DGKα/ζ inhibitors combine with PD-1 checkpoint therapy to promote T cell-mediated antitumor immunity.

Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses. Toward this end, we designed a human primary T cell phenotypic high-throughput screening strategy to identify small molecules with distinct and complementary mechanisms of action to PD-1 checkpoint blockade. Through these efforts, we selected and optimized a chemical series that showed robust potentiation of T cell activation and combinatorial activity with αPD-1 blockade. Target identification was facilitated by chemical proteomic profiling with a lipid-based photoaffinity probe, which displayed enhanced binding to diacylglycerol kinase α (DGKα) in the presence of the active compound, a phenomenon that correlated with the translocation of DGKα to the plasma membrane. We further found that optimized leads within this chemical series were potent and selective inhibitors of both DGKα and DGKζ, lipid kinases that constitute an intracellular T cell checkpoint that blunts T cell signaling through diacylglycerol metabolism. We show that dual DGKα/ζ inhibition amplified suboptimal T cell receptor signaling mediated by low-affinity antigen presentation and low major histocompatibility complex class I expression on tumor cells, both hallmarks of resistance to PD-1 blockade. In addition, DGKα/ζ inhibitors combined with αPD-1 therapy to elicit robust tumor regression in syngeneic mouse tumor models. Together, these findings support targeting DGKα/ζ as a next-generation T cell immune checkpoint strategy.

Long-Acting Tumor-Activated Prodrug of a TGFßR Inhibitor.

Inhibition of TGFß signaling in concert with a checkpoint blockade has been shown to provide improved and durable antitumor immune response in mouse models. However, on-target adverse cardiovascular effects have limited the clinical use of TGFß receptor (TGFßR) inhibitors in cancer therapy. To restrict the activity of TGFßR inhibitors to tumor tissues and thereby widen the therapeutic index, a series of tumor-activated prodrugs of a selective small molecule TGFßR1 inhibitor 1 were prepared by appending 1 to a serine protease substrate and a half-life extension fatty acid carbon chain. The prodrugs were shown to be selectively metabolized in tumor tissues relative to the heart and blood and demonstrated a prolonged favorable increase in the tumor-to-heart ratio of the active drug in tissue distribution studies. Once-weekly administration of the most tissue-selective compound 10 provided anti-tumor efficacy comparable to the parent compound and reduced systemic exposure of the active drug.

Conformational-Analysis-Guided Discovery of 2,3-Disubstituted Pyridine IDO1 Inhibitors.

IDO1 inhibitors have shown promise as immunotherapies for the treatment of a variety of cancers, including metastatic melanoma and renal cell carcinoma. We recently reported the identification of several novel heme-displacing IDO1 inhibitors, including the clinical molecules linrodostat (BMS-986205) and BMS-986242. Both molecules contain quinolines that, while being present in successful medicines, are known to be potentially susceptible to oxidative metabolism. Efforts to swap this quinoline with an alternative aromatic system led to the discovery of 2,3-disubstituted pyridines as suitable replacements. Further optimization, which included lowering ClogP in combination with strategic fluorine incorporation, led to the discovery of compound 29, a potent, selective IDO1 inhibitor with robust pharmacodynamic activity in a mouse xenograft model.

Azatricyclic Inverse Agonists of RORγt That Demonstrate Efficacy in Models of Rheumatoid Arthritis and Psoriasis.

Structure-activity relationship studies directed toward the replacement of the fused phenyl ring of the lead hexahydrobenzoindole RORγt inverse agonist series represented by 1 with heterocyclic moieties led to the identification of three novel aza analogs 5-7. The hexahydropyrrolo[3,2-f]quinoline series 5 (X = N, Y = Z=CH) showed potency and metabolic stability comparable to series 1 but with improved in vitro membrane permeability and serum free fraction. This structural modification was applied to the hexahydrocyclopentanaphthalene series 3, culminating in the discovery of 8e as a potent and selective RORγt inverse agonist with an excellent in vitro profile, good pharmacokinetic properties, and biologic-like in vivo efficacy in preclinical models of rheumatoid arthritis and psoriasis.

