The Antimicrobial Properties of Cannabis and Cannabis-Derived Compounds and Relevance to CB2-Targeted Neurodegenerative Therapeutics.

Cannabinoid receptor 2 (CB2) is of interest as a much-needed target for the treatment or prevention of several neurogenerative diseases. However, CB2 agonists, particularly phytocannabinoids, have been ascribed antimicrobial properties and are associated with the induction of microbiome compositional fluxes. When developing novel CB2 therapeutics, CB2 engagement and antimicrobial functions should both be considered. This review summarizes those cannabinoids and cannabis-informed molecules and preparations (CIMPs) that show promise as microbicidal agents, with a particular focus on the most recent developments. CIMP-microbe interactions and anti-microbial mechanisms are discussed, while the major knowledge gaps and barriers to translation are presented. Further research into CIMPs may proffer novel direct or adjunctive strategies to augment the currently available antimicrobial armory. The clinical promise of CIMPs as antimicrobials, however, remains unrealized. Nevertheless, the microbicidal effects ascribed to several CB2 receptor-agonists should be considered when designing therapeutic approaches for neurocognitive and other disorders, particularly in cases where such regimens are to be long-term. To this end, the potential development of CB2 agonists lacking antimicrobial properties is also discussed.

Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation.

Effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By using complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fueled biosynthetic processes while controlling the ratio of succinate/α-ketoglutarate (α-KG) to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells caused proliferation and survival defects when the TCA cycle was truncated, blocking carbon flux to support nucleoside biosynthesis. Replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. SDH deficiency induced a proinflammatory gene signature in T cells and promoted T helper 1 and T helper 17 lineage differentiation. An increasing succinate/α-KG ratio in SDH-deficient T cells promoted inflammation by changing the pattern of the transcriptional and chromatin accessibility signatures and consequentially increasing the expression of the transcription factor, PR domain zinc finger protein 1. Collectively, our studies revealed a role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.

HIV-1 fusion inhibitors targeting the membrane-proximal external region of Env spikes.

Combination antiretroviral therapy has transformed HIV-1 infection, once a fatal illness, into a manageable chronic condition. Drug resistance, severe side effects and treatment noncompliance bring challenges to combination antiretroviral therapy implementation in clinical settings and indicate the need for additional molecular targets. Here, we have identified several small-molecule fusion inhibitors, guided by a neutralizing antibody, against an extensively studied vaccine target-the membrane proximal external region (MPER) of the HIV-1 envelope spike. These compounds specifically inhibit the HIV-1 envelope-mediated membrane fusion by blocking CD4-induced conformational changes. An NMR structure of one compound complexed with a trimeric MPER construct reveals that the compound partially inserts into a hydrophobic pocket formed exclusively by the MPER residues, thereby stabilizing its prefusion conformation. These results suggest that the MPER is a potential therapeutic target for developing fusion inhibitors and that strategies employing an antibody-guided search for novel therapeutics may be applied to other human diseases.

An open-source drug discovery platform enables ultra-large virtual screens.

On average, an approved drug currently costs US$2-3 billion and takes more than 10 years to develop1. In part, this is due to expensive and time-consuming wet-laboratory experiments, poor initial hit compounds and the high attrition rates in the (pre-)clinical phases. Structure-based virtual screening has the potential to mitigate these problems. With structure-based virtual screening, the quality of the hits improves with the number of compounds screened2. However, despite the fact that large databases of compounds exist, the ability to carry out large-scale structure-based virtual screening on computer clusters in an accessible, efficient and flexible manner has remained difficult. Here we describe VirtualFlow, a highly automated and versatile open-source platform with perfect scaling behaviour that is able to prepare and efficiently screen ultra-large libraries of compounds. VirtualFlow is able to use a variety of the most powerful docking programs. Using VirtualFlow, we prepared one of the largest and freely available ready-to-dock ligand libraries, with more than 1.4 billion commercially available molecules. To demonstrate the power of VirtualFlow, we screened more than 1 billion compounds and identified a set of structurally diverse molecules that bind to KEAP1 with submicromolar affinity. One of the lead inhibitors (iKeap1) engages KEAP1 with nanomolar affinity (dissociation constant (Kd) = 114 nM) and disrupts the interaction between KEAP1 and the transcription factor NRF2. This illustrates the potential of VirtualFlow to access vast regions of the chemical space and identify molecules that bind with high affinity to target proteins.

Discovery of AZ0108, an orally bioavailable phthalazinone PARP inhibitor that blocks centrosome clustering.

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca's collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

Tobacco cessation treatment education for dental students using standardized patients.

The use of standardized patients (SPs) shows promise in tobacco cessation treatment (TCT) training by providing a simulated clinical environment for dental students to practice counseling skills with individuals trained to portray patients. The purpose of this study was to determine if there was a difference in attitudes, perceptions, and knowledge between dental students who received a lecture and practice sessions with SPs and those who received a lecture only. Dental students in an introductory clinical course at one dental school were invited to participate in the study by completing a pre and post questionnaire. The pre questionnaire was administered to all students prior to a tobacco cessation lecture. Students were group-randomized to either the intervention or control group. The intervention group completed the post questionnaire after the lecture and practice sessions with SPs, and the control group completed it after the lecture only. Of ninety-eight students who attended the lecture and were invited to participate in the study, a total of ninety-four from the two groups (96 percent) provided two linkable questionnaires for analysis. In the results, training with lecture and SPs increased the students' understanding of barriers, subjective norms, perceived skills, self-efficacy, and intentions to provide TCT more than those in the lecture only; however, it did not significantly increase their attitudes and knowledge. These findings suggest that using SPs is a valuable educational method to promote the provision of TCT by dental students and graduates.

