A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors.

Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.

Pyrimidone-based series of glucokinase activators with alternative donor-acceptor motif.

Glucokinase activators are a class of experimental agents under investigation as a therapy for Type 2 diabetes mellitus. An X-ray crystal structure of a modestly potent agent revealed the potential to substitute the common heterocyclic amide donor-acceptor motif for a pyridone moiety. We have successfully demonstrated that both pyridone and pyrimidone heterocycles can be used as a potent donor-acceptor substituent. Several sub-micromolar analogs that possess the desired partial activator profile were synthesized and characterized. Unfortunately, the most potent activators suffered from sub-optimal pharmacokinetic properties. Nonetheless, these donor-acceptor motifs may find utility in other glucokinase activator series or beyond.

N-(Pyridin-2-yl) arylsulfonamide inhibitors of 11ß-hydroxysteroid dehydrogenase type 1: strategies to eliminate reactive metabolites.

N-(Pyridin-2-yl) arylsulfonamides 1 and 2 (PF-915275) were identified as potent inhibitors of 11ß-hydroxysteroid dehydrogenase type 1. A screen for bioactivation revealed that these compounds formed glutathione conjugates. This communication presents the results of a risk benefit analysis carried out to progress 2 (PF-915275) to a clinical study and the strategies used to eliminate reactive metabolites in this series of inhibitors. Based on the proposed mechanism of bioactivation and structure-activity relationships, design efforts led to N-(pyridin-2-yl) arylsulfonamides such as 18 and 20 that maintained potent 11ß-hydroxysteroid dehydrogenase type 1 activity, showed exquisite pharmacokinetic profiles, and were negative in the reactive metabolite assay.

Direct control of CAR T cells through small molecule-regulated antibodies.

Antibody-based therapeutics have experienced a rapid growth in recent years and are now utilized in various modalities spanning from conventional antibodies, antibody-drug conjugates, bispecific antibodies to chimeric antigen receptor (CAR) T cells. Many next generation antibody therapeutics achieve enhanced potency but often increase the risk of adverse events. Antibody scaffolds capable of exhibiting inducible affinities could reduce the risk of adverse events by enabling a transient suspension of antibody activity. To demonstrate this, we develop conditionally activated, single-module CARs, in which tumor antigen recognition is directly modulated by an FDA-approved small molecule drug. The resulting CAR T cells demonstrate specific cytotoxicity of tumor cells comparable to that of traditional CARs, but the cytotoxicity is reversibly attenuated by the addition of the small molecule. The exogenous control of conditional CAR T cell activity allows continual modulation of therapeutic activity to improve the safety profile of CAR T cells across all disease indications.

N-(Pyridin-2-yl) arylsulfonamide inhibitors of 11beta-hydroxysteroid dehydrogenase type 1: Discovery of PF-915275.

N-(Pyridin-2-yl) arylsulfonamides are identified as inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1), an enzyme that catalyzes the reduction of the glucocorticoid cortisone to cortisol. Dysregulation of glucocorticoids has been implicated in the pathogenesis of diabetes and the metabolic syndrome. In this Letter, we present the development of an initial lead to an efficient ligand with improved physiochemical properties using a deletion strategy. This strategy allowed for further optimization of potency leading to the discovery of the clinical candidate PF-915275.

A strategy for risk management of drug-induced phospholipidosis.

Drug-induced phospholipidosis (PL) is an excessive accumulation of phospholipids and drug in lysosomes. Phospholipidosis signals a change in cell membrane integrity and accumulation of intracellular drug or metabolite in tissues. The sensitivity and susceptibility of preclinical models to detect PL vary with therapeutic agents, and PL is expected to be reversible after discontinuation of drug treatment. The prevailing scientific opinion is that PL by itself is not adverse; however, some regulatory authorities consider PL to be adverse because a small number of chemicals are able to cause PL and concurrent organ toxicity. Until a greater understanding of PL emerges, a well-thought-out risk management strategy for PL will increase confidence in safety and improve selection and development of new drugs. This paper provides a tiered approach to risk management of drug-induced PL. It begins with use of in silico and in vitro tools to design and select compounds with reduced potential to produce PL. Early in vivo studies in two species are used to better characterize potential for toxicity and PL. Finally, routine risk management tools (i.e., translational biomarkers, assessment of reversibility) are used to support confidence in safety of compounds that induce PL in animals.