TGFß2 and TGFß3 mediate appropriate context-dependent phenotype of rat valvular interstitial cells.

This study focused on characterizing the potential mechanism of valvular toxicity caused by TGFß receptor inhibitors (TGFßRis) using rat valvular interstitial cells (VICs) to evaluate early biological responses to TGFßR inhibition. Three TGFßRis that achieved similar exposures in the rat were assessed. Two dual TGFßRI/-RII inhibitors caused valvulopathy, whereas a selective TGFßRI inhibitor did not, leading to a hypothesis that TGFß receptor selectivity may influence the potency of valvular toxicity. The dual valvular toxic inhibitors had the most profound effect on altering VIC phenotype including altered morphology, migration, and extracellular matrix production. Reduction of TGFß expression demonstrated that combined TGFß2/ß3 inhibition by small interfering RNA or neutralizing antibodies caused similar alterations as TGFßRis. Inhibition of TGFß3 transcription was only associated with the dual TGFßRis, suggesting that TGFßRII inhibition impacts TGFß3 transcriptional regulation, and that the potency of valvular toxicity may relate to alteration of TGFß2/ß3-mediated processes involved in maintaining proper balance of VIC phenotypes in the heart valve.

Discovery of Imidazopyridines as Potent Inhibitors of Indoleamine 2,3-Dioxygenase 1 for Cancer Immunotherapy.

Indoleamine 2,3-dioxygenase 1 (IDO1) has been identified as a target for small-molecule immunotherapy for the treatment of a variety of cancers including renal cell carcinoma and metastatic melanoma. This work focuses on the identification of IDO1 inhibitors containing replacements or isosteres for the amide found in BMS-986205, an amide-containing, IDO1-selective inhibitor currently in phase III clinical trials. Detailed subsequently are efforts to identify a structurally differentiated IDO1 inhibitor via the pursuit of a variety of heterocyclic isosteres, leading to the discovery of highly potent, imidazopyridine-containing IDO1 inhibitors.

Discovery and Preclinical Evaluation of BMS-986242, a Potent, Selective Inhibitor of Indoleamine-2,3-dioxygenase 1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing dioxygenase enzyme implicated in cancer immune response. This account details the discovery of BMS-986242, a novel IDO1 inhibitor designed for the treatment of a variety of cancers including metastatic melanoma and renal cell carcinoma. Given the substantial interest around this target for cancer immunotherapy, we sought to identify a structurally differentiated clinical candidate that performs comparably to linrodostat (BMS-986205) in terms of both in vitro potency and in vivo pharmacodynamic effect in a mouse xenograft model. On the basis of its preclinical profile, BMS-986242 was selected as a candidate for clinical development.

An innovative kinome platform to accelerate small-molecule inhibitor discovery and optimization from hits to leads.

Kinases, accounting for 20% of the human genome, have been the focus of pharmaceutical drug discovery efforts for over three decades. Despite concerns surrounding the tractability of kinases as drug targets, it is evident that kinase drug discovery offers great potential, underscored by the US Food and Drug Administration (FDA) approval of 48 small-molecule kinase inhibitors. Despite these successes, it is challenging to identify novel kinome selective inhibitors with good pharmacokinetic/pharmacodynamic (PK/PD) properties, and resistance to kinase inhibitor treatment frequently arises. A new era of kinase drug discovery predicates the need for diverse and powerful tools to discover the next generation of kinase inhibitors. Here, we outline key tenets of the Bristol Meyers Squibb (BMS) kinase platform, to enable efficient generation of highly optimized kinase inhibitors.

Pharmacodynamics-based approach for efficacious human dose projection of BMS-986260, a small molecule transforming growth factor beta receptor 1 inhibitor.

Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine that has a wide array of biological effects. For decades, tumor biology implicated TGF-ß as an attractive therapeutic target due to its immunosuppressive effects. Toward this end, multiple pharmaceutical companies developed a number of drug modalities that specifically target the TGF-ß pathway. BMS-986260 is a small molecule, selective TGF-ßR1 kinase inhibitor that was under preclinical development for oncology. In vivo studies across mouse, rat, dog, and monkey and cryopreserved hepatocytes predicted human pharmacokinetics (PK) and distribution of BMS-986260. Efficacy studies of BMS-986260 were undertaken in the MC38 murine colon cancer model, and target engagement, as measured by phosphorylation of SMAD2/3, was assessed in whole blood to predict the clinical efficacious dose. The human clearance is predicted to be low, 4.25 ml/min/kg. BMS-986260 provided a durable and robust antitumor response at 3.75 mg/kg daily and 1.88 mg/kg twice-daily dosing regimens. Phosphorylation of SMAD2/3 was 3.5-fold less potent in human monocytes than other preclinical species. Taken together, the projected clinical efficacious dose was 600 mg QD or 210 mg BID for 3 days followed by a 4-day drug holiday. Mechanism-based cardiovascular findings in the rat ultimately led to the termination of BMS-986260. This study describes the preclinical PK characterization and pharmacodynamics-based efficacious dose projection of a novel small molecule TGF-ßR1 inhibitor.

Design, Synthesis, and Structure-Activity Relationships of Novel Tetrahydroisoquinolino Benzodiazepine Dimer Antitumor Agents and Their Application in Antibody-Drug Conjugates.

A series of tetrahydroisoquinoline-based benzodiazepine dimers were synthesized and tested for in vitro cytotoxicity against a panel of cancer cell lines. Structure-activity relationship investigation of various spacers guided by molecular modeling studies helped to identify compounds with picomolar activity. Payload 17 was conjugated to anti-mesothelin and anti-fucosylated monosialotetrahexosylganglioside (FucGM1) antibodies using lysosome-cleavable valine-citrulline dipeptide linkers via heterogeneous lysine conjugation and bacterial transglutaminase-mediated site-specific conjugation. In vitro, these antibody drug conjugates (ADCs) exhibited significant cytotoxic and target-mediated selectivity on human cancer cell lines. The pharmacokinetics and efficacy of these ADCs were further evaluated in gastric and lung cancer xenograft models in mice. Consistent pharmacokinetic profiles, high target specificity, and robust antitumor activity were observed in these models after a single dose of the ADC-46 (0.02 µmol/kg).

Discovery of BMS-986260, a Potent, Selective, and Orally Bioavailable TGFßR1 Inhibitor as an Immuno-oncology Agent.

Novel imidazole-based TGFßR1 inhibitors were identified and optimized for potency, selectivity, and pharmacokinetic and physicochemical characteristics. Herein, we report the discovery, optimization, and evaluation of a potent, selective, and orally bioavailable TGFßR1 inhibitor, 10 (BMS-986260). This compound demonstrated functional activity in multiple TGFß-dependent cellular assays, excellent kinome selectivity, favorable pharmacokinetic properties, and curative in vivo efficacy in combination with anti-PD-1 antibody in murine colorectal cancer (CRC) models. Since daily dosing of TGFßR1 inhibitors is known to cause class-based cardiovascular (CV) toxicities in preclinical species, a dosing holiday schedule in the anti-PD-1 combination efficacy studies was explored. An intermittent dosing regimen of 3 days on and 4 days off allowed mitigation of CV toxicities in one month dog and rat toxicology studies and also provided similar efficacy as once daily dosing.

Discovery of 4-Azaindole Inhibitors of TGFßRI as Immuno-oncology Agents.

