Multiple conformational states of the HPK1 kinase domain in complex with sunitinib reveal the structural changes accompanying HPK1 trans-regulation.

Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a Ser/Thr kinase that operates via the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways to dampen the T-cell response and antitumor immunity. Accordingly, selective HPK1 inhibition is considered a means to enhance antitumor immunity. Sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for the management of gastrointestinal stromal tumors (GISTs), renal cell carcinoma (RCC), and pancreatic cancer, has been reported to inhibit HPK1 in vitro In this report, we describe the crystal structures of the native HPK1 kinase domain in both nonphosphorylated and doubly phosphorylated states, in addition to a double phosphomimetic mutant (T165E,S171E), each complexed with sunitinib at 2.17-3.00-Å resolutions. The native nonphosphorylated cocrystal structure revealed an inactive dimer in which the activation loop of each monomer partially occupies the ATP- and substrate-binding sites of the partner monomer. In contrast, the structure of the protein with a doubly phosphorylated activation loop exhibited an active kinase conformation with a greatly reduced monomer-monomer interface. Conversely, the phosphomimetic mutant cocrystal structure disclosed an alternative arrangement in which the activation loops are in an extended domain-swapped configuration. These structural results indicate that HPK1 is a highly dynamic kinase that undergoes trans-regulation via dimer formation and extensive intramolecular and intermolecular remodeling of the activation segment.

Molecular conformations, interactions, and properties associated with drug efficiency and clinical performance among VEGFR TK inhibitors.

Analyses of compounds in clinical development have shown that ligand efficient-molecules with privileged physical properties and low dose are less likely to fail in the various stages of clinical testing, have fewer postapproval withdrawals, and are less likely to receive black box safety warnings. However, detailed side-by-side examination of molecular interactions and properties within single drug classes are lacking. As a class, VEGF receptor tyrosine kinase inhibitors (VEGFR TKIs) have changed the landscape of how cancer is treated, particularly in clear cell renal cell carcinoma, which is molecularly linked to the VEGF signaling axis. Despite the clear role of the molecular target, member molecules of this validated drug class exhibit distinct clinical efficacy and safety profiles in comparable renal cell carcinoma clinical studies. The first head-to-head randomized phase III comparative study between active VEGFR TKIs has confirmed significant differences in clinical performance [Rini BI, et al. (2011) Lancet 378:193-1939]. To elucidate how fundamental drug potency-efficiency is achieved and impacts differentiation within the VEGFR TKI class, we determined potencies, time dependence, selectivities, and X-ray structures of the drug-kinase complexes using a VEGFR2 TK construct inclusive of the important juxtamembrane domain. Collectively, the studies elucidate unique drug-kinase interactions that are dependent on distinct juxtamembrane domain conformations, resulting in significant potency and ligand efficiency differences. The identified structural trends are consistent with in vitro measurements, which translate well to clinical performance, underscoring a principle that may be broadly applicable to prospective drug design for optimal in vivo performance.

The Discovery of Exarafenib (KIN-2787): Overcoming the Challenges of Pan-RAF Kinase Inhibition.

RAF, a core signaling component of the MAPK kinase cascade, is often mutated in various cancers, including melanoma, lung, and colorectal cancers. The approved inhibitors were focused on targeting the BRAFV600E mutation that results in constitutive activation of kinase signaling through the monomeric protein (Class I). However, these inhibitors also paradoxically activate kinase signaling of RAF dimers, resulting in increased MAPK signaling in normal tissues. Recently, significant attention has turned to targeting RAF alterations that activate dimeric signaling (class II and III BRAF and NRAS). However, the discovery of a potent and selective inhibitor with biopharmaceutical properties suitable to sustain robust target inhibition in the clinical setting has proven challenging. Herein, we report the discovery of exarafenib (15), a highly potent and selective inhibitor that intercepts the RAF protein in the dimer compatible αC-helix-IN conformation and demonstrates anti-tumor efficacy in preclinical models with BRAF class I, II, and III and NRAS alterations.

Discovery of KIN-3248, An Irreversible, Next Generation FGFR Inhibitor for the Treatment of Advanced Tumors Harboring FGFR2 and/or FGFR3 Gene Alterations.

