Inhibition of Macrophage Migration Inhibitory Factor by a Chimera of Two Allosteric Binders.

Human Macrophage Migration Inhibitory Factor (MIF) is a trimeric cytokine implicated in a number of inflammatory and autoimmune diseases and cancer. We previously reported that the dye p425 (Chicago Sky Blue), which bound MIF at the interface of two MIF trimers covering the tautomerase and allosteric pockets, revealed a unique strategy to block MIF's pro-inflammatory activities. Structural liabilities, including the large size, precluded p425 as a medicinal chemistry lead for drug development. We report here a rational design strategy linking only the fragment of p425 that binds over the tautomerase pocket to the core of ibudilast, a known MIF allosteric site-specific inhibitor. The chimeric compound, termed L2-4048, was shown by X-ray crystallography to bind at the allosteric and tautomerase sites as anticipated. L2-4048 retained target binding and blocked MIF's tautomerase CD74 receptor binding, and pro-inflammatory activities. Our studies lay the foundation for the design and synthesis of smaller and more drug-like compounds that retain the MIF inhibitory properties of this chimera.

Selective CB2 receptor agonists. Part 1: the identification of novel ligands through computer-aided drug design (CADD) approaches.

Computer-aided drug design scaffold hopping strategies were utilized to identify new classes of CB2 agonists when compounds of an established series with low nanomolar potency were challenging to optimize for good drug-like properties. Use of ligand-based design strategies through BI Builder (a tool for de novo design) and PharmShape (a virtual screening software package) approaches led to the discovery of new chemotypes. Specifically, compounds containing azetidine-, proline-, and piperidine-based cores were found to have low nanomolar and picomolar CB2 agonist activities with drug-like properties considered appropriate for early profiling.

Selective CB2 receptor agonists. Part 3: the optimization of a piperidine-based series that demonstrated efficacy in an in vivo neuropathic pain model.

A novel class of potent cannabinoid receptor 2 (CB2) agonists based on a (S)-piperidine scaffold was identified using ligand-based pharmacophore models. Optimization of solubility and metabolic stability led to the identification of several potent CB2 agonists (e.g., 30) that displayed selectivity over cannabinoid receptor 1 (CB1) and acceptable drug like properties. In rats, compound 30 demonstrated a favorable pharmacokinetic profile and efficacy in a Streptozotocin-induced diabetic neuropathy model, with full reversal of mechanical hyperalgesia.

Inhibition of mycobacterial alanine racemase activity and growth by thiadiazolidinones.

The genus Mycobacterium includes non-pathogenic species such as M. smegmatis, and pathogenic species such as M. tuberculosis, the causative agent of tuberculosis (TB). Treatment of TB requires a lengthy regimen of several antibiotics, whose effectiveness has been compromised by the emergence of resistant strains. New antibiotics that can shorten the treatment course and those that have not been compromised by bacterial resistance are needed. In this study, we report that thiadiazolidinones, a relatively little-studied heterocyclic class, inhibit the activity of mycobacterial alanine racemase, an essential enzyme that converts l-alanine to d-alanine for peptidoglycan synthesis. Twelve members of the thiadiazolidinone family were evaluated for inhibition of M. tuberculosis and M. smegmatis alanine racemase activity and bacterial growth. Thiadiazolidinones inhibited M. tuberculosis and M. smegmatis alanine racemases to different extents with 50% inhibitory concentrations (IC50) ranging from <0.03 to 28µM and 23 to >150µM, respectively. The compounds also inhibited the growth of these bacteria, including multidrug resistant strains of M. tuberculosis. The minimal inhibitory concentrations (MIC) for drug-susceptible M. tuberculosis and M. smegmatis ranged from 6.25µg/ml to 100µg/ml, and from 1.56 to 6.25µg/ml for drug-resistant M. tuberculosis. The in vitro activities of thiadiazolidinones suggest that this family of compounds might represent starting points for medicinal chemistry efforts aimed at developing novel antimycobacterial agents.

A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF).

Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.

Thiadiazolidinones: a new class of alanine racemase inhibitors with antimicrobial activity against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen and a major cause of hospital-acquired infections. New antibacterial agents that have not been compromised by bacterial resistance are needed to treat MRSA-related infections. We chose the S. aureus cell wall synthesis enzyme, alanine racemase (Alr) as the target for a high-throughput screening effort to obtain novel enzyme inhibitors, which inhibit bacterial growth. Among the 'hits' identified was a thiadiazolidinone with chemical properties attractive for lead development. This study evaluated the mode of action, antimicrobial activities, and mammalian cell cytotoxicity of the thiadiazolidinone family in order to assess its potential for development as a therapeutic agent against MRSA. The thiadiazolidones inhibited Alr activity with 50% inhibitory concentrations (IC50) ranging from 0.36 to 6.4 µM, and they appear to inhibit the enzyme irreversibly. The series inhibited the growth of S. aureus, including MRSA strains, with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 µg/ml. The antimicrobial activity showed selectivity against Gram-positive bacteria and fungi, but not Gram-negative bacteria. The series inhibited human HeLa cell proliferation. Lead development centering on the thiadiazolidinone series would require additional medicinal chemistry efforts to enhance the antibacterial activity and minimize mammalian cell toxicity.