Mannose supplements induce embryonic lethality and blindness in phosphomannose isomerase hypomorphic mice.

Patients with congenital disorder of glycosylation (CDG), type Ib (MPI-CDG or CDG-Ib) have mutations in phosphomannose isomerase (MPI) that impair glycosylation and lead to stunted growth, liver dysfunction, coagulopathy, hypoglycemia, and intestinal abnormalities. Mannose supplements correct hypoglycosylation and most symptoms by providing mannose-6-P (Man-6-P) via hexokinase. We generated viable Mpi hypomorphic mice with residual enzymatic activity comparable to that of patients, but surprisingly, these mice appeared completely normal except for modest (~15%) embryonic lethality. To overcome this lethality, pregnant dams were provided 1-2% mannose in their drinking water. However, mannose further reduced litter size and survival to weaning by 40 and 66%, respectively. Moreover, ~50% of survivors developed eye defects beginning around midgestation. Mannose started at birth also led to eye defects but had no effect when started after eye development was complete. Man-6-P and related metabolites accumulated in the affected adult eye and in developing embryos and placentas. Our results demonstrate that disturbing mannose metabolic flux in mice, especially during embryonic development, induces a highly specific, unanticipated pathological state. It is unknown whether mannose is harmful to human fetuses during gestation; however, mothers who are at risk for having MPI-CDG children and who consume mannose during pregnancy hoping to benefit an affected fetus in utero should be cautious.

Methylene tetrahydrofolate dehydrogenase/cyclohydrolase and the synthesis of 10-CHO-THF are essential in Leishmania major.

10-Formyl tetrahydrofolate (10-CHO-THF) is a key metabolite in C1 carbon metabolism, arising through the action of formate-tetrahydrofolate ligase (FTL) and/or 5,10-methenyltetrahydrofolate cyclohydrolase/5,10-methylene tetrahydrofolate dehydrogenase (DHCH). Leishmania major possesses single DHCH1 and FTL genes encoding exclusively cytosolic proteins, unlike other organisms where isoforms occur in the mitochondrion as well. Recombinant DHCH1 showed typical NADP(+)-dependent methylene tetrahydrofolate DH and 5,10-methenyltetrahydrofolate CH activities, and the DH activity was potently inhibited by a substrate analogue 5,10-CO-THF (K(i) 105 nM), as was Leishmania growth (EC(50) 1.1 microM). Previous studies showed null ftl(-) mutants were normal, raising the possibility that loss of the purine synthetic pathway had rendered 10-CHO-THF dispensable in evolution. We were unable to generate dhch1(-) null mutants by gene replacement, despite using a wide spectrum of nutritional supplements expected to bypass DHCH function. We applied an improved method for testing essential genes in Leishmania, based on segregational loss of episomal complementing genes rather than transfection; analysis of approximately 1400 events without successful loss of DHCH1 again established its requirement. Lastly, we employed 'genetic metabolite complementation' using ectopically expressed FTL as an alternative source of 10-CHO-THF; now dhch1(-) null parasites were readily obtained. These data establish a requirement for 10-CHO-THF metabolism in L. major, and provide genetic and pharmacological validation of DHCH as a target for chemotherapy, in this and potentially other protozoan parasites.

Cost-effectiveness of low-level heat wrap therapy for low back pain.

OBJECTIVES: To evaluate the cost-effectiveness and budget impact of a new heat wrap therapy for low back pain compared to paracetamol and ibuprofen from the perspective of the UK National Health Service (NHS). METHODS: We evaluated cost-effectiveness using data from a phase III trial comparing the three therapies in 371 patients aged 18 to 55 years presenting with acute uncomplicated low back pain. The primary effectiveness measure used was successful treatment, defined as both clinically meaningful pain relief and clinically meaningful reduction in disability. We conducted a simple evaluation using NHS prescription costs and a modeled extrapolation including the costs of further treatment and consultations for patients treated unsuccessfully or with adverse events. Uncertainty was addressed using nonparametric bootstrapping and sensitivity analyses. RESULTS: Successful treatment was reported by 57% of patients treated with heat wrap therapy, 26% treated with paracetamol and 18% treated with ibuprofen (P < 0.05 for heat wrap vs. both other groups). NHS prescription cost per patient was estimated to be 1.35 pounds Sterling for heat wrap therapy, 0.26 pounds Sterling for paracetamol, and 0.28 pounds Sterling for ibuprofen and cost per successful treatment was 3.52 pounds Sterling for heat wrap therapy compared to paracetamol, and 2.72 pounds Sterling compared to ibuprofen. In the modeled extrapolation, NHS cost per patient was 27.77 pounds Sterling for heat wrap therapy, 34.20 pounds Sterling for paracetamol, and 36.04 pounds Sterling for ibuprofen. Sensitivity analyses indicated that the findings were robust to plausible changes in data and assumptions. CONCLUSIONS: Economic evaluation of this study suggests that the NHS cost of introducing heat wrap therapy in place of oral analgesics would be modest and heat wrap therapy might potentially reduce the total cost of managing episodes of lower back pain.