Mark Edward Flanagan

Discovery of CP-690,550: A Potent and Selective Janus Kinase (JAK) Inhibitor for the Treatment of Autoimmune Diseases and Organ Transplant Rejection

There is a critical need for safer and more convenient treatments for organ transplant rejection and autoimmune disorders such as rheumatoid arthritis. Janus tyrosine kinases (JAK1, JAK3) are expressed in lymphoid cells and are involved in the signaling of multiple cytokines important for various T cell functions. Blockade of the JAK1/JAK3-STAT pathway with a small molecule was anticipated to provide therapeutic immunosuppression/immunomodulation. The Pfizer compound library was screened against the catalytic domain of JAK3 resulting in the identification of a pyrrolopyrimidine-based series of inhibitors represented by CP-352,664 (2a). Synthetic analogues of 2a were screened against the JAK enzymes and evaluated in an IL-2 induced T cell blast proliferation assay. Select compounds were evaluated in rodent efficacy models of allograft rejection and destructive inflammatory arthritis. Optimization within this chemical series led to identification of CP-690,550 1, a potential first-in-class JAK inhibitor for treatment of autoimmune diseases and organ transplant rejection.

Discovery and Development of Janus Kinase (JAK) Inhibitors for Inflammatory Diseases

The Janus kinases (JAKs) are a family of intracellular tyrosine kinases that play an essential role in the signaling of numerous cytokines that have been implicated in the pathogenesis of inflammatory diseases. As a consequence, the JAKs have received significant attention in recent years from the pharmaceutical and biotechnology industries as therapeutic targets. Here, we provide a review of the JAK pathways, the structure, function, and activation of the JAK enzymes followed by a detailed look at the JAK inhibitors currently in the clinic or approved for these indications. Finally, a perspective is provided on what the past decade of research with JAK inhibitors for inflammatory indications has taught along with thoughts on what the future may hold in terms of addressing the opportunities and challenges that remain.

Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors.

Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymatic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry additional risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biologically relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.

Discovery of azetidinyl ketolides for the treatment of susceptible and multidrug resistant community-acquired respiratory tract infections.

Respiratory tract bacterial strains are becoming increasingly resistant to currently marketed macrolide antibiotics. The current alternative telithromycin (1) from the newer ketolide class of macrolides addresses resistance but is hampered by serious safety concerns, hepatotoxicity in particular. We have discovered a novel series of azetidinyl ketolides that focus on mitigation of hepatotoxicity by minimizing hepatic turnover and time-dependent inactivation of CYP3A isoforms in the liver without compromising the potency and efficacy of 1.

Preparation, Gram-Negative Antibacterial Activity, and Hydrolytic Stability of Novel Siderophore-Conjugated Monocarbam Diols

A novel series of monocarbam compounds exhibiting promising antibacterial activity against multidrug resistant Gram-negative microorganisms is reported, along with the synthesis of one such molecule MC-1 (1). Also reported are structure-activity relationships associated with the in vitro and in vivo efficacy of 1 and related analogues in addition to the hydrolytic stability of such compounds and possible implications thereof.

Discovery of Dap-3 Polymyxin Analogues for the Treatment of Multidrug-Resistant Gram-Negative Nosocomial Infections

We report novel polymyxin analogues with improved antibacterial in vitro potency against polymyxin resistant recent clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa . In addition, a human renal cell in vitro assay (hRPTEC) was used to inform structure-toxicity relationships and further differentiate analogues. Replacement of the Dab-3 residue with a Dap-3 in combination with a relatively polar 6-oxo-1-phenyl-1,6-dihydropyridine-3-carbonyl side chain as a fatty acyl replacement yielded analogue 5x, which demonstrated an improved in vitro antimicrobial and renal cytotoxicity profiles relative to polymyxin B (PMB). However, in vivo PK/PD comparison of 5x and PMB in a murine neutropenic thigh model against P. aeruginosa strains with matched MICs showed that 5x was inferior to PMB in vivo, suggesting a lack of improved therapeutic index in spite of apparent in vitro advantages.

Novel monobactams utilizing a siderophore uptake mechanism for the treatment of gram-negative infections

Novel siderophore-linked monobactams with in vitro and in vivo anti-microbial activity against MDR Gram-negative pathogens are described.

Intrinsic reactivity profile of electrophilic moieties to guide covalent drug design: N-α-acetyl-L-lysine as an amine nucleophile

Covalent drugs contain a reactive electrophilic moiety or covalent reactive group (CRG), which forms an irreversible bond between the drug and a biological target. Consequently, the intrinsic reactivity of the CRG is an important consideration in the design of irreversible inhibitors. Although reactivity assessments of CRGs with sulfur nucleophiles, such as glutathione and cysteine have been reported, reactivity of these moieties with amine-containing nucleophiles is not well described. In this study, intrinsic reactivities were determined for a series of electrophiles (acrylamides, nitriles, cyanamides, sulfones, and sulfonamides) using N-α-acetyl-L-lysine as a model amine-based nucleophile and compared with results using glutathione (GSH). Since the e-amine of N-α-acetyl-L-lysine is protonated at neutral pH, reactions were carried out at pH 10.2. In addition to reporting rate data for reactions of CRGs with N-α-acetyl-L-lysine, elements of selectivity relative to thiol-containing nucleophiles are also be discussed.

Chiral Sulfoxide-Induced Single Turn Peptide α-Helicity

Inducing α-helicity through side-chain cross-linking is a strategy that has been pursued to improve peptide conformational rigidity and bio-availability. Here we describe the preparation of small peptides tethered to chiral sulfoxide-containing macrocyclic rings. Furthermore, a study of structure-activity relationships (SARs) disclosed properties with respect to ring size, sulfur position, oxidation state, and stereochemistry that show a propensity to induce α-helicity. Supporting data include circular dichroism spectroscopy (CD), NMR spectroscopy, and a single crystal X-ray structure for one such stabilized peptide. Finally, theoretical studies are presented to elucidate the effect of chiral sulfoxides in inducing backbone α-helicity.

New analyses of MIC90 data to aid antibacterial drug discovery

In this work we present a number of statistical and visualization methods derived from MIC90 data designed to aid decision-making in antibacterial drug discovery research. A statistical method known as bootstrapping was applied to MIC90 raw data to uncover data trends and a metric termed Net Percent Superior (NPS) was developed to capture a strain-by-strain analysis of analogs to enable rank-ordering of similar compounds. We also present novel methods of reporting the data using a variety of visualization techniques. Furthermore, the work was cross-validated using experimental results generated with siderophore-conjugated monocarbam analogs to demonstrate the effectiveness of the various parameters and visualization techniques. The methods reported herein have been incorporated in a Scitegic Pipeline Pilot protocol to enable facile, automated generation of MIC90 analyses from experimental raw data to aid prospective medicinal chemistry design as well as retrospective analyses.