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Oncology Epigenetic Therapy SP-2528, an inhibitor of Lysine-Specific Demethylase 1 LSD1 PowerPoint Presentation
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Oncology Epigenetic Therapy SP-2528, an inhibitor of Lysine-Specific Demethylase 1 LSD1

Oncology Epigenetic Therapy SP-2528, an inhibitor of Lysine-Specific Demethylase 1 LSD1

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Oncology Epigenetic Therapy SP-2528, an inhibitor of Lysine-Specific Demethylase 1 LSD1

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    1. Oncology Epigenetic Therapy SP-2528, an inhibitor of Lysine-Specific Demethylase 1 (LSD1) Salarius Pharmaceuticals Non-Confidential Package November, 2011

    2. The Salarius Team August 11, 2012 2

    3. Why SP-2528 is an exciting potential oncology therapeutic August 11, 2012 3

    4. SP-2528, a Potent and Selective LSD1 Inhibitor SP-2528 First-in-class selective and reversible LSD1 inhibitor Novel compound series with strong IP position Low nanomolar potency against LSD1 Demonstrated cell-based activity consistent with target inhibition Excellent drug-like properties August 11, 2012 4 2509 represents a lead compound in the series and we have 3 compounds that meet clinical candidate criteria Ė 2528, 2509, and 2533. these are all from one series and then additionally we do have a backup series. They are novel compounds All the compounds are selective and reversible and do not bind co-valently like MAOi based compounds would They appear to be very selective, and do not hit other proteins which are similar to LSD except LSD2 and we do hit that The compounds are small, with a molecular weight of less than 400, and yet selective and potent Meet our criteria that is showing they can act like a real drug Our top 3 are in animal pharmacology and toxicology2509 represents a lead compound in the series and we have 3 compounds that meet clinical candidate criteria Ė 2528, 2509, and 2533. these are all from one series and then additionally we do have a backup series. They are novel compounds All the compounds are selective and reversible and do not bind co-valently like MAOi based compounds would They appear to be very selective, and do not hit other proteins which are similar to LSD except LSD2 and we do hit that The compounds are small, with a molecular weight of less than 400, and yet selective and potent Meet our criteria that is showing they can act like a real drug Our top 3 are in animal pharmacology and toxicology

    5. LSD1 as a cancer target Epigenetic control of histone methylation is an important driver of cancer LSD1 is overexpressed in cancer tissues compared to normal cells (prostate, breast, small cell lung cancer, bladder, neuroblastoma, others) siRNA knockdown of LSD1 suppresses growth of cancer cells Microarray signature of LSD1 inhibition by small molecules is available August 11, 2012 5 LSD1 is moving to the forefront as a potent target in cancer It functions as an epigenetic regulator There have been quite a few papers about LSD1, and based on gene expression, its interesting profile as overexpressed in many cancers such as lung, breast and prostate It is interesting that activity with inhibiting LSD1 via siRNA knock down is somewhat different than the activity seen when it is inhibited by a small moleculeLSD1 is moving to the forefront as a potent target in cancer It functions as an epigenetic regulator There have been quite a few papers about LSD1, and based on gene expression, its interesting profile as overexpressed in many cancers such as lung, breast and prostate It is interesting that activity with inhibiting LSD1 via siRNA knock down is somewhat different than the activity seen when it is inhibited by a small molecule

    6. Epigenetics August 11, 2012 6 The field of epigenetics and how these methylation marks impact function and regulate gene expression is of great interest today. Histones, core proteins associated with dna, are really important in regulating chromatin structure and gene expression The field of epigenetics and how these methylation marks impact function and regulate gene expression is of great interest today. Histones, core proteins associated with dna, are really important in regulating chromatin structure and gene expression

    7. Epigenetic Regulation of Histone H3 Histone H3 is highly regulated by methylation August 11, 2012 7 We know that histone H3 is highly regulated by methylation and that some of the marks are permissive while others are repressive.We know that histone H3 is highly regulated by methylation and that some of the marks are permissive while others are repressive.

