Background Granulocyte colony-stimulating factor (G-CSF) is the standard of care for mobilization of hematopoietic stem cells (HSCs). G-CSF requires 4-7 days of injections and often multiple aphereses to acquire sufficient CD34+ cells for transplant. The number of CD34+ HSCs mobilized can be variable and patients who fail to mobilize enough CD34+ cells are treated with the combination of G-CSF plus plerixafor. G-CSF use is associated with bone pain, nausea, headaches, fatigue, rare episodes of splenic rupture, and is contraindicated for patients with autoimmune and sickle cell disease. MGTA-145 (GroβT) is a CXCR2 agonist. MGTA-145, in combination with plerixafor, a CXCR4 inhibitor, has the potential to rapidly and reliably mobilize robust numbers of HSCs with a single dose and same-day apheresis for transplant that is free from G-CSF. MGTA-145 plus plerixafor work synergistically to rapidly mobilize HSCs in both mice and non-human primates (Hoggatt, Cell 2018; Goncalves, Blood 2018). Based on these data, Magenta initiated a Phase 1 dose-escalating study to evaluate the safety, PK and PD of MGTA-145 as a single agent and in combination with plerixafor. Methods This study consists of four parts. In Part A, healthy volunteers were dosed with MGTA-145 (0.0075 - 0.3 mg/kg) or placebo. In Part B, MGTA-145 dose levels from Part A were selected for use in combination with a clinically approved dose of plerixafor. In Part C, a single dose MGTA-145 plus plerixafor will be administered on day 1 and day 2. In Part D, MGTA-145 plus plerixafor will be administered followed by apheresis. Results MGTA-145 monotherapy was well tolerated in all subjects dosed (Table 1) with no significant adverse events. Some subjects experienced mild (Grade 1) transient lower back pain that dissipated within minutes. In the ongoing study, the combination of MGTA-145 with plerixafor was well tolerated, with some donors experiencing Grade 1 and 2 gastrointestinal adverse events commonly observed with plerixafor alone. Pharmacokinetic (PK) exposure and maximum plasma concentrations increased dose proportionally and were not affected by plerixafor (Fig 1A). Monotherapy of MGTA-145 resulted in an immediate increase in neutrophils (Fig 1B) and release of plasma MMP-9 (Fig 1C). Neutrophil mobilization plateaued within 1-hour post MGTA-145 at doses greater than 0.03 mg/kg. This plateau was followed by a rebound of neutrophil mobilization which correlated with re-expression of CXCR2 and presence of MGTA-145 at pharmacologically active levels. Markers of neutrophil activation were relatively unchanged (<2-fold vs baseline). A rapid and statistically significant increase in CD34+ cells occurred @ 0.03 and 0.075 mg/kg of MGTA-145 (p < 0.01) relative to placebo with peak mobilization (Fig 1D) 30 minutes post MGTA-145 (7-fold above baseline @ 0.03 mg/kg). To date, the combination of MGTA-145 plus plerixafor mobilized >20/µl CD34s in 92% (11/12) subjects compared to 50% (2/4) subjects receiving plerixafor alone. Preliminary data show that there was a significant increase in fold change relative to baseline in CD34+ cells (27x vs 13x) and phenotypic CD34+CD90+CD45RA- HSCs (38x vs 22x) mobilized by MGTA-145 with plerixafor. Mobilized CD34+ cells were detectable at 15 minutes with peak mobilization shifted 2 - 4 hours earlier for the combination vs plerixafor alone (4 - 6h vs 8 - 12h). Detailed results of single dose administration of MGTA-145 and plerixafor given on one day as well as also on two sequential days will be presented along with fully characterized graft analysis post apheresis from subjects given MGTA-145 and plerixafor. Conclusions MGTA-145 is safe and well tolerated, as a monotherapy and in combination with plerixafor and induced rapid and robust mobilization of significant numbers of HSCs with a single dose in all subjects to date. Kinetics of CD34+ cell mobilization for the combination was immediate (4x increase vs no change for plerixafor alone @ 15 min) suggesting the mechanism of action of MGTA-145 plus plerixafor is different from plerixafor alone. Preliminary data demonstrate that MGTA-145 when combined with plerixafor results in a significant increase in CD34+ fold change relative to plerixafor alone. Magenta Therapeutics intends to develop MGTA-145 as a first line mobilization product for blood cancers, autoimmune and genetic diseases and plans a Phase 2 study in multiple myeloma and non-Hodgkin lymphoma in 2020. Disclosures DiPersio: Magenta Therapeutics: Equity Ownership; NeoImmune Tech: Research Funding; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Consultancy; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Macrogenics: Research Funding, Speakers Bureau; Bioline Rx: Research Funding, Speakers Bureau; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding. Hoggatt:Magenta Therapeutics: Consultancy, Equity Ownership, Research Funding. Devine:Kiadis Pharma: Other: Protocol development (via institution); Bristol Myers: Other: Grant for monitoring support & travel support; Magenta Therapeutics: Other: Travel support for advisory board; My employer (National Marrow Donor Program) has equity interest in Magenta. Biernat:Medpace, Inc.: Employment. Howell:Magenta Therapeutics: Employment, Equity Ownership. Schmelmer:Magenta Therapeutics: Employment, Equity Ownership. Neale:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Goncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Raffel:Magenta Therapeutics: Employment, Equity Ownership. Falahee:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Morrow:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Davis:Magenta Therapeutics: Employment, Equity Ownership.

