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.
Novel pyridine- and pyrimidine-based allosteric inhibitors are reported that achieve PDE4D subtype selectivity through recognition of a single amino acid difference on a key regulatory domain, known ...as UCR2, that opens and closes over the catalytic site for cAMP hydrolysis. The design and optimization of lead compounds was based on iterative analysis of X-ray crystal structures combined with metabolite identification. Selectivity for the activated, dimeric form of PDE4D provided potent memory enhancing effects in a mouse model of novel object recognition with improved tolerability and reduced vascular toxicity over earlier PDE4 inhibitors that lack subtype selectivity. The lead compound, 28 (BPN14770), has entered midstage, human phase 2 clinical trials for the treatment of Fragile X Syndrome.
Objective
Non-pharmacologic treatments (NPTs) are recommended for chronic pain. Information is limited on patient use or perceptions of NPTs. We examined the frequency and correlates of use and ...self-rated helpfulness of NPTs for chronic pain among patients who are prescribed long-term opioid therapy (LTOT).
Methods
Participants (
n
= 517) with musculoskeletal pain who were prescribed LTOT were recruited from two integrated health systems. They rated the frequency and utility of six clinician-directed and five self-directed NPTs for chronic pain. We categorized NPT use at four levels based on number of interventions used and frequency of use (none, low, moderate, high). Analyses examined clinical and demographic factors that differed among groups for both clinician-directed and self-directed NPTs.
Results
Seventy-one percent of participants reported use of any NPT for pain within the prior 6 months. NPTs were rated as being helpful by more than 50% of users for all treatments assessed (range 51–79%). High users of clinician-directed NPTs were younger than non-users or low-frequency users and had the most depressive symptoms. In both clinician-directed and self-directed categories, high NPT users had significantly higher pain disability compared to non-NPT users. No significant group differences were detected on other demographic or clinical variables. In multivariable analyses, clinician-directed NPT use was modestly associated with younger age (OR = 0.97, 95% CI = 0.96–0.98) and higher pain disability (OR = 1.01, 95% CI = 1.00–1.02). Variables associated with greater self-directed NPT use were some college education (OR = 1.80, 95% CI = 1.13–2.84), college graduate or more (OR = 2.02, 95% CI = 1.20–3.40), and higher pain disability (OR = 1.01, 95% CI = 1.01–1.02).
Conclusions
NPT use was associated with higher pain disability and younger age for both clinician-directed and self-directed NPTs and higher education for self-directed NPTs. These strategies were rated as helpful by those that used them. These results can inform intervention implementation and be used to increase engagement in NPTs for chronic pain.
Little is known about co-occurring long-term opioid therapy (LTOT) and medical cannabis use. We compared characteristics of patients prescribed LTOT who endorsed using medical cannabis for pain to ...patients who did not report cannabis use.
Participants (n=371) prescribed LTOT completed self-report measures about pain, substance use, and mental health.
Eighteen percent of participants endorsed using medical cannabis for pain. No significant differences were detected on pain-related variables, depression, or anxiety between those who endorsed medical cannabis use and those who did not. Medical cannabis users had higher scores of risk for prescription opioid misuse (median=17.0 vs. 11.5, p<0.001), rates of hazardous alcohol use (25% vs. 16%, p<0.05), and rates of nicotine use (42% vs. 26%, p=0.01). Multivariable analyses indicated that medical cannabis use was significantly associated with risk of prescription opioid misuse (β=0.17, p=0.001), but not hazardous alcohol use (aOR=1.96, 95% CI=0.96–4.00, p=0.06) or nicotine use (aOR=1.61, 95% CI=0.90–2.88, p=0.11).
There are potential risks associated with co-occurring LTOT and medical cannabis for pain. Study findings highlight the need for further clinical evaluation in this population. Future research is needed to examine the longitudinal impact of medical cannabis use on pain-related and substance use outcomes.
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.
