Axitinib effectively inhibits BCR-ABL1(T315I) with a distinct binding conformation.

The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30-50% of cases of adult acute lymphoblastic leukaemia. Introduction of ABL1 kinase inhibitors (for example, imatinib) has markedly improved patient survival, but acquired drug resistance remains a challenge. Point mutations in the ABL1 kinase domain weaken inhibitor binding and represent the most common clinical resistance mechanism. The BCR-ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR-ABL1-driven leukaemia. Axitinib potently inhibited BCR-ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.

The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase.

The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors.

Recent progress on third generation covalent EGFR inhibitors.

First generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (gefitinib and erlotinib) demonstrate excellent clinical efficacy for NSCLC patients carrying EGFR oncogenic mutations (L858R, del exon 19 deletions between amino acids 746 and 750). Invariable, drug resistance occurs with around 60% of it driven by the EGFR-T790M gatekeeper mutation. To counter the T790M-dependent resistance, third generation covalent EGFR inhibitors have been developed with high potency toward T790M containing mutants and selectivity over WT EGFR. This review provides an overview of the third generation drugs currently in clinical trials and also encompasses novel methodologies developed to discover third generation covalent EGFR drugs.

An algebraic model for the kinetics of covalent enzyme inhibition at low substrate concentrations.

This article describes an integrated rate equation for the time course of covalent enzyme inhibition under the conditions where the substrate concentration is significantly lower than the corresponding Michaelis constant, for example, in the Omnia assays of epidermal growth factor receptor (EGFR) kinase. The newly described method is applicable to experimental conditions where the enzyme concentration is significantly lower than the dissociation constant of the initially formed reversible enzyme-inhibitor complex (no "tight binding"). A detailed comparison with the traditionally used rate equation for covalent inhibition is presented. The two methods produce approximately identical values of the first-order inactivation rate constant (kinact). However, the inhibition constant (Ki), and therefore also the second-order inactivation rate constant kinact/Ki, is underestimated by the traditional method by up to an order of magnitude.

Structure-based design of novel human Pin1 inhibitors (III): optimizing affinity beyond the phosphate recognition pocket.

The design of potent Pin1 inhibitors has been challenging because its active site specifically recognizes a phospho-protein epitope. The de novo design of phosphate-based Pin1 inhibitors focusing on the phosphate recognition pocket and the successful replacement of the phosphate group with a carboxylate have been previously reported. The potency of the carboxylate series is now further improved through structure-based optimization of ligand-protein interactions in the proline binding site which exploits the H-bond interactions necessary for Pin1 catalytic function. Further optimization using a focused library approach led to the discovery of low nanomolar non-phosphate small molecular Pin1 inhibitors. Structural modifications designed to improve cell permeability resulted in Pin1 inhibitors with low micromolar anti-proliferative activities against cancer cells.

A simple and efficient maleimide-based approach for peptide extension with a cysteine-containing peptide phage library.

Although peptide-based molecules are known to have therapeutic potential, the generation of phage focused libraries to optimize peptides is effort-consuming. A chemical method is developed to extend a maleimide-conjugated peptide with a cysteine-containing random-peptide phage display library. As a proof of concept, a 15-mer epidermal growth factor receptor (EGFR)-binding peptide was synthesized with a maleimide group at its C-terminus and then conjugated to the cysteine-containing library. After panning and screening, several extended peptides were discovered and tested to have a higher affinity to EGFR. This strategy can have broad utility to optimize pharmacophores of any modalities (peptides, unnatural peptides, drug conjugates) capable of bearing a maleimide group.

Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth.

Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K(d) = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC(50) = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC(50) = 4.7 +/- 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC(50) value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.

Protein kinase biochemistry and drug discovery.