Background. The majority of bone marrow transplants (BMTs) are performed with granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood (mPB) as the source of hematopoietic stem cells (HSCs) for patients. Up to 80% of mPB allogeneic recipients, however, will experience graft-versus-host disease (GvHD). Despite higher levels of CD3+ T cells in mPB grafts compared to BM, the level of acute GvHD observed following transplant of HLA-matched mPB is comparable to HLA-matched BM. One explanation is that G-CSF mobilized grafts contain myeloid-derived suppressor cells (MDSCs) possessing potent immunosuppressive properties capable of inhibiting T cell proliferation in vitro. The percentage of MDSCs is variable in grafts mobilized with G-CSF and clinical data suggest that patients transplanted with mPB grafts that contain higher numbers of MDSCs may have better outcomes including lower rates of acute GvHD (Vendramin et al., BBMT 2014). Identification of a mobilizing regimen that consistently produces high numbers of HSCs and MDSCs may be preferred.

Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.

The majority of bone marrow transplants (BMT) utilize granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood (mPB) as the source of hematopoietic stem cells (HSCs). However, CD34+ harvest with G-CSF is variable and frequently unpredictable, often requires multiple apheresis sessions and is associated with significant side effects. Identification of a mobilizing regimen that consistently produces high numbers of engraftable CD34+ cells without the need for G-CSF would be ideal. We previously reported that MGTA-145, a CXCR2 agonist, when combined with the CXCR4 inhibitor, plerixafor, rapidly mobilizes CD34+ cells (Blood 2017 130:1920). In the current study, we evaluated the ability of MGTA-145 plus plerixafor to mobilize and effectively engraft HSCs in a pre-clinical nonhuman primate (NHP) stem cell transplantation animal model.   Rhesus macaques were mobilized with a single dose of MGTA-145, plerixafor or MGTA-145 plus plerixafor versus a multi-dose regimen of G-CSF. Detailed immune profiling was performed at 0 through 24 hours post treatment. Within 4 hours of dosing, MGTA-145 plus plerixafor resulted in a 16-fold increase in the number of CD34+CD90+CD45RA- HSCs in the periphery (p=0.0003, n=11), which were previously shown to predict successful transplant as evidenced by rapid neutrophil and platelet recovery in an autologous NHP transplant model (Sci Trans Med 2017 9:1145). Next, NHPs mobilized with MGTA-145 plus plerixafor were leukapheresed and CD34+ cells enriched by positive selection. These cells were genetically modified and transplanted back into the same NHP conditioned with 1080 cGy (4 doses over 48 hours). A representative apheresis yield was 2.3 × 106 CD34+ and 0.9 × 106 HSCs per kg, and the final dose of gene-marked CD34+ and HSCs for transplant following 48 hours in culture was 1.7 × 106 and 0.8 × 106 per kg, respectively. GFP marking in vivo was consistent with the marking on the primitive HSCs prior to transplant. Neutrophil and platelet engraftment occurred at day 10 and 15 post-transplant, respectively (Figure 1). The study is ongoing, and additional follow-up data on all available animals will be presented.   Co-administration of MGTA-145 plus plerixafor leads to both rapid and efficacious mobilization of CD34+ cells in the pre-clinical NHP large animal model. We found that MGTA-145 plus plerixafor mobilized grafts contain large numbers of CD34+CD90+CD45RA- HSCs, which are capable of long-term multilineage reconstitution, and preliminary results from autologous transplantation studies in NHPs demonstrate that MGTA-145 plus plerixafor mobilized HSCs lead to rapid neutrophil and platelet recovery. This study demonstrates, for the first time, that CD34+ cells mobilized with a novel regimen of MGTA-145 plus plerixafor can be collected via leukapheresis, subsequently gene modified and transplanted into NHPs, resulting in rapid engraftment.