Fibroblast growth factor receptor (FGFR) alterations are present as oncogenic drivers and bypass mechanisms in many forms of cancer. These alterations can include fusions, amplifications, rearrangements, and mutations. Acquired drug resistance to current FGFR inhibitors often results in disease progression and unfavorable outcomes for patients. Genomic profiling of tumors refractory to current FGFR inhibitors in the clinic has revealed several acquired driver alterations that could be the target of next generation therapeutics. Herein, we describe how structure-based drug design (SBDD) was used to enable the discovery of the potent and kinome selective pan-FGFR inhibitor KIN-3248, which is active against many acquired resistance mutations. KIN-3248 is currently in phase I clinical development for the treatment of advanced tumors harboring FGFR2 and/or FGFR3 gene alterations.

Structure-based design, SAR analysis and antitumor activity of PI3K/mTOR dual inhibitors from 4-methylpyridopyrimidinone series.

PI3K, AKT and mTOR, key kinases from a frequently dysregulated PI3K signaling pathway, have been extensively pursued to treat a variety of cancers in oncology. Clinical trials of PF-04691502, a highly potent and selective ATP competitive kinase inhibitor of class 1 PI3Ks and mTOR, from 4-methylpyridopyrimidinone series, led to the discovery of a metabolite with a terminal carboxylic acid, PF-06465603. This paper discusses structure-based drug design, SAR and antitumor activity of the MPP derivatives with a terminal alcohol, a carboxylic acid or a carboxyl amide.

Design and Synthesis of Functionally Active 5-Amino-6-Aryl Pyrrolopyrimidine Inhibitors of Hematopoietic Progenitor Kinase 1.

Immune activating agents represent a valuable class of therapeutics for the treatment of cancer. An area of active research is expanding the types of these therapeutics that are available to patients via targeting new biological mechanisms. Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of immune signaling and a target of high interest for the treatment of cancer. Herein, we present the discovery and optimization of novel amino-6-aryl pyrrolopyrimidine inhibitors of HPK1 starting from hits identified via virtual screening. Key components of this discovery effort were structure-based drug design aided by analyses of normalized B-factors and optimization of lipophilic efficiency.

Structural modifications of a 3-methoxy-2-aminopyridine compound to reduce potential for mutagenicity and time-dependent drug-drug interaction.

(S)-1-((4-(3-(6-Amino-5-methoxypyridin-3-yl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)propan-2-ol, 1, was recently identified as a potent inhibitor of the oncogenic kinase bRAF. Compounds containing 3-methoxy-2-aminopyridine, as in 1, comprised a promising lead series because of their high ligand efficiency and excellent ADME profile. However, following metabolic oxidation, compounds in this series also demonstrated two significant safety risks: mutagenic potential and time-dependent drug-drug interaction (TDI). Metabolite identification studies revealed formation of a reactive metabolite. We hypothesized that minimizing or blocking the formation of such a metabolite would mitigate the safety liabilities. Our investigation demonstrated that structural modifications which either reduced the electron density of the 3-methoxy-2-aminopyridine ring or blocked the reactive site following metabolic oxidation were successful in reducing TDI and AMES mutagenicity.

Design strategies to target crystallographic waters applied to the Hsp90 molecular chaperone.

A series of novel and potent small molecule Hsp90 inhibitors was optimized using X-ray crystal structures. These compounds bind in a deep pocket of the Hsp90 enzyme that is partially comprised by residues Asn51 and Ser52. Displacement of several water molecules observed crystallographically in this pocket using rule-based strategies led to significant improvements in inhibitor potency. An optimized inhibitor (compound 17) exhibited potent Hsp90 inhibition in ITC, biochemical, and cell-based assays (K(d)=1.3 nM, K(i)=15 nM, and cellular IC(50)=0.5 µM).

Structure-Based Drug Design and Synthesis of PI3Kα-Selective Inhibitor (PF-06843195).

The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway is a frequently dysregulated pathway in human cancer, and PI3Kα is one of the most frequently mutated kinases in human cancer. A PI3Kα-selective inhibitor may provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family. Here, we describe our efforts to discover a PI3Kα-selective inhibitor by applying structure-based drug design (SBDD) and computational analysis. A novel series of compounds, exemplified by 2,2-difluoroethyl (3S)-3-{[2'-amino-5-fluoro-2-(morpholin-4-yl)-4,5'-bipyrimidin-6-yl]amino}-3-(hydroxymethyl)pyrrolidine-1-carboxylate (1) (PF-06843195), with high PI3Kα potency and unique PI3K isoform and mTOR selectivity were discovered. We describe here the details of the design and synthesis program that lead to the discovery of 1.

Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19.

The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins, which are cleaved at specific sites by a 3C-like cysteine protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.

Characterizing the effects of the juxtamembrane domain on vascular endothelial growth factor receptor-2 enzymatic activity, autophosphorylation, and inhibition by axitinib.

The catalytic domains of protein kinases are commonly treated as independent modular units with distinct biological functions. Here, the interactions between the catalytic and juxtamembrane domains of VEGFR2 are studied. Highly purified preparations of the receptor tyrosine kinase VEGFR2 catalytic domain without (VEGFR2-CD) and with (VEGFR2-CD/JM) the juxtamembrane (JM) domain were characterized by kinetic, biophysical, and structural methods. Although the catalytic parameters for both constructs were similar, the autophosphorylation rate of VEGFR2-CD/JM was substantially faster than VEGFR2-CD. The first event in the autophosphorylation reaction was phosphorylation of JM residue Y801 followed by phosphorylation of activation loop residues in the CD. The rates of activation loop autophosphorylation for the two constructs were determined to be similar. The autophosphorylation rate of Y801 was invariant on enzyme concentration, which is consistent with an intramolecular reaction. In addition, the first biochemical characterization of the advanced clinical compound axitinib is reported. Axitinib was found to have 40-fold enhanced biochemical potency toward VEGFR2-CD/JM (K(i) = 28 pM) compared to VEGFR2-CD, which correlates better with cellular potency. Calorimetric studies, including a novel ITC compound displacement method, confirmed the potency and provided insight into the thermodynamic origin of the potency differences. A structural model for the VEGFR2-CD/JM is proposed based on the experimental findings reported here and on the JM position in c-Kit, FLT3, and CSF1/cFMS. The described studies identify potential functions of the VEGFR2 JM domain with implications to both receptor biology and inhibitor design.

Nonclinical antiangiogenesis and antitumor activities of axitinib (AG-013736), an oral, potent, and selective inhibitor of vascular endothelial growth factor receptor tyrosine kinases 1, 2, 3.

PURPOSE: Axitinib (AG-013736) is a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases 1 to 3 that is in clinical development for the treatment of solid tumors. We provide a comprehensive description of its in vitro characteristics and activities, in vivo antiangiogenesis, and antitumor efficacy and translational pharmacology data. EXPERIMENTAL DESIGN: The potency, kinase selectivity, pharmacologic activity, and antitumor efficacy of axitinib were assessed in various nonclinical models. RESULTS: Axitinib inhibits cellular autophosphorylation of VEGF receptors (VEGFR) with picomolar IC(50) values. Counterscreening across multiple kinase and protein panels shows it is selective for VEGFRs. Axitinib blocks VEGF-mediated endothelial cell survival, tube formation, and downstream signaling through endothelial nitric oxide synthase, Akt and extracellular signal-regulated kinase. Following twice daily oral administration, axitinib produces consistent and dose-dependent antitumor efficacy that is associated with blocking VEGFR-2 phosphorylation, vascular permeability, angiogenesis, and concomitant induction of tumor cell apoptosis. Axitinib in combination with chemotherapeutic or targeted agents enhances antitumor efficacy in many tumor models compared with single agent alone. Dose scheduling studies in a human pancreatic tumor xenograft model show that simultaneous administration of axitinib and gemcitabine without prolonged dose interruption or truncation of axitinib produces the greatest antitumor efficacy. The efficacious drug concentrations predicted in nonclinical studies are consistent with the range achieved in the clinic. Although axitinib inhibits platelet-derived growth factor receptors and KIT with nanomolar in vitro potencies, based on pharmacokinetic/pharmacodynamic analysis, axitinib acts primarily as a VEGFR tyrosine kinase inhibitor at the current clinical exposure. CONCLUSIONS: The selectivity, potency for VEGFRs, and robust nonclinical activity may afford broad opportunities for axitinib to improve cancer therapy.