Protein kinases are fascinating biological catalysts with a rapidly expanding knowledge base, a growing appreciation in cell regulatory control, and an ascendant role in successful therapeutic intervention. To better understand protein kinases, the molecular underpinnings of phosphoryl group transfer, protein phosphorylation, and inhibitor interactions are examined. This analysis begins with a survey of phosphate group and phosphoprotein properties which provide context to the evolutionary selection of phosphorylation as a central mechanism for biological regulation of most cellular processes. Next, the kinetic and catalytic mechanisms of protein kinases are examined with respect to model aqueous systems to define the elements of catalysis. A brief structural biology overview further delves into the molecular basis of catalysis and regulation of catalytic activity. Concomitant with a prominent role in normal physiology, protein kinases have important roles in the disease state. To facilitate effective kinase drug discovery, classic and emerging approaches for characterizing kinase inhibitors are evaluated including biochemical assay design, inhibitor mechanism of action analysis, and proper kinetic treatment of irreversible inhibitors. As the resulting protein kinase inhibitors can modulate intended and unintended targets, profiling methods are discussed which can illuminate a more complete range of an inhibitor's biological activities to enable more meaningful cellular studies and more effective clinical studies. Taken as a whole, a wealth of protein kinase biochemistry knowledge is available, yet it is clear that a substantial extent of our understanding in this field remains to be discovered which should yield many new opportunities for therapeutic intervention.

Molecular Characteristics of Repotrectinib That Enable Potent Inhibition of TRK Fusion Proteins and Resistant Mutations.

NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to TRK inhibitors (larotrectinib and entrectinib) but often mutate, limiting the durability of response for NTRK + patients. Next-generation inhibitors with compact macrocyclic structures (repotrectinib and selitrectinib) were designed to avoid resistance mutations. Head-to-head potency comparisons of TRK inhibitors and molecular characterization of binding interactions are incomplete, obscuring a detailed understanding of how molecular characteristics translate to potency. Larotrectinib, entrectinib, selitrectinib, and repotrectinib were characterized using cellular models of wild-type TRKA/B/C fusions and resistance mutant variants with a subset evaluated in xenograft tumor models. Crystal structures were determined for repotrectinib bound to TRKA (wild-type, solvent-front mutant). TKI-naïve and pretreated case studies are presented. Repotrectinib was the most potent inhibitor of wild-type TRKA/B/C fusions and was more potent than selitrectinib against all tested resistance mutations, underscoring the importance of distinct features of the macrocycle structures. Cocrystal structures of repotrectinib with wild-type TRKA and the TRKAG595R SFM variant elucidated how differences in macrocyclic inhibitor structure, binding orientation, and conformational flexibility affect potency and mutant selectivity. The SFM crystal structure revealed an unexpected intramolecular arginine sidechain interaction. Repotrectinib caused tumor regression in LMNA-NTRK1 xenograft models harboring GKM, SFM, xDFG, and GKM + SFM compound mutations. Durable responses were observed in TKI-naïve and -pretreated patients with NTRK + cancers treated with repotrectinib (NCT03093116). This comprehensive analysis of first- and second-generation TRK inhibitors informs the clinical utility, structural determinants of inhibitor potency, and design of new generations of macrocyclic inhibitors.

TPX-0131, a Potent CNS-penetrant, Next-generation Inhibitor of Wild-type ALK and ALK-resistant Mutations.

Since 2011, with the approval of crizotinib and subsequent approval of four additional targeted therapies, anaplastic lymphoma kinase (ALK) inhibitors have become important treatments for a subset of patients with lung cancer. Each generation of ALK inhibitor showed improvements in terms of central nervous system (CNS) penetration and potency against wild-type (WT) ALK, yet a key continued limitation is their susceptibility to resistance from ALK active-site mutations. The solvent front mutation (G1202R) and gatekeeper mutation (L1196M) are major resistance mechanisms to the first two generations of inhibitors while patients treated with the third-generation ALK inhibitor lorlatinib often experience progressive disease with multiple mutations on the same allele (mutations in cis, compound mutations). TPX-0131 is a compact macrocyclic molecule designed to fit within the ATP-binding boundary to inhibit ALK fusion proteins. In cellular assays, TPX-0131 was more potent than all five approved ALK inhibitors against WT ALK and many types of ALK resistance mutations, e.g., G1202R, L1196M, and compound mutations. In biochemical assays, TPX-0131 potently inhibited (IC50 <10 nmol/L) WT ALK and 26 ALK mutants (single and compound mutations). TPX-0131, but not lorlatinib, caused complete tumor regression in ALK (G1202R) and ALK compound mutation-dependent xenograft models. Following repeat oral administration of TPX-0131 to rats, brain levels of TPX-0131 were approximately 66% of those observed in plasma. Taken together, preclinical studies show that TPX-0131 is a CNS-penetrant, next-generation ALK inhibitor that has potency against WT ALK and a spectrum of acquired resistance mutations, especially the G1202R solvent front mutation and compound mutations, for which there are currently no effective therapies.