Neuronal voltage-gated potassium channels, K(v)7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule K(v)7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. K(v)7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual K(v)7 isoforms was investigated in human detrusor tissue using a panel of K(v)7 openers with distinct activity profiles among K(v)7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric K(v)7.4 did not reduce detrusor contraction. This may suggest that the homomeric K(v)7.4 channel plays a less significant role in bladder contraction and further investigation is needed.

Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROβ, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.

PURPOSE:Degenerative diseases of the retina, such as retinitis pigmentosa and age-related macular degeneration, are characterized by the irreversible loss of photoreceptors. Several growth factors, including glial cell derived neurotrophic factor (GDNF), have been shown to rescue retinal neurons. An alternative strategy to direct GDNF administration is its induction in host retina by small molecules. Here we studied the ability of a novel small molecule GSK812 to induce GDNF in vitro/in vivo and rescue photoreceptors. METHODS:GDNF induction in vitro was assessed in human ARPE-19, human retinal progenitor cells (RPCs) and mouse pluripotent cell-derived eyecups. For time course pharmacokinetic and GDNF induction studies in C57Bl/6 mice, GSK812 sustained release formulation was injected intravitreally. The same delivery approach was used in the rhodopsin knockout mice and Royal College of Surgeon (RCS) rats to assess long-term GDNF induction and photoreceptor rescue. RESULTS:The suspension provided sustained GSK812 delivery with 28 μg of drug remaining in the eye 2 weeks after a single injection. GSK812 suspension injection in C57Bl/6 mice resulted in significant upregulation of GDNF mRNA (>1.8-fold) and protein levels (>2.8-fold). Importantly, GSK812 treatment resulted in outer nuclear layer preservation in rho-/- mice with a 2-fold difference in photoreceptor number. In the RCS rat, the GSK812 injection provided long-term rescue of photoreceptors and outer segments, accompanied by function preservation as well. CONCLUSIONS:GSK812 is a potent neuroprotectant that can induce GDNF in normal and diseased retina. This induction results in photoreceptor rescue in 2 models of retinal degeneration.

Introduction: The integrin very-late antigen-4 (VLA-4) is comprised of an alpha4 chain (CD49d) that is non-covalently associated with the beta1 integrin chain (CD29). VLA-4 plays an important role in the homing and retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. We previously reported (Karpova et al. Blood 2016;128:659) a novel strategy and pathway for rapid and potent mobilization of HSPCs in mice using a combination of a VLA-4 inhibitor and the truncated isoform of the CXCR2 agonist Gro-beta (tGro-β, SB-251353). These studies were completed with a novel VLA-4 inhibitor, CWHM-823, that exhibited increased potency and/or solubility relative to the previously described comparators BIO5192 (α4β1-specific) and firategrast (α4β1 and α4β7 dual-specific). Here we extended these findings by generating and characterizing additional novel VLA-4 inhibitors.

