METHODS AND COMPOSITIONS FOR TREATING EUKARYOTIC INFECTIONS VIA ALTERING AGGREGATION DYNAMICS OF RAPTOR/KOG1

Abstract

Methods and compositions for activating TORC1 aggregate formation in a fungi or a parasite, wherein TORC1 aggregate formation inhibits growth of the fungi or parasite. TORC1 aggregate formation may be activated using small molecules or other agents, and said agents may be used to treat or prevent a disease or condition associated with the fungi and parasite. The agent may target Kog1 of TORC1, e.g., the agent may directly or indirectly inhibit Kog1 leading to aggregation of the TORC1 complex.

Claims

1. A method of treating or preventing a disease or a condition associated with a fungi or a parasite in a subject in need thereof, said method comprising administering an agent that activates TORC1 aggregate formation to the subject that has been identified as having a disease or condition associated with a fungi or parasite, the TORC1 aggregate formation inhibits growth of the fungi or parasite.

2. The method of claim 1, wherein the subject includes a human, an animal or a plant.

3. The method of claim 1, wherein the agent that activates TORC1 aggregate formation targets Kog1 of TORC1.

4. The method of claim 3, wherein the agent binds to Kog1.

5. The method of claim 3, wherein the agent inhibits Kog1 indirectly.

6. The method of claim 1, wherein the disease or condition associated with the fungi or the parasites is parasitic worms, malaria, leishmaniasis, giardia infection, or a candidiasis infection.

7. The method of any one of claim 1, wherein the agent comprises a small molecule.

8. The method of claim 7, wherein the small molecule comprises metformin, PT 1, salicylsalicylic acid, Phenformin, AICAR, A-769662, Acadesine, orsomorphin, 1,1-Dimethylbiguanide hydrochloride, or BML-275.

9. The method of claim 1, wherein the agent comprises an antibody or a fragment thereof.

10. The method of claim 9, wherein the antibody or fragment thereof is monoclonal or polyclonal, nanobody.

11. The method of claim 1, wherein the parasite is selected from the group consisting of Helminths, Plasmodium, Leishmania, and Giardia.

12. The method of claim 1, wherein the method further comprises co-treating the subject with an antifungal drug or an anti-parasitic drug.

13. A method of inhibiting fungal or parasitic survival or replication in vitro, said method comprising applying to a cell or an in vitro system an agent that activates TORC1 aggregate formation, the TORC1 aggregate formation inhibits growth of the fungi or parasite.

14. A method of inhibiting fungal or parasitic survival or replication in an animal or a plant in need thereof, said method comprising administering to the animal or applying to the plant an agent that activates TORC1 aggregate formation, the TORC1 aggregate formation inhibits growth of the fungi or parasite.

15-19. (canceled)

20. The method of claim 13, wherein the agent comprises a small molecule that activates TORC1 aggregate formation by binding to and inhibiting Kog1.

21. The method of claim 16, wherein the small molecule comprises metformin, PT 1, salicylsalicylic acid, Phenformin, AICAR, A-769662, Acadesine, orsomorphin, 1,1-Dimethylbiguanide hydrochloride, or BML-275.

22. The method of claim 13, wherein the agent comprises an antibody or a fragment thereof.

23. The method of claim 14, wherein the agent comprises a small molecule that activates TORC1 aggregate formation by binding to and inhibiting Kog1.

24. The method of claim 19, wherein the small molecule comprises metformin, PT 1, salicylsalicylic acid, Phenformin, AICAR, A-769662, Acadesine, orsomorphin, 1,1-Dimethylbiguanide hydrochloride, or BML-275.

