Discovery of 2-amino-3-amido-5-aryl-pyridines as highly potent, orally bioavailable, and efficacious PERK kinase inhibitors.

The protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is one of the three endoplasmic reticulum (ER) transmembrane sensors of the unfolded protein response (UPR) that regulates protein synthesis, alleviates cellular ER stress and has been implicated in tumorigenesis and prolonged cancer cell survival. In this study, we report a series of 2-amino-3-amido-5-aryl-pyridines that we have identified as potent, selective, and orally bioavailable PERK inhibitors. Amongst the series studied herein, compound (28) a (R)-2-Amino-5-(4-(2-(3,5-difluorophenyl)-2-hydroxyacetamido)-2-ethylphenyl)-N-isopropylnicotinamide has demonstrated potent biochemical and cellular activity, robust pharmacokinetics and 70% oral bioavailability in mice. Given these data, this compound (28) was studied in the 786-O renal cell carcinoma xenograft model. We observed dose-dependent, statistically significant tumor growth inhibition, supporting the use of this tool compound in additional mechanistic studies.

Inhibition of the eukaryotic initiation factor-2-α kinase PERK decreases risk of autoimmune diabetes in mice.

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing beta cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved ß cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in ß cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein PD-L1 in ß cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-ßH1 human ß cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances ß cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

Inhibition of the Eukaryotic Initiation Factor-2-α Kinase PERK Decreases Risk of Autoimmune Diabetes in Mice.

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing ß cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves ß cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in ß cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in ß cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-ßH1 human ß cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances ß cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

Discovery of HC-7366: An Orally Bioavailable and Efficacious GCN2 Kinase Activator.

A series of activators of GCN2 (general control nonderepressible 2) kinase have been developed, leading to HC-7366, which has entered the clinic as an antitumor therapy. Optimization resulted in improved permeability compared to that of the original indazole hinge binding scaffold, while maintaining potency at GCN2 and selectivity over PERK (protein kinase RNA-like endoplasmic reticulum kinase). The improved ADME properties of this series led to robust in vivo compound exposure in both rats and mice, allowing HC-7366 to be dosed in xenograft models, demonstrating that activation of the GCN2 pathway by this compound leads to tumor growth inhibition.

Development of Potent Mcl-1 Inhibitors: Structural Investigations on Macrocycles Originating from a DNA-Encoded Chemical Library Screen.

Evasion of apoptosis is critical for the development and growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family, associated with tumor aggressiveness, poor survival, and drug resistance. Development of Mcl-1 inhibitors implies blocking of protein-protein interactions, generally requiring a lengthy optimization process of large, complex molecules. Herein, we describe the use of DNA-encoded chemical library synthesis and screening to directly generate complex, yet conformationally privileged macrocyclic hits that serve as Mcl-1 inhibitors. By applying a conceptual combination of conformational analysis and structure-based design in combination with a robust synthetic platform allowing rapid analoging, we optimized in vitro potency of a lead series into the low nanomolar regime. Additionally, we demonstrate fine-tuning of the physicochemical properties of the macrocyclic compounds, resulting in the identification of lead candidates 57/59 with a balanced profile, which are suitable for future development toward therapeutic use.

Discovery of potent, selective and orally bioavailable imidazo[1,5-a]pyrazine derived ACK1 inhibitors.

This Letter describes the medicinal chemistry effort towards a series of novel imidazo[1,5-a]pyrazine derived inhibitors of ACK1. Virtual screening led to the discovery of the initial hit, and subsequent exploration of structure-activity relationships and optimization of drug metabolism and pharmacokinetic properties led to the identification of potent, selective and orally bioavailable ACK1 inhibitors.

Discovery of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1: optimization of kinase selectivity and pharmacokinetics.

The kinase selectivity and pharmacokinetic optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. The intersection of insights from molecular modeling, computational prediction of metabolic sites, and in vitro metabolite identification studies resulted in a simple and unique solution to both of these problems. These efforts culminated in the discovery of compound 13a, a potent, relatively selective inhibitor of TAK1 with good pharmacokinetic properties in mice, which was active in an in vivo model of ovarian cancer.

Discovery and optimization of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1.

The discovery and potency optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. Micromolar hits taken from high-throughput screening were optimized for biochemical and cellular mechanistic potency to ~10nM, as exemplified by compound 12az. Application of structure-based drug design aided by co-crystal structures of TAK1 with inhibitors significantly shortened the number of iterations required for the optimization.

Novel 6-aminofuro[3,2-c]pyridines as potent, orally efficacious inhibitors of cMET and RON kinases.

A series of novel 6-aminofuro[3,2-c]pyridines as kinase inhibitors is described, most notably, OSI-296 (6). We discuss our exploration of structure-activity relationships and optimization leading to OSI-296 and disclose its pharmacological activity against cMET and RON in cellular assays. OSI-296 is a potent and selective inhibitor of cMET and RON kinases that shows in vivo efficacy in tumor xenografts models upon oral dosing and is well tolerated.