    8. Methylation of Lysine August 11, 2012 8 Methylation of lysine happens in a multi-step process. There is mono, di and tri methylation. So it is important to look at the dynamics of how this happens. LSD1 vs. a jerad inhibitor This marks are quite dynamic and there is a lot of movement which has to happen to reach the tri-methylation state. We have seen that LSD1 demethylation can effect all of these methylated states. LSD1 tends to act more on the mono/di methylation and Jerad tends to act more on the di/tri methylated states. There could be a lot of potential to combine these agents with other epigenetic agents and with chemo therapies.Methylation of lysine happens in a multi-step process. There is mono, di and tri methylation. So it is important to look at the dynamics of how this happens. LSD1 vs. a jerad inhibitor This marks are quite dynamic and there is a lot of movement which has to happen to reach the tri-methylation state. We have seen that LSD1 demethylation can effect all of these methylated states. LSD1 tends to act more on the mono/di methylation and Jerad tends to act more on the di/tri methylated states. There could be a lot of potential to combine these agents with other epigenetic agents and with chemo therapies.

    9. Lysine-Specific Demethylase 1 LSD1 is highly conserved across species, suggesting it is a highly important player C-terminal 2/3 similarities to FAD-dependent amine oxidases N-terminal SWIRM domain Ė unknown function characteristic of proteins involved in chromatin regulation August 11, 2012 9 From a structural biology perspective, LSD1 is a highly conserved gene across species. It has a regulatory domain called SWIRM (at the N terminal) and the business end is the amine oxidase domain at the C terminal. From a structural biology perspective, LSD1 is a highly conserved gene across species. It has a regulatory domain called SWIRM (at the N terminal) and the business end is the amine oxidase domain at the C terminal.

    10. Regulation of Gene Expression by LSD1 Epigenetics is different than merely repressing or activating one biological process Because LSD1 demethylates H3K4 and H3K9 it can regulate gene expression in both directions Removing methyl groups on H3K4 represses expression Removing methyl groups on H3K9 enhances expression Binding partners to LSD1 determine specificity and activity August 11, 2012 10 LSD1 has interesting biology and can act as both a repressor and an activiator depending on the histone it interacts with. LSD1 has interesting biology and can act as both a repressor and an activiator depending on the histone it interacts with.

    11. LSD1 inhibitor in prostate cancer would: Reactivate silenced tumor suppressor genes to make the cancer androgen responsive again Repress AR-dependent (and ER-dependent) transcription through decreasing H3K9 methylation in ARE Reverse the repression of genes silenced by H3K4 hypomethylation (re-express tumor suppressor genes) August 11, 2012 11 LSD1 inhibitor would suppressor hormonal response and the main enzyme that mediated androgen receptor independence Cartoon shows the reprogramming of tumor suppressor genes from LSD1LSD1 inhibitor would suppressor hormonal response and the main enzyme that mediated androgen receptor independence Cartoon shows the reprogramming of tumor suppressor genes from LSD1

    12. Other Known Inhibitors of LSD1 Due to homology with monoamine oxidase enzymes and their dependence on FAD, MAOIs also inhibit LSD1, but with toxic side effects Examples: Due to homology to FAD-dependent polyamine oxidases, polyamine analogues also inhibit LSD1, but are poor drugs Examples: All these molecules have major issues with either their specificity and side effects, or with their ability to be acceptable drugs for people. SP-2528 has no such downsides 12 August 11, 2012 Known inhibitors all work because of the dependence of the enzyme on FAD and amine oxidase, but the MAOiís while effective, have several downsides We do not feel this is the best way to go to get activity based on amines and polyamines, although other companies are working in this space. We wanted to focus on a completely different MOA and take advantage of what was know about the crystal structures. Polyamine oxidase inhibitors are not very clean and have other activities besides LSD1 inhibitionKnown inhibitors all work because of the dependence of the enzyme on FAD and amine oxidase, but the MAOiís while effective, have several downsides We do not feel this is the best way to go to get activity based on amines and polyamines, although other companies are working in this space. We wanted to focus on a completely different MOA and take advantage of what was know about the crystal structures. Polyamine oxidase inhibitors are not very clean and have other activities besides LSD1 inhibition