25. The method of claim 14, wherein the agent comprises an antibody or a fragment thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 shows the TOR pathway in budding yeast. (a) The activity of the TOR kinase (Tor1 and 2) depends on its participation in one of two protein complexes (TORC1 and TORC2). Only TORC1 activity is repressed by the small molecule rapamycin and this inhibition depends on an interaction with the prolyl isomerase Fpr1/Fkb12. The proteins in TORC1 and TORC2, except Tco89, are conserved across the eukaryotes. (b) TORC1 is regulated, in response to stress and a range of nutrients, through unknown pathways and mechanisms. Amino acids act on TOR through the RAG GTPases. When activated, TORC1 up-regulates growth through a variety of mechanisms (blue genes), and down regulates anabolic metabolism via TAP42/PP2A, stress genes (organge genes) and autophagy (not shown). Hexagons-kinases and phosphates: squares-transcription factors (TF): circle the directly regulated TF Sfp1.

[0028] FIG. 2 shows TORC1 regulation in KCl stress. (a) DNA microarray data comparing the gene expression program activated by KCl stress and the TORC1 inhibitor rapamycin. The rapamycin experiments compare the mRNA levels from cells grown in the conditions indicated on the diagram prior to, and 20 minutes after, treatment with 200 ng/ml rapamycin (aa=amino acid, glu=glucose). The KCl stress experiment compares the mRNA levels from cells in log growth conditions (YEPD, OD=0.6) prior to, and 20 min after, treatment with 0.4M KCl. In all of these experiments cDNA from cells treated with a stimuli are labeled with Cy5 while untreated cells are labeled with Cy3. Thus, genes activated by KCl or rapamycin are red on the heat map while genes repressed in these stimuli are green. The heat map shows data for all 700 genes that are up- or down-regulated >4-fold in one of the experiments. Four major gene sets were found by clustering; (1) genes down-regulated in rapamycin but not KCl, (2) genes down-regulated in both KCl and rapamycin, (3) genes activated in rapamycin but not KCl, and (4) genes activated primarily in KCl stress. The major ontology groups for each set are listed, along with their statistical significance. Experiments were performed using Agilent 60-mer oligonucleotide arrays, and were repeated at least two-times. (b) Sch9 is dephosphorylated in KCl stress. A phosphorylation-induced shift in the electrophoretic mobility of Sch9-3HA was measured by performing a western blot to a SDS-PAGE gel loaded with extract from cells collected before and after treatment with 0.4M KCl, using an anti-HA antibody. To increase the magnitude of the band shift for this large protein (110 kDa with tag), and ensure that we measure phosphorylation at the 6 C-terminal TORC1 target sites, proteins in the extract were cut into fragments using 2-Nitro-5-thiocyanatobenzoic acid, prior to loading on the gel. The image shows the smallest detected fragment, approximately 50 kDa (⅓ of protein without tag). Previous experiments identified the shift in this fragment and showed that it is due to dephosphorylation of Sch9 at the TORC1 sites. (c) Hog1/p38 inhibits TORC1 activity in KCl stress. The expression data for genes repressed more than 4-fold by rapamycin treatment in the log growth and KCl stress experiments from (a) are shown together with data from arrays comparing mRNA from 1) hog1Δ cells in KCl and rapamycin for 20 min (Cy5) to hog1Δ cells in KCl alone for 20 min (Cy3) and 2) hog1Δ in 0.4M KCl for 20 min (Cy5) to wt cells in the same conditions (Cy3). (d) Sch9 dephosphorylation in stress is partially blocked by deletion of Hog1. The experiment described in (b) was carried out in triplicate in wild-type and hog1Δ strains and the fraction of protein in the phosphorylated state was quantified using densitometry. The fraction of Sch9 phosphorylated at each time-point was normalized using the data at time 0 to eliminate noise from to prep-to-prep variation in the total level of Sch9 phosphorylation (which varied from 80 to 90%, at time=0, in hog1Δ and wt cells). The graph shows the average and standard deviation of each time-point from the three normalized data sets.