Nile tilapia (Oreochromis niloticus) is a globally significant aquaculture species rapidly gaining status as a farmed commodity. In West Africa, wild Nile tilapia genetic resources are abundant yet ...knowledge of fine-scale population structure and patterns of natural genetic variation are limited. Coinciding with this is a burgeoning growth in tilapia aquaculture in Ghana and other countries within the region underpinned by locally available genetic resources. Using 192 single nucleotide polymorphism (SNP) markers this study conducted a genetic survey of Nile tilapia throughout West Africa, sampling 23 wild populations across eight countries (Benin, Burkina Faso, Côte d'Ivoire, Ghana, Togo, Mali, Gambia and Senegal), representing the major catchments of the Volta, Niger, Senegal and Gambia River basins. A pattern of isolation-by-distance and significant spatial genetic structure was identified throughout West Africa (Global F
= 0.144), which largely corresponds to major river basins and, to a lesser extent, sub-basins. Two populations from the Gambia River (Kudang and Walekounda), one from the western Niger River (Lake Sélingué) and one from the upper Red Volta River (Kongoussi) showed markedly lower levels of diversity and high genetic differentiation compared to all other populations, suggesting genetically isolated populations occurring across the region. Genetic structure within the Volta Basin did not always follow the pattern expected for sub-river basins. This study identifies clear genetic structuring and differentiation amongst West African Nile tilapia populations, which concur with broad patterns found in previous studies. In addition, we provide new evidence for fine-scale genetic structuring within the Volta Basin and previously unidentified genetic differences of populations in Gambia. The 192 SNP marker suite used in this study is a useful tool for differentiating tilapia populations and we recommend incorporating this marker suite into future population screening of O. niloticus. Our results form the basis of a solid platform for future research on wild tilapia genetic resources in West Africa, and the identification of potentially valuable germplasm for use in ongoing breeding programs for aquaculture.
Serial crystallography of membrane proteins often employs high‐viscosity injectors (HVIs) to deliver micrometre‐sized crystals to the X‐ray beam. Typically, the carrier medium is a lipidic cubic ...phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self‐assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X‐ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The reservoir pressure in the HVI was tracked to determine if there is any correlation with the phase behaviour of the LCP. The results indicated that, even though the reservoir pressure underwent (at times) significant variation, this did not appear to correlate with observed phase changes in the sample stream or correspond to shifts in the LCP lattice parameter. During vacuum injection, there was a three‐way coexistence of the gyroid cubic phase, diamond cubic phase and lamellar phase. During injection at atmospheric pressure, the coexistence of a cubic phase and lamellar phase in the monoolein/water mixtures was also observed. The degree to which the lamellar phase is formed was found to be strongly dependent on the co‐flowing gas conditions used to stabilize the LCP stream. A combination of laboratory‐based optical polarization microscopy and simulation studies was used to investigate these observations.
This is a study of the phase changes detected in monoolein samples under constant flow using a high‐viscousity injector. The sample behaviour was studied using X‐ray techniques while light microscopy and modelling studies were used to help interpret some of the effects observed in the data.
Abnormal proliferation mediated by disruption of the normal cell cycle mechanisms is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDK) and ...cyclins, such as CDK2/cyclin A and CDK2/cyclin E, and inhibiting their kinase activity are regarded as promising antitumor agents to complement the existing therapies. From a high-throughput screening effort, we identified a new class of CDK2/cyclin A/E inhibitors. The hit-to-lead expansion of this class is described. X-ray crystallographic data of early compounds in this series, as well as in vitro testing funneled for rapidly achieving in vivo efficacy, led to a nanomolar inhibitor of CDK2/cyclin A (N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2-naphthyl)acetamide (41), PNU-292137, IC50 = 37 nM) with in vivo antitumor activity (TGI > 50%) in a mouse xenograft model at a dose devoid of toxic effects.
We aimed to develop radioligands for PET imaging of brain phosphodiesterase subtype 4D (PDE4D), a potential target for developing cognition enhancing or antidepressive drugs. Exploration of several ...chemical series gave four leads with high PDE4D inhibitory potency and selectivity, optimal lipophilicity, and good brain uptake. These leads featured alkoxypyridinyl cores. They were successfully labeled with carbon-11 (t 1/2 = 20.4 min) for evaluation with PET in monkey. Whereas two of these radioligands did not provide PDE4D-specific signal in monkey brain, two others, 11CT1660 and 11CT1650, provided sizable specific signal, as judged by pharmacological challenge using rolipram or a selective PDE4D inhibitor (BPN14770) and subsequent biomathematical analysis. Specific binding was highest in prefrontal cortex, temporal cortex, and hippocampus, regions that are important for cognitive function. 11CT1650 was progressed to evaluation in humans with PET, but the output measure of brain enzyme density (V T) increased with scan duration. This instability over time suggests that radiometabolite(s) were accumulating in the brain. BPN14770 blocked PDE4D uptake in human brain after a single dose, but the percentage occupancy was difficult to estimate because of the unreliability of measuring V T. Overall, these results show that imaging of PDE4D in primate brain is feasible but that further radioligand refinement is needed, most likely to avoid problematic radiometabolites.
A novel series of potent benzoxazole mPGES-1 inhibitors has been derived from a hit from a high throughput screen. Compound 37 displays mPGES-1 inhibition in an enzyme assay (0.018μM) and PGE-2 ...inhibition in a cell-based assay (0.034μM). It demonstrates 500- and 2500-fold selectivity for mPGES-1 over COX-2 and 6-keto PGF-1α, respectively. In vivo PK studies in dogs demonstrate 55% oral bioavailability and an 7h half-life.
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