The multifunctional cytokine TGFß plays a central role in regulating antitumor immunity. It has been postulated that inhibition of TGFß signaling in concert with checkpoint blockade will provide improved and durable immune response against tumors. Herein, we describe a novel series of 4-azaindole TGFß receptor kinase inhibitors with excellent selectivity for TGFß receptor 1 kinase. The combination of compound 3f and an antimouse-PD-1 antibody demonstrated significantly improved antitumor efficacy compared to either treatment alone in a murine tumor model.

Immune-modulating enzyme indoleamine 2,3-dioxygenase is effectively inhibited by targeting its apo-form.

For cancer cells to survive and proliferate, they must escape normal immune destruction. One mechanism by which this is accomplished is through immune suppression effected by up-regulation of indoleamine 2,3-dioxygenase (IDO1), a heme enzyme that catalyzes the oxidation of tryptophan to N-formylkynurenine. On deformylation, kynurenine and downstream metabolites suppress T cell function. The importance of this immunosuppressive mechanism has spurred intense interest in the development of clinical IDO1 inhibitors. Herein, we describe the mechanism by which a class of compounds effectively and specifically inhibits IDO1 by targeting its apo-form. We show that the in vitro kinetics of inhibition coincide with an unusually high rate of intrinsic enzyme-heme dissociation, especially in the ferric form. X-ray crystal structures of the inhibitor-enzyme complexes show that heme is displaced from the enzyme and blocked from rebinding by these compounds. The results reveal that apo-IDO1 serves as a unique target for inhibition and that heme lability plays an important role in posttranslational regulation.

Heterobicyclic inhibitors of transforming growth factor beta receptor I (TGFßRI).

The TGFß-TGFßR signaling pathway has been reported to play a protective role in the later stages of tumorigenesis via increasing immunosuppressive Treg cells and facilitating the epithelial to mesenchymal transition (EMT). Therefore, inhibition of TGFßR has the potential to enhance antitumor immunity. Herein we disclose the identification and optimization of novel heterobicyclic inhibitors of TGFßRI that demonstrate potent inhibition of SMAD phosphorylation. Application of structure-based drug design to the novel pyrrolotriazine chemotype resulted in improved binding affinity (Ki apparent = 0.14 nM), long residence time (T1/2 > 120 min) and significantly improved potency in the PSMAD cellular assay (IC50 = 24 nM). Several analogs inhibited phosphorylation of SMAD both in vitro and in vivo. Additionally, inhibition of TGFß-stimulated phospho-SMAD was observed in primary human T cells.

Macrocyclic pyrrolobenzodiazepine dimers as antibody-drug conjugate payloads.

Macrocyclic pyrrolobenzodiazepine dimers were designed and evaluated for use as antibody-drug conjugate payloads. Initial structure-activity exploration established that macrocyclization could increase the potency of PBD dimers compared with non-macrocyclic analogs. Further optimization overcame activity-limiting solubility issues, leading to compounds with highly potent (picomolar) activity against several cancer cell lines. High levels of in vitro potency and specificity were demonstrated with an anti-mesothelin conjugate.

Decarboxylative Peptide Macrocyclization through Photoredox Catalysis.

A method for the decarboxylative macrocyclization of peptides bearing N-terminal Michael acceptors has been developed. This synthetic method enables the efficient synthesis of cyclic peptides containing γ-amino acids and is tolerant of functionalities present in both natural and non-proteinogenic amino acids. Linear precursors ranging from 3 to 15 amino acids cyclize effectively under this photoredox method. To demonstrate the preparative utility of this method in the context of bioactive molecules, we synthesized COR-005, a somatostatin analogue that is currently in clinical trials.

Antroquinonol A: Scalable Synthesis and Preclinical Biology of a Phase 2 Drug Candidate.