Subtypes in patients with opioid misuse: A prognostic enrichment strategy using electronic health record data in hospitalized patients.

BACKGROUND: Approaches are needed to better delineate the continuum of opioid misuse that occurs in hospitalized patients. A prognostic enrichment strategy with latent class analysis (LCA) may facilitate treatment strategies in subtypes of opioid misuse. We aim to identify subtypes of patients with opioid misuse and examine the distinctions between the subtypes by examining patient characteristics, topic models from clinical notes, and clinical outcomes. METHODS: This was an observational study of inpatient hospitalizations at a tertiary care center between 2007 and 2017. Patients with opioid misuse were identified using an operational definition applied to all inpatient encounters. LCA with eight class-defining variables from the electronic health record (EHR) was applied to identify subtypes in the cohort of patients with opioid misuse. Comparisons between subtypes were made using the following approaches: (1) descriptive statistics on patient characteristics and healthcare utilization using EHR data and census-level data; (2) topic models with natural language processing (NLP) from clinical notes; (3) association with hospital outcomes. FINDINGS: The analysis cohort was 6,224 (2.7% of all hospitalizations) patient encounters with opioid misuse with a data corpus of 422,147 clinical notes. LCA identified four subtypes with differing patient characteristics, topics from the clinical notes, and hospital outcomes. Class 1 was categorized by high hospital utilization with known opioid-related conditions (36.5%); Class 2 included patients with illicit use, low socioeconomic status, and psychoses (12.8%); Class 3 contained patients with alcohol use disorders with complications (39.2%); and class 4 consisted of those with low hospital utilization and incidental opioid misuse (11.5%). The following hospital outcomes were the highest for each subtype when compared against the other subtypes: readmission for class 1 (13.9% vs. 10.5%, p<0.01); discharge against medical advice for class 2 (12.3% vs. 5.3%, p<0.01); and in-hospital death for classes 3 and 4 (3.2% vs. 1.9%, p<0.01). CONCLUSIONS: A 4-class latent model was the most parsimonious model that defined clinically interpretable and relevant subtypes for opioid misuse. Distinct subtypes were delineated after examining multiple domains of EHR data and applying methods in artificial intelligence. The approach with LCA and readily available class-defining substance use variables from the EHR may be applied as a prognostic enrichment strategy for targeted interventions.

Small molecule inhibitors reveal PTK6 kinase is not an oncogenic driver in breast cancers.

Protein tyrosine kinase 6 (PTK6, or BRK) is aberrantly expressed in breast cancers, and emerging as an oncogene that promotes tumor cell proliferation, migration and evasion. Both kinase-dependent and -independent functions of PTK6 in driving tumor growth have been described, therefore targeting PTK6 kinase activity by small molecule inhibitors as a therapeutic approach to treat cancers remains to be validated. In this study, we identified novel, potent and selective PTK6 kinase inhibitors as a means to investigate the role of PTK6 kinase activity in breast tumorigenesis. We report here the crystal structures of apo-PTK6 and inhibitor-bound PTK6 complexes, providing the structural basis for small molecule interaction with PTK6. The kinase inhibitors moderately suppress tumor cell growth in 2D and 3D cell cultures. However, the tumor cell growth inhibition shows neither correlation with the PTK6 kinase activity inhibition, nor the total or activated PTK6 protein levels in tumor cells, suggesting that the tumor cell growth is independent of PTK6 kinase activity. Furthermore, in engineered breast tumor cells overexpressing PTK6, the inhibition of PTK6 kinase activity does not parallel the inhibition of tumor cell growth with a >500-fold shift in compound potencies (IC50 values). Overall, these findings suggest that the kinase activity of PTK6 does not play a significant role in tumorigenesis, thus providing important evidence against PTK6 kinase as a potential therapeutic target for breast cancer treatment.

Optimization of Orally Bioavailable Enhancer of Zeste Homolog 2 (EZH2) Inhibitors Using Ligand and Property-Based Design Strategies: Identification of Development Candidate (R)-5,8-Dichloro-7-(methoxy(oxetan-3-yl)methyl)-2-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3,4-dihydroisoquinolin-1(2H)-one (PF-06821497).