[0029] FIG. 3 shows Kog1 aggregation in glucose starvation. (a) Images of Kog1-YFP cells grown in synthetic medium with 2% glucose (SD medium, top row), or grown in SD medium and transferred to synthetic medium without glucose (bottom row) taken at 60× magnification on an inverted fluorescence microscope. The fraction of cells with a Kog1 aggregate at each time point are shown in the graph below. Each point shows the average and standard deviation from at least three separate experiments. (b) The formation of Kog1-YFP aggregates is inhibited strongly in cells missing the AMP activated protein kinase AMPK/Snf1 and in cells where a stretch of glutamine repeats has been replaced by alanine (see FIG. 2 for details).

[0030] FIG. 4 shows Kog1 contains two glutamine repeat regions, similar to those found in prion proteins (see the top sequence of FIG. 4 showing LQSRKSISLQSSQQQLQQQQQQQQQFTGFFEQNLTAFELW (SEQ ID NO: 1) and QLHSQLQHLQNQSHLQQQQSQQQQQHLEQQQMKIEKQIRH (SEQ ID NO: 2)). To probe the function of these regions, the core of these repeats was replaced with stretches of alanine (see bottom sequence of FIG. 4 showing LQSRKSISLQSSAAALAAAAAAAAAFTGFFEQNLTAFELW (SEQ ID NO:3) and QLHSQLQHLQNQSHLAAAASAAAAQHLEQQQMKIEKQIRH (SEQ ID NO:4)).

[0031] FIG. 5 shows TOR kinase activity is inhibited by Kog1 aggregation. To measure TORC1 activity the phosphorylation of the s6 kinase, Sch9, was followed using a bandshift assay (as described in Hughes Hallet et al., 2014). To perform this assay cells carrying Sch9 with an HA epitope tag at the C-terminus were grown in synthetic medium containing 2% glucose, and then transferred to synthetic medium containing either 0% or 0.02% glucose, as indicated on the gel (left hand images). The proteins in the cell were then precipitated using trichloroacetic acid, the cells lysed, and the protein extracted by bead beating in urea buffer. Proteins were then cut into fragments using 2-nitro-5-thiocyanatobenzoic acid (NTCB), and Sch9 detected by SDS-PAGE and western blotting using an anti-HA antibody. The 6 TORC1 phosphorylation sites in Sch9 are all located in the C-terminus of the protein and thus the lowest bands on the gel show changes in Sch9 phosphorylation at these sites (as the HA tag is also at the C-terminus). Examining phosphorylation in the wild-type (left gel) and glutamine mutant Kog1 (right gel) it is seen that Sch9 is fully phosphorylated in 2% glucose (upper band/arrow), and fully dephosphorylated after 60 min in 0% glucose (lower band/arrow). However, once cells starved for glucose are exposed to low concentrations of glucose, the two strains behave differently (see quantitation graphs on the right). In the wild-type strain, 0.02% glucose triggers phosphorylation of approximately 40% of the Sch9 in the population. This matches the fraction of the cells that do not have a Kog1 aggregate in glucose starvation conditions. In contrast, we see that 90-100% of the Sch9 is activated in cells with mutated glutamine repeats. Thus, cells with Kog1 aggregates are unable to activate TORC-Sch9 and grown in low concentrations of glucose. Nearly identical results are seen in 0.1% glucose.

[0032] FIG. 6 shows the distribution of Kog1 bodies in WT cells following acute glucose starvation (top) and the frequency distribution of Kog1 bodies in WT cells following acute glucose starvation (middle). The bottom panel shows the distribution of Kog1 bodies in WT cells following natural/chronic glucose starvation.

[0033] FIG. 7 shows Kog1 body formation in additional stress/starvation conditions (acute) in WT cells (top), and the distribution of Kog1 bodies in mutant cells following acute glucose starvation (bottom).

[0034] FIG. 8 compares the fraction of cells containing 1 or more Kog1 bodies in various strains.

[0035] FIG. 9 shows the dynamics of Kog1 foci (Kog1 bodies) resolution following glucose repletion in WT cells (top). The bottom panels show that cycloheximide (CHX) alone (a TORC1 activator) does not trigger disaggregation of Kog1.