    13. Competitive Landscape EPI Pharmaceuticals Virtual program in LSD1 polyamine biosynthesis inhibitors No public announcements since divestment by Progen into Epi (May, 2011) PG11047and PH11144 are analogs of the natural polyamine, spermine, that lowers cellular endogenous polyamine levels and competitively inhibits natural polyamine functions As such, their activity is broader than just LSD1 inhibition PG11047 in phase 1b, PG11144 in preclinical The phase 1 trials of PG11047 20 patient trial in lymphomas done by the NIH, listed as completed in 2008, We are unaware of any published readout of results An Advanced Solid Tumors or Lymphoma Combo trial done in combinations with 1) Gemcitabine or 2) Docetaxel or 3) Bevacizumab or 4) Erlotinib or 5) Cisplatin or 6) 5-Flurouracil or 7) Sunitinib Previously Progen (2010), previously Cellgate (2008), previously SLIL Biomedical Johns Hopkins University/Wayne State University and Wisconsin Alumni Research Foundation (WARF) licenses Oryzon Genomics, Barcelona Spanish diagnostics and therapeutics biotech LSD1 program is preclinical, and may be focused on neuroblastoma in oncology and the regulation of the expression of genes involved in the progression of Alzheimer's disease (AD), Parkinson's and Huntington's diseases. OG-98 is the lead compound for LSD1 inhibition in late preclinical development. 13 August 11, 2012

    14. LSD1 Discovery Approach 14 August 11, 2012 In our efforts, we took a structural and fragment based screening approach. We were able to start with solved crystal structures. The purple is the enzymatic domain, the green in the Swirm domain, and the yellow is the tower domain, which serves as a scaffolding function to bring transcriptional complexes together and therefore there are slight differences between the RNAi knock down and small molecule inhibition. Knock downs and biochemical inhibition seem to give a different phenotype probably because of this tower domain. We have beam time in Berkley in September to continue to solve the co-crystal structure. Our compounds do not hit the amine oxidase domain. Reversibility Ė we have done enzyme kinetics and we see a biochemical profile that shows they are competitive and reversible inhibitors, they donít act like covalent inhibitors, donít have chemistry on them to form covalent bonds Our compounds bind in a unique way which drives their selectivity and activity In our efforts, we took a structural and fragment based screening approach. We were able to start with solved crystal structures. The purple is the enzymatic domain, the green in the Swirm domain, and the yellow is the tower domain, which serves as a scaffolding function to bring transcriptional complexes together and therefore there are slight differences between the RNAi knock down and small molecule inhibition. Knock downs and biochemical inhibition seem to give a different phenotype probably because of this tower domain. We have beam time in Berkley in September to continue to solve the co-crystal structure. Our compounds do not hit the amine oxidase domain. Reversibility Ė we have done enzyme kinetics and we see a biochemical profile that shows they are competitive and reversible inhibitors, they donít act like covalent inhibitors, donít have chemistry on them to form covalent bonds Our compounds bind in a unique way which drives their selectivity and activity

    15. Identification of SP-0665 15 August 11, 2012 For early hits, we were able to make compounds with nice potency and 0665 quickly separated with an IC-50 around 100 nanomolar and could also effect lysine methylation. Jump in activity to 50-100 nanomolar activityFor early hits, we were able to make compounds with nice potency and 0665 quickly separated with an IC-50 around 100 nanomolar and could also effect lysine methylation. Jump in activity to 50-100 nanomolar activity

