Aib3 ATL(Aib)E(Aib)LI 21 LA19 ATLKDQLIYNLLKEEQTPQ 22 LA8 ATLKDQLI 23 LB8-A1 AALKEKLI 24 LB8-A6 ATLKEKAI 25 LB8-AL7 ATLKEKL 26 LBc Ac-LIAPVAE-NH2 27 LB8-AcTI7 Ac-TLKEKLI 28 LB8-TI7 TLKEKLI 29 LB8-G3 ATGKEKLI 30 VS-142-BisAlk ACLKECLI 31 LT018 CTLKCKLI 32 LT020 ATLKCKLIC 33 LT021 ACTLKCKLI 34 LT022 CATLCEKLI 35 CB-09 ATCKEKCI 36 Antennapedia RQIKWFQNRRMKWKK Penetratin CCP 37 TAT CCP YGRKKRRQRRR 38 SynB1 CCP RGGRLSYSRRRFSTSTGR 39 SynB3 CCP RRLSYSRRRF 40 PTD-4 CCP PIRRRKKLRRLK 41 PTD-5 CCP RRQRRTSKLMKR 42 FHV Coat-(35- RRRRNRTRRNRRRVR 49) CCP 43 BMV Gag-(7- KMTRAQRRAAARRNRWTAR 25) CCP 44 HTLV-II Rex- TRRQRTRRARRNR (4-16) CCP 45 D-Tat CCP GRKKRRQRRRPPQ 46 R9-Tat CCP GRRRRRRRRRPPQ 47 Transportan GWTLNSAGYLLGKINLKALAALAKKIL CCP 48 MAP CCP KLALKLALKLALALKLA 49 SBP CCP MGLGLHLLVLAAALQGAWSQPKKKRKV 50 FBP CCP GALFLGWLGAAGSTMGAWSQPKKKRKV 51 MPG CCP GALFLGFLGAAGSTMGAWSQPKKKRKV 52 MPG(ΔNLS) GALFLGFLGAAGSTMGAWSQPKSKRKV CCP 53 PEP-1 CCP KETWWETWWTEWSQPKKKRKV 54 PEP-2 CCP KETWEETWFTEWSQPKKKRKV 55 MP1 CTLKCKLI 56 MP2 ACTLKCKLI 57 MP3 ACLKECLI 58 MP4 ATLKCKLIC 59 MPS ATLKECLIAC 60 MP6 CATLCEKLI 61 MP7 CTLKCKLI 62 MP8 CTLKCKLI 63 MP9 CTLKCKLI 64 MP10 Ac-KTLKDKLI 65 MP11 ATLKEKLI 66 MP12 ATLKEKLI 67 CT-44 CT(mlL)KCKLI 68 CT-45 CTLKCK(cpA)I
REFERENCES
[0296] Aihara et al. (2015). Synthesis of lactam-bridged cyclic peptides using sequential olefin metathesis and diimide reduction reactions. Tetrahedron 71(24): 4183-4191. [0297] Beckert et al. (2006). Lactate stimulates endothelial cell migration. Wound Repair Regen 14(3):321-324. [0298] Billiard et al. (2013). Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells. Cancer Metab 1(1):19. [0299] Brisson et al. (2016). Lactate Dehydrogenase B Controls Lysosome Activity and Autophagy in Cancer. Cancer Cell 30(3):418-431. [0300] Colegio et al. (2014). Functional polarization of tumour-associated macrophages by tumour-derived lactic acid. Nature 513(7519):559-563. [0301] Colen et al. (2011). Metabolic Targeting of Lactate Efflux by Malignant Glioma Inhibits Invasiveness and Induces Necrosis: An In Vivo Study. Neoplasia 13(7):620-632. [0302] Derossi et al. (1994). The third helix of the Antennapedia homeodomain translocates through biological membranes. Biol. Chem., 269, 10444-10450. [0303] de Saedeleer et al. (2012). Lactate Activates HIF-1 in Oxidative but Not in Warburg-Phenotype Human Tumor Cells. PLoS One 7(10). [0304] Döbeli et al. (1982). Isoenzyme specific inhibition of the reactivation of in vitro dissociated lactic dehydrogenase isoenzymes by two different peptides isolated from human liver”, Peptides, 3(2):167-174. [0305] Döbeli et al. (1987). Inhibition of the reactivation of acid-dissociated lactate dehydrogenase isoenzymes by their aminoterminal CNBr fragments. Peptides 8(5):773-778. [0306] Doherty and Cleveland (2013). Targeting lactate metabolism for cancer therapeutics. J Clin Invest 123(9):3685-3692. [0307] Eszes et al. (1996). Removal of substrate inhibition in a lactate dehydrogenase from human muscle by a single residue change. FEBS Lett 399(3):193-197. [0308] Fischer et al. (2001). Cellular delivery of impermeable effector molecules in the form of conjugates with peptides capable of mediating membrane translocation. Bioconjug. Chem., 12, 825-841. [0309] Fiume et al. (2014). Inhibition of lactate dehydrogenase activity as an approach to cancer therapy. Futur Med Chem 6(4):429-445. [0310] Hewitt et al. (1999). A general method for relieving substrate inhibition in lactate dehydrogenases. Protein Eng 12(6):491-496. [0311] Hill et al. (2014). Constraining cyclic peptides to mimic protein structure motifs. Angew Chemie—Int Ed 53(48):13020-13041. [0312] Izumi et al. (2011). Monocarboxylate transporters 1 and 4 are involved in the invasion activity of human lung cancer cells. Cancer Sci 102(5):1007-1013. [0313] Jafary et al. (2019). Novel Peptide Inhibitors for Lactate Dehydrogenase A (LDHA): A Survey to Inhibit LDHA Activity via Disruption of Protein-Protein Interaction”, Scientific Reports, 9(1):4686. [0314] Jo et al. (2012). Development of alpha-helical calpain probes by mimicking a natural protein-protein interaction. J Am Chem Soc 134(42):17704-17713. [0315] Kohlmann et al. (2013). Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors. J Med Chem 56(3):1023-1040. [0316] Labadie et al. (2015). Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enones as potent inhibitors of human lactate dehydrogenase. Bioorganic Med Chem Lett 25(1):75-82. [0317] Lacroix et al. (1998). Elucidating the folding problem of a-helices: Local motifs, long-range electrostatics, ionic strength dependence and prediction of NMR parameters. J. Mol. Biol. 284, 173-191. [0318] Muñoz and Serrano. (1994a). Elucidating the folding problem of helical peptides using empirical parameters. Nature: Struct. Biol. 1, 399-409. [0319] Muñoz and Serrano. (1994b). Elucidating the folding problem of a-helical peptides using empirical parameters, II. Helix macrodipole effects and rational modification of the helical content of natural peptides. J. Mol. Biol 245, 275-296. [0320] Muñoz and Serrano. (1994c). Elucidating the folding problem of a-helical peptides using empirical parameters III: Temperature and pH dependence. J. Mol. Biol 245, 297-308. [0321] Muñoz and Serrano. (1997). Development of the Multiple Sequence Approximation within the Agadir Model of a-Helix Formation. Comparison with Zimm-Bragg and Lifson-Roig Formalisms. Biopolymers 41, 495-509. [0322] Purkey et al. (2016) Cell Active Hydroxylactam Inhibitors of Human Lactate Dehydrogenase with Oral Bioavailability in Mice. ACS Med Chem Lett 7(10):896-901. [0323] Rai et al. (2017). Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH). J Med Chem 60(22):9184-9204. [0324] Rani and Kumar (2016). Recent Update on Human Lactate Dehydrogenase Enzyme 5 (hLDH5) Inhibitors: A Promising Approach for Cancer Chemotherapy. J Med Chem 59(2):487-496. [0325] Rao and Rossmann (1973). Comparison of super-secondary structures in proteins. J Mol Biol 76(2):241-250. [0326] Remington's Pharmaceutical Sciences 17th Ed., Mack Publishing Co. (A. R. Gennaro ed. 1985). [0327] Rudolph and Jaenicke (1976). Kinetics of reassociation and reactivation of pig-muscle lactic dehydrogenase after acid dissociation. Eur J Biochem 63(2):409-17. [0328] Sonveaux et al. (2008). Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. J Clin Invest 118(12):3930-3942. [0329] Spokoyny et al. (2013). Pentelute, A perfluoroaryl-cysteine SNAr chemistry approach to unprotected peptide stapling, J. Am. Chem. Soc. 135:5946-5949. [0330] Taylor (2002). The synthesis and study of side-chain lactam-bridged peptides. Biopolymers. 66(1):49-75. [0331] Végran et al. (2011). Lactate influx through the endothelial cell monocarboxylate transporter MCT1 supports an NF-kB/IL-8 pathway that drives tumor angiogenesis. Cancer Res 71(7):2550-2560. [0332] Vives et al. (1997). A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem., 272, 16010-16017. [0333] Ward et al. (2012). Design and Synthesis of Novel Lactate Dehydrogenase A Inhibitors by Fragment-Based Lead Generation. J Med Chem 55:3285-3306. [0334] Yachdav et al. (2014). PredictProtein—an open resource for online prediction of protein structural and functional features. Nucleic acids research, gku366. [0335] Ždralević et al. (2018). Double genetic disruption of lactate dehydrogenase A and B is required to ablate the “Warburg effect” restricting tumor growth to oxidative metabolism. J Biol Chem (49).