Tyzoon K. Nomanbhoy

Mechanistic and Pharmacological Characterization of PF-04457845: A Highly Potent and Selective Fatty Acid Amide Hydrolase Inhibitor That Reduces Inflammatory and Noninflammatory Pain

The endogenous cannabinoid (endocannabinoid) anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH). Pharmacological blockade of FAAH has emerged as a potentially attractive strategy for augmenting endocannabinoid signaling and retaining the beneficial effects of cannabinoid receptor activation, while avoiding the undesirable side effects, such as weight gain and impairments in cognition and motor control, observed with direct cannabinoid receptor 1 agonists. Here, we report the detailed mechanistic and pharmacological characterization of N-pyridazin-3-yl-4-(3-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzylidene)piperidine-1-carboxamide (PF-04457845), a highly efficacious and selective FAAH inhibitor. Mechanistic studies confirm that PF-04457845 is a time-dependent, covalent FAAH inhibitor that carbamylates FAAHs catalytic serine nucleophile. PF-04457845 inhibits human FAAH with high potency (kinact/Ki = 40,300 M−1s−1; IC50 = 7.2 nM) and is exquisitely selective in vivo as determined by activity-based protein profiling. Oral administration of PF-04457845 produced potent antinociceptive effects in both inflammatory [complete Freunds adjuvant (CFA)] and noninflammatory (monosodium iodoacetate) pain models in rats, with a minimum effective dose of 0.1 mg/kg (CFA model). PF-04457845 displayed a long duration of action as a single oral administration at 1 mg/kg showed in vivo efficacy for 24 h with a concomitant near-complete inhibition of FAAH activity and maximal sustained elevation of anandamide in brain. Significantly, PF-04457845-treated mice at 10 mg/kg elicited no effect in motility, catalepsy, and body temperature. Based on its exceptional selectivity and in vivo efficacy, combined with long duration of action and optimal pharmacokinetic properties, PF-04457845 is a clinical candidate for the treatment of pain and other nervous system disorders.

Transfer RNA–Dependent Translocation of Misactivated Amino Acids to Prevent Errors in Protein Synthesis

Misactivation of amino acids by aminoacyl-tRNA synthetases can lead to significant errors in protein synthesis that are prevented by editing reactions. As an example, discrete sites in isoleucyl-tRNA synthetase for amino acid activation and editing are about 25 A apart. The details of how misactivated valine is translocated from one site to the other are unknown. Here, we present a kinetic study in which a fluorescent probe is used to monitor translocation of misactivated valine from the active site to the editing site. Isoleucine-specific tRNA, and not other tRNAs, is essential for translocation of misactivated valine. Misactivation and translocation occur on the same enzyme molecule, with translocation being rate limiting for editing. These results illustrate a remarkable capacity for a specific tRNA to enhance amino acid fine structure recognition by triggering a unimolecular translocation event.

Mutational Separation of Two Pathways for Editing by a Class I tRNA Synthetase

Aminoacyl tRNA synthetases (aaRSs) catalyze the first step in protein biosynthesis, establishing a connection between codons and amino acids. To maintain accuracy, aaRSs have evolved a second active site that eliminates noncognate amino acids. Isoleucyl tRNA synthetase edits valine by two tRNA(Ile)-dependent pathways: hydrolysis of valyl adenylate (Val-AMP, pretransfer editing) and hydrolysis of mischarged Val-tRNA(Ile) (posttransfer editing). Not understood is how a single editing site processes two distinct substrates--an adenylate and an aminoacyl tRNA ester. We report here distinct mutations within the center for editing that alter adenylate but not aminoacyl ester hydrolysis, and vice versa. These results are consistent with a molecular model that shows that the single editing active site contains two valyl binding pockets, one specific for each substrate.

Benzothiophene piperazine and piperidine urea inhibitors of fatty acid amide hydrolase (FAAH)

The synthesis and structure-activity relationships (SAR) of a series of benzothiophene piperazine and piperidine urea FAAH inhibitors is described. These compounds inhibit FAAH by covalently modifying the enzymes active site serine nucleophile. Activity-based protein profiling (ABPP) revealed that these urea inhibitors were completely selective for FAAH relative to other mammalian serine hydrolases. Several compounds showed in vivo activity in a rat complete Freunds adjuvant (CFA) model of inflammatory pain.