The fungal-derived Taiwanese natural product antroquinonol A has attracted both academic and commercial interest due to its reported exciting biological properties. This reduced quinone is currently in phase II trials (USA and Taiwan) for the treatment of non-small-cell lung carcinoma (NSCLC) and was recently granted orphan drug status by the FDA for the treatment of pancreatic cancer and acute myeloid leukemia. Pending successful completion of human clinical trials, antroquinonol is expected to be commercialized under the trade name Hocena. A synthesis-enabled biological re-examination of this promising natural product, however, reveals minimal in vitro and in vivo antitumor activity in preclinical models.

Building homogeneous time-resolved fluorescence resonance energy transfer assays for characterization of bivalent inhibitors of an inhibitor of apoptosis protein target.

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is a central apoptosis regulator that blocks cell death by inhibiting caspase-3, caspase-7, and caspase-9 via binding interactions with the XIAP BIR2 and BIR3 domains (where BIR is baculovirus IAP repeat). Smac protein, in its dimeric form, effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Here we describe the development of highly sensitive homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assays to measure binding affinities of potent bivalent peptidomimetic inhibitors of XIAP. Our results indicate that these assays can differentiate Smac-mimetic inhibitors with a wide range of binding affinities down to the picomolar range. Furthermore, we demonstrate the utility of these fluorescent tools for characterization of inhibitor off-rates, which as a crucial determinant of target engagement and cellular potency is another important parameter to guide optimization in a structure-based drug discovery effort. Our study also explores how increased inhibitor valency can lead to enhanced potency at multimeric proteins such as IAP.

Ultrasensitive quantitative LC-MS/MS of an inhibitor of apoptosis protein's antagonist in plasma using protein target affinity extraction.

BACKGROUND: A target protein-based affinity extraction LC-MS/MS method was developed to enable plasma level determination following ultralow dosing (0.1-3 µg/kg) of an inhibitor of apoptosis proteins molecule. Methodology & results: Affinity extraction (AE) utilizing immobilized target protein BIR2/BIR3 was used to selectively capture the inhibitor of apoptosis proteins molecule from dog plasma and enable removal of background matrix components. Pretreatment of plasma samples using protein precipitation was found to provide an additional sensitivity gain. A LLOQ of 7.8 pM was achieved by combining protein precipitation with AE. The method was used to support an ultralow dose dog toxicity study. CONCLUSION: AE-LC-MS/MS, utilizing target protein, is a highly sensitive methodology for small molecule quantification with potential for broader applicability.

Pharmacology of smac mimetics; chemotype differentiation based on physical association with caspase regulators and cellular transport.

Cellular levels of inhibitor of apoptosis (IAP) proteins are elevated in multiple human cancers and their activities often play a part in promoting cancer cell survival by blocking apoptotic pathways, controlling signal transduction pathways and contributing to resistance. These proteins function through interactions of their BIR (baculoviral IAP repeat) protein domains with pathway components and these interactions are endogenously antagonized by Smac/Diablo (second mitochondrial activator of caspases/direct IAP binding protein with low isoelectric point). This report describes development of synthetic smac mimetics (SM) and compares their binding, antiproliferative and anti-tumor activities. All dimeric antagonists inhibit in vitro smac tetrapeptide binding to recombinant IAP proteins, rescue IAP-bound caspase-3 activity and show anti-proliferative activity against human A875 melanoma cells. One heterodimeric SM, SM3, binds tightly to IAP proteins in vitro and slowly dissociates (greater than two hours) from these protein complexes compared to the other antagonists. In addition, in vitro SM anti-proliferation potency is influenced by ABCB1 transporter (ATP-binding cassette, sub-family B; MDR1, P-gp) activities and one antagonist, SM5, does not appear to be an ABCB1 efflux pump substrate. All dimeric smac mimetics inhibit the growth of human melanoma A875 tumors implanted in athymic mice at well-tolerated doses. One antagonist, SM4, shows broad spectrum in vivo anti-tumor activity and modulates known pharmacodynamic markers of IAP antagonism. These data taken together demonstrate the range of diverse dimeric IAP antagonist activities and supports their potential as anticancer agents.