Discovery of PF-06873600, a CDK2/4/6 Inhibitor for the Treatment of Cancer.

Control of the cell cycle through selective pharmacological inhibition of CDK4/6 has proven beneficial in the treatment of breast cancer. Extending this level of control to additional cell cycle CDK isoforms represents an opportunity to expand to additional tumor types and potentially provide benefits to patients that develop tumors resistant to selective CDK4/6 inhibitors. However, broad-spectrum CDK inhibitors have a long history of failure due to safety concerns. In this approach, we describe the use of structure-based drug design and Free-Wilson analysis to optimize a series of CDK2/4/6 inhibitors. Further, we detail the use of molecular dynamics simulations to provide insights into the basis for selectivity against CDK9. Based on overall potency, selectivity, and ADME profile, PF-06873600 (22) was identified as a candidate for the treatment of cancer and advanced to phase 1 clinical trials.

Structure-based design of novel human Pin1 inhibitors (II).

Following the discovery of a novel series of phosphate-containing small molecular Pin1 inhibitors, the drug design strategy shifted to replacement of the phosphate group with an isostere with potential better pharmaceutical properties. The initial loss in potency of carboxylate analogs was likely due to weaker charge-charge interactions in the putative phosphate binding pocket and was subsequently recovered by structure-based optimization of ligand-protein interactions in the proline binding site, leading to the discovery of a sub-micromolar non-phosphate small molecular Pin1 inhibitor.

Symmetric Arginine Dimethylation Is Selectively Required for mRNA Splicing and the Initiation of Type I and Type III Interferon Signaling.

Alternative splicing is well understood to enhance proteome diversity as cells respond to stimuli. However, mechanistic understanding for how the spliceosome processes precursor messenger RNA (mRNA) transcripts to achieve template diversification is incomplete. We use recently developed enzymatic inhibitors of protein arginine methyltransferase 5 (PRMT5) and human naive T lymphocyte activation as a model system to uncover a precise set of mRNA transcripts that require symmetric arginine dimethylation. This methylation-dependent splicing selectivity is associated with a limited set of signaling pathways that are affected when PRMT5 is inhibited. Specifically, we identify a conserved role for symmetric arginine dimethylation in the induction of antiviral type I and type III interferon signaling following T cell receptor and pattern recognition receptor stimulation in human T lymphocytes and undifferentiated human THP-1 monocytes. Altogether, these findings reveal a mechanism by which cells may be enabled to precisely modulate transcript heterogeneity to orchestrate specific functional outcomes.

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.

Enzymatic characterization of c-Met receptor tyrosine kinase oncogenic mutants and kinetic studies with aminopyridine and triazolopyrazine inhibitors.