Blocking site-to-site translocation of a misactivated amino acid by mutation of a class I tRNA synthetase.

The genetic code is established by the aminoacylation reactions of tRNA synthetases. Its accuracy depends on editing reactions that prevent amino acids from being assigned to incorrect codons. A group of class I synthetases share a common insertion that encodes a distinct site for editing that is about 30 Å from the active site. Both misactivated aminoacyl adenylates and mischarged amino acids attached to tRNA are translocated to this site, which, in turn, is divided into subsites—one for the adenylate and one for the aminoacyl moiety attached to tRNA. Here we report that a specific mutation in isoleucyl-tRNA synthetase prevents editing by blocking translocation. The mutation alters a widely conserved residue that is believed to tether the amino group of mischarged tRNA to its subsite for editing. These and other data support a model where editing is initiated by translocation of the misacylated amino acid attached to tRNA to create an “editing complex” that facilitates subsequent rounds of editing by translocation of the misactivated adenylate.

Simultaneous binding of two proteins to opposite sides of a single transfer RNA.

Transfer RNA (tRNA) is a small nucleic acid (typically 76 nucleotides) that forms binary complexes with proteins, such as aminoacyl tRNA synthetases (RS) and Trbp111. The latter is a widely distributed structure-specific tRNA-binding protein that is incorporated into cell signaling molecules. The structure of Trbp111 was modeled onto to the outer, convex side of the L-shaped tRNA. Here we present RNA footprints that are consistent with this model. This binding mode is in contrast to that of tRNA synthetases, which bind to the inside, or concave side, of tRNA. These opposite locations of binding for these two proteins suggest the possibility of a ternary complex. The formation of a tRNA synthetase–tRNA–Trbp111 ternary complex was detected by two independent methods. The results indicate that the tRNA is sandwiched between the two protein molecules. A thermodynamic and functional analysis is consistent with the tRNA retaining its native structure in the ternary complex. These results may have implications for how the translation apparatus is linked to other cellular machinery.

Functional interrogation of kinases and other nucleotide-binding proteins

The largest mammalian enzyme family is the kinases. Kinases and other nucleotide‐binding proteins are key regulators of signal transduction pathways and the mutation or overexpression of these proteins is often the difference between health and disease. As a result, a massive research effort has focused on understanding how these proteins function and how to inhibit them for therapeutic benefit. Recent advances in chemical biological tools have enabled functional interrogation of these enzymes to provide a deeper understanding of their physiological roles. In addition, these innovative platforms have paved the way for a new generation of drugs whose properties have been guided by functional profiling.

Synthesis and structure-activity relationship of 4-quinolone-3-carboxylic acid based inhibitors of glycogen synthase kinase-3β.

The synthesis, GSK-3β inhibitory activity, and anti-microbial activity of bicyclic and tricyclic derivatives of the 5,7-diamino-6-fluoro-4-quinolone-3-carboxylic acid scaffold were studied. Kinase selectivity profiling indicated that members of this class were potent and highly selective GSK-3 inhibitors.

Hit-to-lead optimization and kinase selectivity of imidazo[1,2-a]quinoxalin-4-amine derived JNK1 inhibitors

As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 μM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC50=160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC50=47 nM) was a highly specific JNK inhibitor.

ERK5 kinase activity is dispensable for cellular immune response and proliferation

Significance Whole protein deletion and pharmacological inhibition are frequently used to functionally annotate enzymes. Each has limitations: whole protein deletion removes both enzymatic and nonenzymatic functions, and small molecule inhibitors can have unrecognized off-target activities. When both approaches agree, it’s nearly incontrovertible support for protein function. Here we describe a counterexample. ERK5 knockdown and inhibition supported a role for this kinase in a number of biological processes. We show that previously reported ERK5 compounds inhibit bromodomain-containing proteins (BRDs) sufficiently to account for their phenotypic effects. We describe highly specific inhibitors of ERK5 that do not inhibit BRDs. With these, we show that cellular inflammation and proliferation are not dependent on ERK5 catalytic activity, thus making ERK5 unique among the MAP kinases. Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.