[0036] FIG. 10 shows that Kog1 distribution/localization is distinct from Tor1 following acute glucose starvation.

[0037] FIG. 11 shows Sch9-HA bandshifts (measuring TORC1 activity) following glucose repletion, indicating that Kog1 foci (Kog1 bodies) formation affects TORC1 signaling.

[0038] FIG. 12 shows that Kog1 body formation plays a role in cell survival during long-term starvation.

DETAILED DESCRIPTION OF THE INVENTION

[0039] Referring now to FIG. 1-12, the present invention features methods and compositions or agents for treating or preventing diseases or conditions associated with eukaryotic organisms, e.g., fungi, parasites. Without wishing to limit the present invention to any theory or mechanism, the methods of the present invention may feature (a) directly or indirectly triggering/permitting Kog1 aggregation so as to inhibit TOR1 function, (b) directly or indirectly inhibiting Kog1 formation, or (c) another appropriate mechanism. The present invention also features methods for screening for such compositions or agents. The compositions may be used to directly or indirectly trigger/permit Kog1 aggregation to inhibit TORC1 function, or to inhibit Kog1 aggregation rendering the cells more susceptible to stress and starvation. The compositions may be used alone or in combination with other drugs/treatments to fight infection. Without wishing to limit the present invention to any theory or mechanism, it is believed that because the features of the signaling pathway that is the functional basis for these compositions (e.g., Kog1 aggregation determinants) is not present in human cells, it is expected that these compositions may have a low side effect profile and may be safe to use in conjunction with existing treatments.

[0040] Non-limiting examples of human diseases caused by eukaryotes include fungal infections (e.g. diseases caused by candida albicans, candida glabrata and other species) and parasitic diseases (e.g., diseases caused by helminths (parasitic worms), diseases caused by protozoans such as Plasmodium (e.g., malaria), diseases caused by Leishmania (e.g., leishmaniasis), giardia, etc.).

[0041] Non-limiting examples of other formulations that may be used in combination with compositions of the present invention include but are not limited to azoles, echinocandins and polyenes. Azole antifungals include but are not limited to fluconazole (Diflucan), itraconazole (Sporanox, Sporanox PulsePak), clotrimazole (Mycelex Troche), ketoconazole (Nizoral), posaconazole (Noxafil), voriconazole (Vfend), butoconazole (Femstat, Femstat 3, Gynazole-1) efinaconazole (Jublia), luliconazole 1% cream (Luzu), econazole, miconazole, oxiconazole, sulconazole, terconazole and tioconazole. Echinocandins include but are not limited to Caspofungin (Cancidas), anidulafungin (Eraxis) and micafungin (Mycamine). Polyenes include but are not limited to AMB lipid complex (Abelcet), AMB liposomal (AmBisome), AMB cholesteryl sulfate (Amphotec), nystatin (Bio-Statin, Mycostatin, Mycostatin Pastilles), AMB (Fungizone) and pimaricin. Allylamines include but are not limited to Amorolfine, Butenafine, Naftifine and Terbinafine (Lamisil). Other antifungals include but are not limited to Griseofulvin (Fulvicin U/F, Gris-Peg, Fulvicin P/G, Grifulvin V, Grisactin 250, Grisactin 500, Grisactin Ultra), flucytosine (Ancobon), ciprofloxacin 0.3%+dexamethasone 0.1% (Ciprodex), sertaconazole nitrate (Eratczo), ciclopirox (Loprox), Pentamidine isethionate (Nebupent, pentacarinat, pentam), haloprogin, tolnaftate and undecylenate. Other antifungal therapeutics include but are not limited to chitan synthase inhibitors, and drugs that disrupt mitotic spindle (e.g., griseofulvin).