PERK Inhibition by HC-5404 Sensitizes Renal Cell Carcinoma Tumor Models to Antiangiogenic Tyrosine Kinase Inhibitors.

PURPOSE: Tumors activate protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK, also called EIF2AK3) in response to hypoxia and nutrient deprivation as a stress-mitigation strategy. Here, we tested the hypothesis that inhibiting PERK with HC-5404 enhances the antitumor efficacy of standard-of-care VEGF receptor tyrosine kinase inhibitors (VEGFR-TKI). EXPERIMENTAL DESIGN: HC-5404 was characterized as a potent and selective PERK inhibitor, with favorable in vivo properties. Multiple renal cell carcinoma (RCC) tumor models were then cotreated with both HC-5404 and VEGFR-TKI in vivo, measuring tumor volume across time and evaluating tumor response by protein analysis and IHC. RESULTS: VEGFR-TKI including axitinib, cabozantinib, lenvatinib, and sunitinib induce PERK activation in 786-O RCC xenografts. Cotreatment with HC-5404 inhibited PERK in tumors and significantly increased antitumor effects of VEGFR-TKI across multiple RCC models, resulting in tumor stasis or regression. Analysis of tumor sections revealed that HC-5404 enhanced the antiangiogenic effects of axitinib and lenvatinib by inhibiting both new vasculature and mature tumor blood vessels. Xenografts that progress on axitinib monotherapy remain sensitive to the combination treatment, resulting in ∼20% tumor regression in the combination group. When tested across a panel of 18 RCC patient-derived xenograft (PDX) models, the combination induced greater antitumor effects relative to monotherapies. In this single animal study, nine out of 18 models responded with ≥50% tumor regression from baseline in the combination group. CONCLUSIONS: By disrupting an adaptive stress response evoked by VEGFR-TKI, HC-5404 presents a clinical opportunity to improve the antitumor effects of well-established standard-of-care therapies in RCC.

A PERK-Specific Inhibitor Blocks Metastatic Progression by Limiting Integrated Stress Response-Dependent Survival of Quiescent Cancer Cells.

PURPOSE: The integrated stress response (ISR) kinase PERK serves as a survival factor for both proliferative and dormant cancer cells. We aim to validate PERK inhibition as a new strategy to specifically eliminate solitary disseminated cancer cells (DCC) in secondary sites that eventually reawake and originate metastasis. EXPERIMENTAL DESIGN: A novel clinical-grade PERK inhibitor (HC4) was tested in mouse syngeneic and PDX models that present quiescent/dormant DCCs or growth-arrested cancer cells in micro-metastatic lesions that upregulate ISR. RESULTS: HC4 significantly blocks metastasis, by killing quiescent/slow-cycling ISRhigh, but not proliferative ISRlow DCCs. HC4 blocked expansion of established micro-metastasis that contained ISRhigh slow-cycling cells. Single-cell gene expression profiling and imaging revealed that a significant proportion of solitary DCCs in lungs were indeed dormant and displayed an unresolved ER stress as revealed by high expression of a PERK-regulated signature. In human breast cancer metastasis biopsies, GADD34 expression (PERK-regulated gene) and quiescence were positively correlated. HC4 effectively eradicated dormant bone marrow DCCs, which usually persist after rounds of therapies. Importantly, treatment with CDK4/6 inhibitors (to force a quiescent state) followed by HC4 further reduced metastatic burden. In HNSCC and HER2+ cancers HC4 caused cell death in dormant DCCs. In HER2+ tumors, PERK inhibition caused killing by reducing HER2 activity because of sub-optimal HER2 trafficking and phosphorylation in response to EGF. CONCLUSIONS: Our data identify PERK as a unique vulnerability in quiescent or slow-cycling ISRhigh DCCs. The use of PERK inhibitors may allow targeting of pre-existing or therapy-induced growth arrested "persister" cells that escape anti-proliferative therapies.

Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor.

The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.

Optimization of a Novel Mandelamide-Derived Pyrrolopyrimidine Series of PERK Inhibitors.

The protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is one of three endoplasmic reticulum (ER) transmembrane sensors of the unfolded protein response (UPR) responsible for regulating protein synthesis and alleviating ER stress. PERK has been implicated in tumorigenesis, cancer cell survival as well metabolic diseases such as diabetes. The structure-based design and optimization of a novel mandelamide-derived pyrrolopyrimidine series of PERK inhibitors as described herein, resulted in the identification of compound 26, a potent, selective, and orally bioavailable compound suitable for interrogating PERK pathway biology in vitro and in vivo, with pharmacokinetics suitable for once-a-day oral dosing in mice.

Potent and selective cyclohexyl-derived imidazopyrazine insulin-like growth factor 1 receptor inhibitors with in vivo efficacy.

