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-yloxy}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 FAAH's catalytic serine nucleophile. PF-04457845 inhibits human FAAH with high potency (k(inact)/K(i) = 40,300 M(-1)s(-1); IC(50) = 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 Freund's 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.
Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or ...pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (k inact/K i and IC50 values of 40300 M−1 s−1 and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.
A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as psoriasis and inflammatory bowel diseases (IBD), by selective targeting of TYK2. Hit triage, following a ...high-throughput screen for TYK2 inhibitors, revealed pyridine 1 as a promising starting point for lead identification. Initial expansion of 3 separate regions of the molecule led to eventual identification of cyclopropyl amide 46, a potent lead analog with good kinase selectivity, physicochemical properties, and pharmacokinetic profile. Analysis of the binding modes of the series in TYK2 and JAK2 crystal structures revealed key interactions leading to good TYK2 potency and design options for future optimization of selectivity.
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•Biological rationale for targeting selective TYK2 inhibition is given.•We report lead identification of potent and efficient TYK2 inhibitors.•Analogs afford modest selectivity against other Janus Family kinases.•We obtained crystal structures of key compounds bound to both TYK2 and JAK2.•ADME properties of the lead compound are favorable.
The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH ...produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-Å crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.
The structure–activity relationships of T0901317 analogs were explored as RORc inverse agonists using the principles of property- and structure-based drug design. An X-ray co-crystal structure of ...T0901317 and RORc was obtained and provided molecular insight into why T0901317 functioned as an inverse agonist of RORc; whereas, the same ligand functioned as an agonist of FXR, LXR, and PXR. The structural data was also used to design inhibitors with improved RORc biochemical and cellular activities. The improved inhibitors possessed enhanced selectivity profiles (rationalized using the X-ray crystallographic data) against other nuclear receptors.
The Type 1 PI3Kinases comprise a family of enzymes, which primarily phosphorylate PIP2 to give the second messenger PIP3, a key player in many intracellular signaling processes
Science,
2002,
296, ...1655;
Trends Pharmacol. Sci.
2003,
24, 366. Of the four type 1 PI3Ks, the γ-isoform, which is expressed almost exclusively in leukocytes
Curr. Biol.,
1997,
7, R470, is of particular interest with respect to its role in inflammatory diseases such as rheumatoid arthritis (RA) and chronic obstructive pulmonary disease (COPD)
Mol. Med. Today,
2000,
6, 347. Investigation of a series of 4,6-disubstituted-4
H-benzo1,4oxazin-3-ones has led to the identification of single-digit nanomolar inhibitors of PI3Kγ, several of which had good cell based activity and were shown to be active in vivo in an aspectic peritonitis model of inflammatory cell migration.
Expression of the insulin-like growth factor-binding protein 5 (IGFBP-5) gene in vascular smooth muscle cells is up-regulated by IGF-I through an IGF-I receptor-mediated mechanism. In this study, we ...studied the possible involvement of the mitogen-activated protein kinase (MAPK) and PI 3-kinase signaling pathways in mediating IGF-I-regulated IGFBP-5 gene expression. The addition of Des(1-3)IGF-I, an IGF analog with reduced affinity to IGFBPs, resulted in a transient activation of p44 and p42 MAPK. Inhibition of the MAPK activation by PD98059, however, did not affect IGF-I-stimulated IGFBP-5 expression. Des(1-3)IGF-I treatment also strongly activated PI 3-kinase. This activation was probably mediated through IRS-1, because IGF-I stimulation resulted in a significant increase in IRS-1- but not IRS-2-associated PI 3-kinase activity. This activation occurred within 5 min and was sustained at high levels for over 6 h. Likewise, Des(1-3)IGF-I caused a long lasting activation of PKB/Akt and p70(s6k). When LY294002 and wortmannin, two specific inhibitors of PI 3-kinase, were added with Des(1-3)IGF-I, the IGF-I-regulated IGFBP-5 expression was negated. The addition of rapamycin, which inhibits IGF-I-induced p70(s6k) activation, significantly inhibited IGF-I-regulated IGFBP-5 gene expression. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI 3-kinase-PKB/Akt-p70(s6k) pathway but not the MAPK pathway in vascular smooth muscle cells.
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Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members ...of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50’s in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).
Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological ...inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system disorders. However, the FAAH inhibitors reported to date lack drug-like pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750) and N-phenyl-4-(quinolin-2-ylmethyl)piperazine-1-carboxamide (PF-622) as a novel mechanistic class of FAAH inhibitors. PF-750 and PF-622 show higher in vitro potencies than previously established classes of FAAH inhibitors. Rather unexpectedly based on the high chemical stability of the urea functional group, PF-750 and PF-622 were found to inhibit FAAH in a time-dependent manner by covalently modifying the enzyme's active site serine nucleophile. Activity-based proteomic profiling revealed that PF-750 and PF-622 were completely selective for FAAH relative to other mammalian serine hydrolases. We hypothesize that this remarkable specificity derives, at least in part, from FAAH's special ability to function as a C(O)−N bond hydrolase, which distinguishes it from the vast majority of metabolic serine hydrolases in mammals that are restricted to hydrolyzing esters and/or thioesters. The piperidine/piperazine urea may thus represent a privileged chemical scaffold for the synthesis of FAAH inhibitors that display an unprecedented combination of potency and selectivity for use as potential analgesic and anxiolytic/antidepressant agents.
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