[0042] The present invention features methods and systems (e.g., compositions) for treating or preventing a disease or a condition associated with a fungus/yeast or a parasite (e.g., in an animal such as a human or other animal). Or the methods and systems may inhibit fungal or parasitic survival or replication in an animal or a plant, or the methods and systems may inhibit fungal or parasitic survival or replication in vitro. In some embodiments, the method comprises administering to an animal, e.g., a human, mouse, rat, fish, etc.), or a plant or other appropriate system, an agent that sensitizes the TORC1 pathway to stress/starvation/treatment via inhibition of Kog1 aggregate formation or inhibits the TORC1 pathway (and growth) by triggering Kog1 aggregate formation.

[0043] In some embodiments, the agent comprises a small molecule. Small molecules may include but are not limited to AMPK activators and repressors such as metformin, PT 1, salicylsalicylic acid, Phenformin, AICAR, A-769662, Acadesine, orsomorphin, 1,1-Dimethylbiguanide hydrochloride, BML-275, etc. In some embodiments, the agent binds to Kog1 to inhibit Kog1/TORC1 function. In some embodiments, the agent inhibits Kog1 or Kog1 aggregate formation indirectly. For example, in some embodiments, the agent functions to hyperactive Snf1. In some embodiments, the agent functions to inactivate Snf1.

[0044] In some embodiments, the dose of metformin is from 50 mg to 5 g. In some embodiments, the dose of metformin is from 0.1 mg to 10 g. In some embodiments, the dose of metformin is from 1 mg to 100 mg. In some embodiments, the dose of metformin is from 100 mg to 2 g.

[0045] In some embodiments, the dose of phenformin is from 50 mg to 5 g. In some embodiments, the dose of phenformin is from 0.1 mg to 10 g. In some embodiments, the dose of phenformin is from 1 mg to 100 mg. In some embodiments, the dose of phenformin is from 100 mg to 2 g.

[0046] In some embodiments, the dose of PT1 is from 50 mg to 5 g. In some embodiments, the dose of PT1 is from 0.1 mg to 10 g. In some embodiments, the dose of PT1 is from 1 mg to 100 mg. In some embodiments, the dose of PT1 is from 100 mg to 2 g.

[0047] In some embodiments, the dose of salicylsalicylic acid is from 50 mg to 5 g. In some embodiments, the dose of salicylsalicylic acid is from 0.1 mg to 10 g. In some embodiments, the dose of salicylsalicylic acid is from 1 mg to 100 mg. In some embodiments, the dose of salicylsalicylic acid is from 100 mg to 2 g.

[0048] In some embodiments, the dose of AICAR is from 50 mg to 5 g. In some embodiments, the dose of AICAR is from 0.1 mg to 10 g. In some embodiments, the dose of AICAR is from 1 mg to 100 mg. In some embodiments, the dose of AICAR is from 100 mg to 2 g.

[0049] In some embodiments, the dose of A-769662 is from 50 mg to 5 g. In some embodiments, the dose of A-769662 is from 0.1 mg to 10 g. In some embodiments, the dose of A-769662 is from 1 mg to 100 mg. In some embodiments, the dose of A-769662 is from 100 mg to 2 g.

[0050] In some embodiments, the dose of Acadesine is from 50 mg to 5 g. In some embodiments, the dose of Acadesine is from 0.1 mg to 10 g. In some embodiments, the dose of Acadesine is from 1 mg to 100 mg. In some embodiments, the dose of Acadesine is from 100 mg to 2 g.

[0051] In some embodiments, the dose of 1,1-Dimethylbiguanide hydrochloride is from 50 mg to 5 g. In some embodiments, the dose of 1,1-Dimethylbiguanide hydrochloride is from 0.1 mg to 10 g. In some embodiments, the dose of 1,1-Dimethylbiguanide hydrochloride is from 1 mg to 100 mg. In some embodiments, the dose of 1,1-Dimethylbiguanide hydrochloride is from 100 mg to 2 g.