Preclinical and emerging clinical evidence suggests that inhibiting insulin-like growth factor 1 receptor (IGF-1R) signaling may offer a promising therapeutic strategy for the treatment of several types of cancer. This Letter describes the medicinal chemistry effort towards a series of 8-amino-imidazo[1,5-a]pyrazine derived inhibitors of IGF-1R which features a substituted quinoline moiety at the C1 position and a cyclohexyl linking moiety at the C3 position. Lead optimization efforts which included the optimization of structure-activity relationships and drug metabolism and pharmacokinetic properties led to the identification of compound 9m, a potent, selective and orally bioavailable inhibitor of IGF-1R with in vivo efficacy in an IGF-driven mouse xenograft model.

Imidazo[1,5-a]pyrazines: orally efficacious inhibitors of mTORC1 and mTORC2.

The discovery and optimization of a series of imidazo[1,5-a]pyrazine inhibitors of mTOR is described. HTS hits were optimized for potency, selectivity and metabolic stability to provide the orally bioavailable proof of concept compound 4c that demonstrated target inhibition in vivo and concomitant inhibition of tumor growth in an MDA-MB-231 xenograft model.

Discovery of 2,4-1H-Imidazole Carboxamides as Potent and Selective TAK1 Inhibitors.

Herein we report the discovery of 2,4-1H-imidazole carboxamides as novel, biochemically potent, and kinome selective inhibitors of transforming growth factor ß-activated kinase 1 (TAK1). The target was subjected to a DNA-encoded chemical library (DECL) screen. After hit analysis a cluster of compounds was identified, which was based on a central pyrrole-2,4-1H-dicarboxamide scaffold, showing remarkable kinome selectivity. A scaffold-hop to the corresponding imidazole resulted in increased biochemical potency. Next, X-ray crystallography revealed a distinct binding mode compared to other TAK1 inhibitors. A benzylamide was found in a perpendicular orientation with respect to the core hinge-binding imidazole. Additionally, an unusual amide flip was observed in the kinase hinge region. Using structure-based drug design (SBDD), key substitutions at the pyrrolidine amide and the glycine resulted in a significant increase in biochemical potency.

The crystal structure of a constitutively active mutant RON kinase suggests an intramolecular autophosphorylation hypothesis.

A complex of RON(M1254T) with AMP-PNP and Mg(2+) reveals a substratelike positioning of Tyr1238 as well as likely catalysis-competent placement of the AMP-PNP and Mg(2+) components and indicates a tendency for cis phosphorylation. The structure shows how the oncogenic mutation may cause the constitutive activation and suggests a mechanistic hypothesis for the autophosphorylation of receptor tyrosine kinases.

Novel Autotaxin Inhibitor for the Treatment of Idiopathic Pulmonary Fibrosis: A Clinical Candidate Discovered Using DNA-Encoded Chemistry.

The activity of the secreted phosphodiesterase autotaxin produces the inflammatory signaling molecule LPA and has been associated with a number of human diseases including idiopathic pulmonary fibrosis (IPF). We screened a single DNA-encoded chemical library (DECL) of 225 million compounds and identified a series of potent inhibitors. Optimization of this series led to the discovery of compound 1 (X-165), a highly potent, selective, and bioavailable small molecule. Cocrystallization of compound 1 with human autotaxin demonstrated that it has a novel binding mode occupying both the hydrophobic pocket and a channel near the autotaxin active site. Compound 1 inhibited the production of LPA in human and mouse plasma at nanomolar levels and showed efficacy in a mouse model of human lung fibrosis. After successfully completing IND-enabling studies, compound 1 was approved by the FDA for a Phase I clinical trial. These results demonstrate that DECL hits can be readily optimized into clinical candidates.

Palladium-catalyzed direct heck arylation of dual pi-deficient/pi-excessive heteroaromatics. Synthesis of C-5 arylated imidazo[1,5-a]pyrazines.

A general and efficient synthesis of 5-aryl imidazo[1,5- a]pyrazines by palladium-catalyzed coupling of the corresponding 8-substituted derivatives with aryl halides is described. The scope of this new reaction for the imidazo[1,5- a]pyrazine ring system was explored using three readily available 8-substituted precursors, X = NH2, NMe2, and OMe, as well as 8-aryl derivatives, X = Ar'. On the basis of these results as well as studies using a deuterated derivative, a Heck-like mechanism is proposed for this transformation.

Novel 2-phenylquinolin-7-yl-derived imidazo[1,5-a]pyrazines as potent insulin-like growth factor-I receptor (IGF-IR) inhibitors.

A series of novel, potent quinolinyl-derived imidazo[1,5-a]pyrazine IGF-IR (IGF-1R) inhibitors--most notably, cis-3-(3-azetidin-1-ylmethylcyclobutyl)-1-(2-phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (AQIP)--is described. Synthetic details, structure-activity relationships, and in vitro biological activity are reported for the series. Key in vitro and in vivo biological results for AQIP are reported, including: inhibition of ligand-stimulated autophosphorylation of IGF-IR and downstream pathways in 3T3/huIGFIR cells; inhibition of proliferation and induction of DNA fragmentation in human tumor cell lines; a pharmacokinetic profile suitable for once-per-day oral dosing; antitumor activity in a 3T3/huIGFIR xenograft model; and effects on insulin and glucose levels.