1,4-Diazepane compounds as potent and selective CB2 agonists: optimization of metabolic stability.

A high-throughput screening campaign has identified 1,4-diazepane compounds which are potent Cannabinoid receptor 2 agonists with excellent selectivity against the Cannabinoid receptor 1. This class of compounds suffered from low metabolic stability. Following various strategies, compounds with a good stability in liver microsomes and rat PK profile have been identified.

Discovery and characterization of the N-phenyl-N'-naphthylurea class of p38 kinase inhibitors.

An effort aimed at exploring structural diversity in the N-pyrazole-N'-naphthylurea class of p38 kinase inhibitors led to the synthesis and characterization of N-phenyl-N'-naphthylureas. Examples of these compounds displayed excellent inhibition of TNF-alpha production in vitro, as well as efficacy in a mouse model of lipopolysaccharide induced endotoxemia. In addition, perspective is provided on the role of a sulfonamide functionality in defining inhibitor potency.

New modifications to the area of pyrazole-naphthyl urea based p38 MAP kinase inhibitors that bind to the adenine/ATP site.

Discovery of the pyrazole-naphthyl urea class of p38 MAP kinase inhibitors typified by the clinical candidate BIRB 796 has encouraged further exploration of this particular scaffold. Modification to the part of the inhibitor that occupies the adenine/ATP binding site has resulted in a new way to obtain potent inhibitors that possess favorable in vitro and in vivo properties.

Structure-activity relationships of the p38alpha MAP kinase inhibitor 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naph- thalen-1-yl]urea (BIRB 796).

We report on the structure-activity relationships (SAR) of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl]urea (BIRB 796), an inhibitor of p38alpha MAP kinase which has advanced into human clinical trials for the treatment of autoimmune diseases. Thermal denaturation was used to establish molecular binding affinities for this class of p38alpha inhibitors. The tert-butyl group remains a critical binding element by occupying a lipophilic domain in the kinase which is exposed upon rearrangement of the activation loop. An aromatic ring attached to N-2 of the pyrazole nucleus provides important pi-CH(2) interactions with the kinase. The role of groups attached through an ethoxy group to the 4-position of the naphthalene and directed into the ATP-binding domain is elucidated. Pharmacophores with good hydrogen bonding potential, such as morpholine, pyridine, and imidazole, shift the melting temperature of p38alpha by 16-17 degrees C translating into K(d) values of 50-100 pM. Finally, we describe several compounds that potently inhibit TNF-alpha production when dosed orally in mice.

The kinetics of binding to p38MAP kinase by analogues of BIRB 796.

BIRB 796, a member of the N-pyrazole-N'-naphthly urea class of p38MAPK inhibitors, binds to the kinase with both slow association and dissociation rates. Prior to binding, the kinase undergoes a reorganization of the activation loop exposing a critical binding domain. We demonstrate that, independent of the loop movement, association rates are governed by low energy conformations of the inhibitor and polar functionality on the tolyl ring. As anticipated, the dissociation rates of the inhibitors from the kinase are slowed by lipophilic and hydrogen bond interactions. The value of structure-kinetic relationships (SKR) in drug design is discussed.

The non-diaryl heterocycle classes of p38 MAP kinase inhibitors.

The p38 mitogen activated protein (MAP) kinase is an integral enzyme involved in the production of a wide variety of pro-inflammatory cytokines from various cell types. The identification of this kinase and of the diaryl imidazole containing inhibitor, SB203580, initiated an intense discovery effort in this field. Numerous inhibitors were subsequently produced containing replacements for the imidazole, as well as some of the pharmacophores attached to it. During this time many other classes of potent p38 inhibitors emerged containing scaffolds and binding components not found in the diaryl imidazole group. This review summarizes nine of those classes. At least one of these classes requires the kinase to undergo reorganization prior to binding. From this diverse set of inhibitors four compounds have been reported advancing into human clinical trials.

Pyrazole urea-based inhibitors of p38 MAP kinase: from lead compound to clinical candidate.

We report on a series of N-pyrazole, N'-aryl ureas and their mode of binding to p38 mitogen activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine 5'-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen bonding interactions between this class of inhibitors and the protein. We describe the correlation of the structure-activity relationships and crystallographic structures of these inhibitors with p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond at the ATP binding site. This modification affords significant improvements in binding, cellular, and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the treatment of inflammatory diseases.

Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site.

The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.