    16. Early Lead Inhibitor Increases H3K4 and K9 me2 Levels 16 August 11, 2012 Took the compound quickly into cell lines. We have about 500 we can screen pretty quickly. EWS-502 and SK-ES-1 are Ewing Sarcoma cell lines and LNCaP is a prostate line and SK-N-MC is a sarcoma line. And these cell lines had sensitivity at 50-100 micromolar to the compounds The effect of the compounds on e-cahedrin expression was also very profound. This has been pretty consistent across cell lines. Gene expression and the increased methylation, treatment time was 24 hours We can show e-cahedrin is heavily repressed with lysine 9 methylation, and was the gene most consistently changed with our compounds and this gene seems to be very sensitive to our compound and we would see lysine 4 methylation increases in the e-cadherin promoter Q: Did you look at the androgen receptor regulated genes and see a very profound effect? We have shown at specific gene loci we can see profound effects and this is what we have done rather than looking globally. This is looking at global effects of lysine 4 and lysine 9, exposed for 24 hours With our current compounds, we have done a lot on methylation and we have gone away from global methylation and are focused on a subset of genes which are moving more dramatically, and looking specifically at titration with dose and in the sensitive cell lines we see methyl marks moving around at low concentrations.Took the compound quickly into cell lines. We have about 500 we can screen pretty quickly. EWS-502 and SK-ES-1 are Ewing Sarcoma cell lines and LNCaP is a prostate line and SK-N-MC is a sarcoma line. And these cell lines had sensitivity at 50-100 micromolar to the compounds The effect of the compounds on e-cahedrin expression was also very profound. This has been pretty consistent across cell lines. Gene expression and the increased methylation, treatment time was 24 hours We can show e-cahedrin is heavily repressed with lysine 9 methylation, and was the gene most consistently changed with our compounds and this gene seems to be very sensitive to our compound and we would see lysine 4 methylation increases in the e-cadherin promoter Q: Did you look at the androgen receptor regulated genes and see a very profound effect? We have shown at specific gene loci we can see profound effects and this is what we have done rather than looking globally. This is looking at global effects of lysine 4 and lysine 9, exposed for 24 hours With our current compounds, we have done a lot on methylation and we have gone away from global methylation and are focused on a subset of genes which are moving more dramatically, and looking specifically at titration with dose and in the sensitive cell lines we see methyl marks moving around at low concentrations.

    17. SP-2509 Does Not Inhibit Monoamine Oxidases 17 August 11, 2012 Our best leads are not potent MAOiís and we donít have this MAOi activity, which we are quite excited about. We do probably effect LSD2. Far less is known about what LSD2 activity means and what LSD2 is doing. Our inhibitors are probably pan LSD inhibitors. We are currently looking at the consequences of LSD2 inhibition to see if it can be separated out. Also in our early animal work we have been able to dose to mid micromolar levels and we have yet to see any neurotoxicity so far. Our compounds are around 400 molecular weight so they have the potential to cross the blood brain barrier. We have not yet proved this but it is looking very good. Our intital oral bioavailability study in mice, it is 35-40% without any formulation at all Ė dry powder in dextrose. We are doing formal bioavailability studies in rats and dogs Our best leads are not potent MAOiís and we donít have this MAOi activity, which we are quite excited about. We do probably effect LSD2. Far less is known about what LSD2 activity means and what LSD2 is doing. Our inhibitors are probably pan LSD inhibitors. We are currently looking at the consequences of LSD2 inhibition to see if it can be separated out. Also in our early animal work we have been able to dose to mid micromolar levels and we have yet to see any neurotoxicity so far. Our compounds are around 400 molecular weight so they have the potential to cross the blood brain barrier. We have not yet proved this but it is looking very good. Our intital oral bioavailability study in mice, it is 35-40% without any formulation at all Ė dry powder in dextrose. We are doing formal bioavailability studies in rats and dogs