Introduction: Mobilized peripheral blood grafts are currently the predominant source of hematopoietic stem and progenitor cells (HSPC) for both autologous and allogeneic transplantation. The most common clinical hematopoietic stem cell mobilization protocol is five days of Filgrastim (G-CSF). This regimen requires daily injections, has been associated with bone pain and often results in unpredictably low yields. A rapid mobilization method that ideally only required a single treatment and had robust and predictable kinetics would be a significant improvement over the current standard of care. In mice, a unique CXCR2 agonist, GROβ, induces rapid mobilization of stem and progenitor cells 15 minutes after a single injection. When co-administered with plerixafor (AMD3100), an inhibitor of CXCR4, a synergistic increase in mobilization results, with a graft enriched in highly engraftable hematopoietic stem cells (Hoggatt et al., Blood 2016 128:368). Here, we present data demonstrating that combination treatment with GROβ and AMD3100 synergistically mobilizes CD34+ cells and colony forming units (CFU) in nonhuman primates (NHP). Methods: Cynomolgus macaques were injected with G-CSF (10 µg/kg/day, s.c. for four days), AMD3100 (1 mg/kg, s.c. once) or GROβ (various doses and routes) alone and in combination with AMD3100. Blood was collected at various times post treatment and analyzed by multicolor flow cytometry to quantitate HSPC numbers. Additional aliquots of mobilized blood were plated in methylcellulose and CFU were enumerated seven days later. Results: We tested the effect of GROβ alone or in combination with AMD3100 in Cynomolgus monkeys. As published previously, administration of G-CSF, AMD3100 or G-CSF+AMD3100 induced significant mobilization of hematopoietic stem and progenitor cells. In comparison, a single injection each of GROβ+AMD3100 yielded 4-fold more CD34+ cells compared to four days of G-CSF (p Conclusions: We describe a rapid mobilization method that within four hours of a single treatment results in robust hematopoietic mobilization in nonhuman primates. The GROβ+AMD3100 regimen enriches for primitive CD34+ CD90+ CD45RA- stem and progenitor cells with increased colony forming capacity compared to CD34+ cells mobilized with G-CSF+AMD3100, suggesting a significant graft quality difference with the new regimen. The ability to rapidly mobilize primitive HSC with increased CFU frequency may allow effective single day mobilization of HPSCs for patients requiring a HSCT and be especially useful for HSC based gene therapy and gene editing protocols where HSPC grafts enriched for primitive HSC may reduce manufacturing costs and ensure durability of the gene modified HSC. The ability to achieve increased HSPC numbers without dramatically increasing WBC and neutrophils may be especially advantageous in patient populations where G-CSF mobilization is not well tolerated such as Sickle Cell Disease, or where G-CSF leads to increased disease activity such as in autologous transplant for Multiple Sclerosis. A single treatment that results in robust mobilization that is equivalent to G-CSF may allow for a one-day collection method of donor stem cells, and these findings in nonhuman primates support clinical exploration. Disclosures Falahee: Magenta Therapeutics: Employment, Equity Ownership. Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Hoggatt: Magenta Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Morrow: Magenta Therapeutics: Employment, Equity Ownership. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Abstract Introduction : Since the first description of hematopoietic stem and progenitor cell (HSPC) mobilization over forty years ago, it has become the standard of care for both autologous and allogeneic transplantation. A five-day course of G-CSF represents the most commonly used mobilization regimen today. The CXCR4 inhibitor, plerixafor, is a more rapid but weak mobilizer when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. Methods : Given the critical role of VLA4/VCAM1 signaling for migration and retention of HSPC, we were seeking to identify small molecule antagonists of VLA4 with improved potency and bioavailability. Relative to previously described comparators Bio5192 (▢4▢1-specific) and firategrast (▢4▢1 and ▢4▢7 dual-specific), our lead candidate, CWHM-823, exhibited increased aqueous solubility and ~10-100 fold better activity in blocking VLA4 and mobilizing HSPC in mice. CWHM-823 pharmacokinetics and mobilization were assessed in BALB/c and DBA/2 mice at different doses (3 to 15 mg/kg) and time points (15 to 240 min) when administered alone or in combination with the truncated isoform of the CXCR2 agonist Gro-beta (tGro-β, 2.5 mg/kg, generously provided by GlaxoSmithKline). HSPC mobilization was monitored using flow cytometry and clonogenic in vitro assays. "True" stem cells were measured in a serial competitive transplantation assay. The combination of tGro-β and VLA4 antagonist was further tested in diabetic mice in comparison to G-CSF (9 x 100μg/kg, q12h). RNA profiling of flow-sorted HSPC was performed via microarray analysis. Results : The combination of tGro-β with each VLA4 antagonist resulted in a dramatic synergistic increase in circulating HSPC numbers when compared to steady state (50-70-fold) or treatment with single agents (3-10 fold) including tGroβ. Mobilization with tGro-β + CWHM-823 was rapid, peaking at 15-30 minutes after injection. In a model of streptozotocin-induced diabetes, the mobilopathy (reduction in stem cell mobilization compared to wild type mice) was considerably less pronounced with the combination tGro-β + CWHM-823 (~1.5-fold lower CFU mobilization in diabetic mice) versus the 5-day course of G-CSF (~3-fold reduction). Despite the superior progenitor cell mobilization achieved with G-CSF (~2-fold more CFU and LSK/ml), the concentration of serially repopulating units (RU) was equally high in the tGro-β + CWHM-823 and G-CSF mobilized grafts suggesting a higher HSC frequency (1 RU out of 200 vs. 1 RU out of 400 LSK/CFU) in the tGro-β + CWHM-823 mobilized grafts (Figure 1). RNA profiling demonstrated close similarity between the expression profile of tGro-β + CWHM-823 mobilized, BM resident, and G-CSF mobilized LSK, with less than 0.5% of genes found to be significantly up- or downregulated. CXCR2 chemokine receptor stimulation was critical for the observed synergistic response, as pretreatment ("priming") or simultaneous treatment with tGro-β resulted in subsequent enhanced mobilization using VLA4 inhibitors, whereas reversed administration (VLA4 antagonist followed by tGro-β) had no effect on potency of either agent. Lack of surface CXCR2 expression on HSPC suggested that a rapidly acting effector molecule released from tGro-β-stimulated mature myeloid cells may subsequently influence VLA4-mediated HSPC adhesion/retention. Consistent with this theory, we observed increased protease MMP-9 in plasma within minutes after treatment with tGro-β + CWHM-823. Conclusions: We describe a novel strategy for rapid, reliable, and potent mobilization of HSPC in mice using a combination of VLA4 blockade (via novel and potent ▢4▢1 inhibitors) and CXCR2 activation (via tGro-β). The combination of tGro-β + VLA4 inhibitors or tGro-β followed by VLA4 inhibitors results in synergistic and rapid HSPC mobilization with quantity and quality of repopulating units similar to optimal mobilization with G-CSF. These data suggest further development of tGro-β + VLA4 inhibitor combinations for clinical testing is warranted. Figure 1. Mobilization of repopulating units (RU) (n=8-10 recipients per group, mean±SEM) Figure 1. Mobilization of repopulating units (RU) (n=8-10 recipients per group, mean±SEM) Disclosures No relevant conflicts of interest to declare.