The c-Met receptor tyrosine kinase (RTK) is a key regulator in cancer, in part, through oncogenic mutations. Eight clinically relevant mutants were characterized by biochemical, biophysical, and cellular methods. The c-Met catalytic domain was highly active in the unphosphorylated state (k(cat) = 1.0 s(-1)) and achieved 160-fold enhanced catalytic efficiency (k(cat)/K(m)) upon activation to 425000 s(-1) M(-1). c-Met mutants had 2-10-fold higher basal enzymatic activity (k(cat)) but achieved maximal activities similar to those of wild-type c-Met, except for Y1235D, which underwent a reduction in maximal activity. Small enhancements of basal activity were shown to have profound effects on the acquisition of full enzymatic activity achieved through accelerating rates of autophosphorylation. Biophysical analysis of c-Met mutants revealed minimal melting temperature differences indicating that the mutations did not alter protein stability. A model of RTK activation is proposed to describe how a RTK response may be matched to a biological context through enzymatic properties. Two c-Met clinical candidates from aminopyridine and triazolopyrazine chemical series (PF-02341066 and PF-04217903) were studied. Biochemically, each series produced molecules that are highly selective against a large panel of kinases, with PF-04217903 (>1000-fold selective relative to 208 kinases) being more selective than PF-02341066. Although these prototype inhibitors have similar potencies against wild-type c-Met (K(i) = 6-7 nM), significant differences in potency were observed for clinically relevant mutations evaluated in both biochemical and cellular contexts. In particular, PF-02341066 was 180-fold more active against the Y1230C mutant c-Met than PF-04217903. These highly optimized inhibitors indicate that for kinases susceptible to active site mutations, inhibitor design may need to balance overall kinase selectivity with the ability to inhibit multiple mutant forms of the kinase (penetrance).

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.

Structure-based design of novel human Pin1 inhibitors (I).

Pin1 is a member of the cis-trans peptidyl-prolyl isomerase family with potential anti-cancer therapeutic value. Here we report structure-based de novo design and optimization of novel Pin1 inhibitors. Without a viable lead from internal screenings, we designed a series of novel Pin1 inhibitors by interrogating and exploring a protein crystal structure of Pin1. The ligand efficiency of the initial concept molecule was optimized with integrated SBDD and parallel chemistry approaches, resulting in a more attractive lead series.

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.

Analysis of Cysteine Redox Post-Translational Modifications in Cell Biology and Drug Pharmacology.

Reversible cysteine oxidation is an emerging class of protein post-translational modification (PTM) that regulates catalytic activity, modulates conformation, impacts protein-protein interactions, and affects subcellular trafficking of numerous proteins. Redox PTMs encompass a broad array of cysteine oxidation reactions with different half-lives, topographies, and reactivities such as S-glutathionylation and sulfoxidation. Recent studies from our group underscore the lesser known effect of redox protein modifications on drug binding. To date, biological studies to understand mechanistic and functional aspects of redox regulation are technically challenging. A prominent issue is the lack of tools for labeling proteins oxidized to select chemotype/oxidant species in cells. Predictive computational tools and curated databases of oxidized proteins are facilitating structural and functional insights into regulation of the network of oxidized proteins or redox proteome. In this chapter, we discuss analytical platforms for studying protein oxidation, suggest computational tools currently available in the field to determine redox sensitive proteins, and begin to illuminate roles of cysteine redox PTMs in drug pharmacology.

Discovery of a Novel and Selective Indoleamine 2,3-Dioxygenase (IDO-1) Inhibitor 3-(5-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (EOS200271/PF-06840003) and Its Characterization as a Potential Clinical Candidate.

Tumors use tryptophan-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO-1) to induce an immunosuppressive environment. IDO-1 is induced in response to inflammatory stimuli and promotes immune tolerance through effector T-cell anergy and enhanced Treg function. As such, IDO-1 is a nexus for the induction of a key immunosuppressive mechanism and represents an important immunotherapeutic target in oncology. Starting from HTS hit 5, IDO-1 inhibitor 6 (EOS200271/PF-06840003) has been developed. The structure-activity relationship around 6 is described and rationalized using the X-ray crystal structure of 6 bound to human IDO-1, which shows that 6, differently from most of the IDO-1 inhibitors described so far, does not bind to the heme iron atom and has a novel binding mode. Clinical candidate 6 shows good potency in an IDO-1 human whole blood assay and also shows a very favorable ADME profile leading to favorable predicted human pharmacokinetic properties, including a predicted half-life of 16-19 h.