    18. Induced Expression of Silenced Tumor Suppressor Genes with SP-2509 18 August 11, 2012

    19. SP-2509 Kills Primary Triple Negative Breast Cancer Cells 19 August 11, 2012 These are primary effusion cells for a triple negative breast cancer and these are stem like cells in breast cancer. In these cells we see profound activity and with the population of cells which are more like cancer stem cells we have strong activity. Collect cells and fractionate them for the CD44+ fraction, and those cells are much more sensitive. There are additional transcription factors that we have identified and when those pathways are active, cancers seem to be much more sensitive to our compounds. We are currently collecting human cancer cells and we are trying to predict response and then see if they are in fact sensitive. We have seen the same thing with Her2Neu +, and even the ER+ breast cancers are more sensitive So far our predictive signature has held up pretty well. Most of our work is in using primary xenograph lines rather than with cell lines and we are profiling those for p53, cmyc, and other gene signatures. We are concentrating on breast cancer, prostate cancer in the unresponsive state and in sarcomas. This is where we feel the opportunity might be. We believe the primary activity is the induction of apoptosis Speroid 3d culture and then exposed for 72 hoursThese are primary effusion cells for a triple negative breast cancer and these are stem like cells in breast cancer. In these cells we see profound activity and with the population of cells which are more like cancer stem cells we have strong activity. Collect cells and fractionate them for the CD44+ fraction, and those cells are much more sensitive. There are additional transcription factors that we have identified and when those pathways are active, cancers seem to be much more sensitive to our compounds. We are currently collecting human cancer cells and we are trying to predict response and then see if they are in fact sensitive. We have seen the same thing with Her2Neu +, and even the ER+ breast cancers are more sensitive So far our predictive signature has held up pretty well. Most of our work is in using primary xenograph lines rather than with cell lines and we are profiling those for p53, cmyc, and other gene signatures. We are concentrating on breast cancer, prostate cancer in the unresponsive state and in sarcomas. This is where we feel the opportunity might be. We believe the primary activity is the induction of apoptosis Speroid 3d culture and then exposed for 72 hours

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    22. Excellent PK and Bioavailability 22 August 11, 2012 This was just a direct slurry in 5% dextrose and saw 30% bioavailability We are currently up over 250mg/kg without seeing any really toxicity Compounds are small, good properties, not greasy or anything like that. Around 95% protein bound in albumin assaysThis was just a direct slurry in 5% dextrose and saw 30% bioavailability We are currently up over 250mg/kg without seeing any really toxicity Compounds are small, good properties, not greasy or anything like that. Around 95% protein bound in albumin assays

    23. Summary We have developed a highly selective and potent LSD1 inhibitor that possess excellent drug-like properties Displays potent and reversible activity in biochemical assays, potent on-target activity in cell-based systems, and performs well in in vivo systems Pharmacology shows good PK properties Animal safety studies so far show remarkable lack of toxicity Salarius is looking for a collaborative partnership to advance SP-2528 into human clinical testing 23 August 11, 2012 We have done initial p450 inhibition assays and we do not inhibit any isoform, we are > 5 micromolar We have done hERG and they are not hERG offenders, > 10 micromolar We have done some ADME characteristics, CACO-2 Working on getting the compound properly formulated Doing some repeat dose and acute toxicity We know we can go 10-50x higher than effective doses and not see any overall toxicity 2528 Ė we know we can make good salt forms and it may have the best pharmaceutical properties Potency is very good Ė both biochem and cell based, We see double digit to single digit potency with NCCIT cells (stem cell like cells) at 9 nanomolar being the most sensitive. Most cell lines range at 50-200 micromolar and we feel we can get to that range These are 72-96 hour cell proliferation assays We have done initial p450 inhibition assays and we do not inhibit any isoform, we are > 5 micromolar We have done hERG and they are not hERG offenders, > 10 micromolar We have done some ADME characteristics, CACO-2 Working on getting the compound properly formulated Doing some repeat dose and acute toxicity We know we can go 10-50x higher than effective doses and not see any overall toxicity 2528 Ė we know we can make good salt forms and it may have the best pharmaceutical properties Potency is very good Ė both biochem and cell based, We see double digit to single digit potency with NCCIT cells (stem cell like cells) at 9 nanomolar being the most sensitive. Most cell lines range at 50-200 micromolar and we feel we can get to that range These are 72-96 hour cell proliferation assays