Abnormal accumulation of β-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, β-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the β-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to β-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/β-catenin is mainly mediated by protein–protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect β-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated β-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce β-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of β-catenin degradation. This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential.

Epigenetic regulation by histone methylation is crucial for proper programming of the genome during development. Homeostasis of histone methylation is balanced by the activities of histone methyltransferases and demethylases. Although these methyltransferases and demethylases represent logical targets for potential drug discovery, the activities of methyltransferases and demethylases regulated in response to a complex biological stimulus are also important and not yet clear. To manipulate and study histone methylation in biological systems, we screened a Biologically Diverse Compound Set (BDCS) utilizing a phenotypic assay system that directly measures the Histone 3 K27 tri-methylation (H3K27me3) level in cells. The BDCS is a unique set of target-annotated chemical probes, containing a total of 5853 compounds targeting 736 unique proteins with multiple maximally selective compounds for each target. A number of targets, with multiple hits against each target, were identified in the screen. This gave us confidence that these targets and pathways may be relevant, and included the identification of non-methyltransferase/demethylase targets as potential upstream regulators of H3K27me3. Our study suggests that a systematically designed chemical probe library can serve as a powerful drug discovery tool when combined with phenotypic screening. Follow-up studies using these findings may reveal novel therapeutically useful pathways and targets of H3K27me3 regulation.