A new series of lactam-derived EZH2 inhibitors was designed via ligand-based and physicochemical-property-based strategies to address metabolic stability and thermodynamic solubility issues associated with previous lead compound 1. The new inhibitors incorporated an sp3 hybridized carbon atom at the 7-position of the lactam moiety present in lead compound 1 as a replacement for a dimethylisoxazole group. This transformation enabled optimization of the physicochemical properties and potency compared to compound 1. Analysis of relationships between calculated log D (clogD) values and in vitro metabolic stability and permeability parameters identified a clogD range that afforded an increased probability of achieving favorable ADME data in a single molecule. Compound 23a exhibited the best overlap of potency and pharmaceutical properties as well as robust tumor growth inhibition in vivo and was therefore advanced as a development candidate (PF-06821497). A crystal structure of 23a in complex with the three-protein PRC2 complex enabled understanding of the key structural features required for optimal binding.

Structure-based design and synthesis of (5-arylamino-2H-pyrazol-3-yl)-biphenyl-2',4'-diols as novel and potent human CHK1 inhibitors.

The cocrystal structure of a library hit was used to design a novel series of CHK1 inhibitors. The new series retained the critical hydrogen-bonding groups of the resorcinol moiety for binding but lacked the phenolic anilide moiety. The newly designed compounds exhibited similar enzymatic activity, while demonstrating increased cellular potency. Compound 10c, showing no single agent effect, potentiated the antiproliferative effect of Gemcitabine in both prostate and breast cancer cell lines.

Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Discovery of 1-{(3R,4R)-3-[({5-Chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1-one (PF-06459988), a Potent, WT Sparing, Irreversible Inhibitor of T790M-Containing EGFR Mutants.

First generation EGFR TKIs (gefitinib, erlotinib) provide significant clinical benefit for NSCLC cancer patients with oncogenic EGFR mutations. Ultimately, these patients' disease progresses, often driven by a second-site mutation in the EGFR kinase domain (T790M). Another liability of the first generation drugs is severe adverse events driven by inhibition of WT EGFR. As such, our goal was to develop a highly potent irreversible inhibitor with the largest selectivity ratio between the drug-resistant double mutants (L858R/T790M, Del/T790M) and WT EGFR. A unique approach to develop covalent inhibitors, optimization of reversible binding affinity, served as a cornerstone of this effort. PF-06459988 was discovered as a novel, third generation irreversible inhibitor, which demonstrates (i) high potency and specificity to the T790M-containing double mutant EGFRs, (ii) minimal intrinsic chemical reactivity of the electrophilic warhead, (iii) greatly reduced proteome reactivity relative to earlier irreversible EGFR inhibitors, and (iv) minimal activity against WT EGFR.

Discovery of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), a macrocyclic inhibitor of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) with preclinical brain exposure and broad-spectrum potency against ALK-resistant mutations.

Although crizotinib demonstrates robust efficacy in anaplastic lymphoma kinase (ALK)-positive non-small-cell lung carcinoma patients, progression during treatment eventually develops. Resistant patient samples revealed a variety of point mutations in the kinase domain of ALK, including the L1196M gatekeeper mutation. In addition, some patients progress due to cancer metastasis in the brain. Using structure-based drug design, lipophilic efficiency, and physical-property-based optimization, highly potent macrocyclic ALK inhibitors were prepared with good absorption, distribution, metabolism, and excretion (ADME), low propensity for p-glycoprotein 1-mediated efflux, and good passive permeability. These structurally unusual macrocyclic inhibitors were potent against wild-type ALK and clinically reported ALK kinase domain mutations. Significant synthetic challenges were overcome, utilizing novel transformations to enable the use of these macrocycles in drug discovery paradigms. This work led to the discovery of 8k (PF-06463922), combining broad-spectrum potency, central nervous system ADME, and a high degree of kinase selectivity.

Design of potent and selective inhibitors to overcome clinical anaplastic lymphoma kinase mutations resistant to crizotinib.

Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients. Under pressure of crizotinib treatment, point mutations arise in the kinase domain of ALK, resulting in resistance and progressive disease. The successful application of both structure-based and lipophilic-efficiency-focused drug design resulted in aminopyridine 8e, which was potent across a broad panel of engineered ALK mutant cell lines and showed suitable preclinical pharmacokinetics and robust tumor growth inhibition in a crizotinib-resistant cell line (H3122-L1196M).