Protein tyrosine phosphatase 1B inhibitors for diabetes.

Increased incidence of type 2 diabetes mellitus and obesity has elevated the medical need for new agents to treat these disease states. Resistance to the hormones insulin and leptin are hallmarks of both type 2 diabetes and obesity. Drugs that can ameliorate this resistance should be effective in treating type 2 diabetes and possibly obesity. Protein tyrosine phosphatase 1B (PTP1B) is thought to function as a negative regulator of insulin and leptin signal transduction. This article reviews PTP1B as a novel target for type 2 diabetes, and looks at the challenges in developing small-molecule inhibitors of this phosphatase.

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.

Structure-based design of a parallel synthetic array directed toward the discovery of irreversible inhibitors of human rhinovirus 3C protease.

Utilizing the tools of parallel synthesis and structure-based design, a new class of Michael acceptor-containing, irreversible inhibitors of human rhinovirus 3C protease (HRV 3CP) was discovered. These inhibitors are shown to inhibit HRV-14 3CP with rates of inactivation ranging from 886 to 31 400 M(-1) sec(-1). These inhibitors exhibit antiviral activity when tested against HRV-14 infected H1-HeLa cells, with EC(50) values ranging from 1.94 to 0.15 microM. No cytotoxicity was observed at the limits of the assay concentration. A crystal structure of one of the more potent inhibitors covalently bound to HRV-2 3CP is detailed. These compounds were also tested against HRV serotypes other than type 14 and were found to have highly variable activities.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 8. Pharmacological optimization of orally bioavailable 2-pyridone-containing peptidomimetics.

The optimization of the pharmacokinetic performance of various 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors following oral administration to either beagle dogs or CM-monkeys is described. The molecules described in this work are composed of a 2-pyridone-containing peptidomimetic binding determinant and an alpha,beta-unsaturated ester Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. Modification of the ester contained within these compounds is detailed along with alteration of the P(2) substituent present in the peptidomimetic portion of the inhibitors. The pharmacokinetics of several inhibitors in both dogs and monkeys are described (7 h plasma concentrations after oral administration) along with their human plasma stabilities, stabilities in incubations with human, dog, and monkey microsomes and hepatocytes, Caco-2 permeabilities, and aqueous solubilities. Compounds containing an alpha,beta-unsaturated ethyl ester fragment and either an ethyl or propargyl P(2) moiety displayed the most promising combination of 3C enzyme inhibition (k(obs)/[I] 170 000-223 000 M(-1) s(-1)), antiviral activity (EC(50) = 0.047-0.058 microM, mean vs seven HRV serotypes), and pharmacokinetics following oral administration (7 h dog plasma levels = 0.248-0.682 microM; 7 h CM-monkey plasma levels = 0.057-0.896 microM).

Evaluation of a published in silico model and construction of a novel Bayesian model for predicting phospholipidosis inducing potential.

The identification of phospholipidosis (PPL) during preclinical testing in animals is a recognized problem in the pharmaceutical industry. Depending on the intended indication and dosing regimen, PPL can delay or stop development of a compound in the drug discovery process. Therefore, for programs and projects where a PPL finding would have adverse impact on the success of the project, it would be desirable to be able to rapidly identify and screen out those compounds with the potential to induce PPL as early as possible. Currently, electron microscopy is the gold standard method for identifying phospholipidosis, but it is low-throughput and resource-demanding. Therefore, a low-cost, high-throughput screening strategy is required to overcome these limitations and be applicable in the drug discovery cycle. A recent publication by Ploemen et al. (Exp. Toxicol. Pathol. 2004, 55, 347-55) describes a method using the computed physicochemical properties pKa and ClogP as part of a simple calculation to determine a compound's potential to induce PPL. We have evaluated this method using a set of 201 compounds, both public and proprietary, with known in vivo PPL-inducing ability and have found the overall concordance to be 75%. We have proposed simple modifications to the model rules, which improve the model's concordance to 80%. Finally, we describe the development of a Bayesian model using the same compound set and found its overall concordance to be 83%.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. Part 7: structure-activity studies of bicyclic 2-pyridone-containing peptidomimetics.

The structure-based design, chemical synthesis, and biological evaluation of bicyclic 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. An optimized compound is shown to exhibit antiviral activity when tested against a variety of HRV serotypes (EC(50)'s ranging from 0.037 to 0.162 microM).