Lead optimization of piperidine amide HTS hits, based on an anilino-thiazole core, led to the identification of analogs which displayed low nanomolar blocking activity at the canonical transient receptor channels 3 and 6 (TRPC3 & 6) based on FLIPR (carbachol stimulated) and electrophysiology (OAG stimulated) assays. In addition, the anilino-thiazole amides displayed good selectivity over other TRP channels (TRPA1, TRPV1, and TRPV4), as well as against cardiac ion channels (CaV1.2, hERG, and NaV1.5). The high oxidation potential of the aliphatic piperidine and aniline groups, as well as the lability of the thiazole amide group contributed to the high clearance observed for this class of compounds. Conversion of an isoquinoline amide to a naphthyridine amide markedly reduced clearance for the bicyclic piperidines, and improved oral bioavailability for this compound series, however TRPC3 and TRPC6 blocking activity was reduced substantially. Although the most potent anilino-thiazole amides ultimately lacked oral exposure in rodents and were not suitable for chronic dosing, analogs such as 14-19, 22, and 23 are potentially valuable in vitro tool compounds for investigating the role of TRPC3 and TRPC6 in cardiovascular disease.

Multiple regions of the 3-oxazolidinedione-6-naphthyl-pyridinone series identified via high throughput screening were explored. SAR studies of these regions including the left-hand side oxazolidinedione moiety, α-substituent on the oxazolidinedione ring, central pyridinone core, and substituents on the central pyridinone core led to the discovery of potent EP(3) receptor antagonists such as compound 29 which possesses outstanding rat pharmacokinetic properties. Synthesis and SAR of these novel compounds and DMPK properties of representative compounds are discussed.

Benzofuran-substituted urea analogs have been identified as novel P2Y(1) receptor antagonists. Structure-activity relationship studies around the urea and the benzofuran moieties resulted in compounds having improved potency. Several analogs were shown to inhibit ADP-mediated platelet activation.

High-throughput screening and subsequent optimization led to the discovery of novel 3-oxazolidinedione-6-aryl-pyridinones exemplified by compound 2 as potent and selective EP3 antagonists with excellent pharmacokinetic properties. Compound 2 was orally active and showed robust in vivo activities in overactive bladder models. To address potential bioactivation liabilities of compound 2, further optimization resulted in compounds 9 and 10, which maintained excellent potency, selectivity, and pharmacokinetic properties and showed no bioactivation liability in glutathione trapping studies. These highly potent, selective, and orally active EP3 antagonists are excellent tool compounds for investigating and validating potential therapeutic benefits from selectively inhibiting the EP3 receptor.

A series of 3-urea-1-(phenylmethyl)-pyridones was discovered as novel EP(3) antagonists via high-throughput screening and subsequent optimization. The synthesis, structure-activity relationships, and optimization of the initial hit that resulted in potent and selective EP(3) receptor antagonists such as 11g are described.

This Letter discloses a series of 2-aminothiadiazole amides as selective EP(3) receptor antagonists. SAR optimization resulted in compounds with excellent functional activity in vitro. In addition, efforts to optimize DMPK properties in the rat are discussed. These efforts have resulted in the identification of potent, selective EP(3) receptor antagonists with excellent DMPK properties suitable for in vivo studies.

Exploration of multiple regions of a bi-aryl amine template led to the identification of highly potent M-3 muscarinic acetylcholine receptor antagonists such as 14 (pA(2) = 11.0) possessing good sub-type selectivity for M-3 over M-2. The structure-activity relationships (SAR) and optimization of the bi-aryl amine series are described. (C) 2009 Elsevier Ltd. All rights reserved.