[0052] In some embodiments, the dose of BML-275 is from 50 mg to 5 g. In some embodiments, the dose of BML-275 is from 0.1 mg to 10 g. In some embodiments, the dose of BML-275 is from 1 mg to 100 mg. In some embodiments, the dose of BML-275 is from 100 mg to 2 g.

[0053] In some embodiments, the dose of orsomorphin is from 50 mg to 5 g. In some embodiments, the dose of orsomorphin is from 0.1 mg to 10 g. In some embodiments, the dose of orsomorphin is from 1 mg to 100 mg. In some embodiments, the dose of orsomorphin is from 100 mg to 2 g.

[0054] In some embodiments, the agent comprises an antibody or a fragment thereof (e.g., monoclonal, polyclonal antibody/fragment, etc.). The present invention is not limited to the aforementioned agents (e.g., small molecules, drugs, antibodies or antibody fragments).

[0055] In some embodiments, the agent comprises a polypeptide adapted to prevent Kog1 aggregate formation. The agent could be a non-antibody peptide that terminally participates in the Kog1 aggregate to block or inhibit aggregate formation (e.g., blocks templating or aggregate extension thus limiting loss of the functional TORC1 complex).

[0056] The methods and compositions of the present invention may further comprise the administration of a secondary drug or compound, e.g., an anti-fungal drug, an anti-parasitic drug, or any other appropriate drug or compound. For example, cells that cannot accommodate proper Kog1 aggregation are likely more susceptible to stress, e.g., drug treatments.

[0057] The present invention also features methods of screening in vitro for a compound that inhibits Kog1 aggregate formation. In some embodiments, the method comprises introducing a test compound to a first cell system and a second cell system, wherein the first cell system comprising test cells expressing Kog1 labeled with a tag, and the second cell system comprising control cells expressing Kog1 labeled with a tag. The cell systems may then be subjected to a particular stress (e.g., glucose starvation, nitrogen starvation or oxidative stress, etc.). The tags of the cell systems may then be visualized, and the amount of Kog1 aggregates (e.g., the number of foci of the tags (Kog1 bodies), or the size of the foci (signal intensity)) may then be calculated. In some embodiments, if the amount of Kog1 aggregates formed is less in the first cell system than in the second cell system, the compound inhibits Kog1 aggregate formation. If the amount of Kog1 aggregates formed is more in the first cell system than in the second cell system, the compound promotes Kog1 aggregate formation. The tag may comprise a fluorescent protein tag including but not limited to yellow fluorescent protein (YFP), green fluorescent protein (GFP), or red fluorescent protein (RFP). Immunohistochemistry may be used to visualize the Kog1 (e.g., via the tag). In some embodiments, the tag could be an epitope or the native protein and visualized via immunohistochemistry.

Example 1

[0058] A 40 year old woman (height of 158 cm, weight of 50 kg) presents to the emergency department exhibiting the following symptoms: difficulty swallowing and sore throat. The physician notes the presence of white plaques on the patient's tongue. The physician diagnoses esophageal candidiasis. The physician administers an oral pharmaceutical containing 200 mg of phenformin, an agent that activates TORC1 aggregate formation. After one day of treatment, the patient shows signs of improvement with the absence of white plaques on the tongue and no difficulty swallowing. By two days of treatment, her sore throat is gone.

Example 2

[0059] A 15 year old boy (height of 165 cm, weight of 55 kg) presents to his primary care physician with a red and itchy rash on his forearm. The rash is in a circular pattern. The physician diagnoses the patient with ringworm. The physician administers a dose of a topical pharmaceutical containing 3 g of AICAR, an agent that activates TORC1 aggregate formation. After two days of treatment, the patient shows signs of improvement with the absence of itch on his forearm. By four days of treatment, the rash is gone.

[0060] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[0061] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. For example, the present compositions and methods may be effectively used on termites. Therefore, the scope of the invention is only to be limited by the following claims. Reference numbers recited in the claims are exemplary and for ease of review by the patent office only, and are not limiting in any way. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting of” is met.

[0062] The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.