Distinguishing Binders from False Positives by Free Energy Calculations: Fragment Screening Against the Flap Site of HIV Protease

Molecular docking is a powerful tool used in drug discovery and structural biology for predicting the structures of ligand–receptor complexes. However, the accuracy of docking calculations can be limited by factors such as the neglect of protein reorganization in the scoring function; as a result, ligand screening can produce a high rate of false positive hits. Although absolute binding free energy methods still have difficulty in accurately rank-ordering binders, we believe that they can be fruitfully employed to distinguish binders from nonbinders and reduce the false positive rate. Here we study a set of ligands that dock favorably to a newly discovered, potentially allosteric site on the flap of HIV-1 protease. Fragment binding to this site stabilizes a closed form of protease, which could be exploited for the design of allosteric inhibitors. Twenty-three top-ranked protein–ligand complexes from AutoDock were subject to the free energy screening using two methods, the recently developed binding energy analysis method (BEDAM) and the standard double decoupling method (DDM). Free energy calculations correctly identified most of the false positives (≥83%) and recovered all the confirmed binders. The results show a gap averaging ≥3.7 kcal/mol, separating the binders and the false positives. We present a formula that decomposes the binding free energy into contributions from the receptor conformational macrostates, which provides insights into the roles of different binding modes. Our binding free energy component analysis further suggests that improving the treatment for the desolvation penalty associated with the unfulfilled polar groups could reduce the rate of false positive hits in docking. The current study demonstrates that the combination of docking with free energy methods can be very useful for more accurate ligand screening against valuable drug targets

Daily steps and all-cause mortality: a meta-analysis of 15 international cohorts

Background Although 10000 steps per day is widely promoted to have health benefits, there is little evidence to support this recommendation. We aimed to determine the association between number of steps per day and stepping rate with all-cause mortality. Methods In this meta-analysis, we identified studies investigating the effect of daily step count on all-cause mortality in adults (aged ≥18 years), via a previously published systematic review and expert knowledge of the field. We asked participating study investigators to process their participant-level data following a standardised protocol. The primary outcome was all-cause mortality collected from death certificates and country registries. We analysed the dose– response association of steps per day and stepping rate with all-cause mortality. We did Cox proportional hazards regression analyses using study-specific quartiles of steps per day and calculated hazard ratios (HRs) with inversevariance weighted random effects models. Findings We identified 15 studies, of which seven were published and eight were unpublished, with study start dates between 1999 and 2018. The total sample included 47 471 adults, among whom there were 3013 deaths (10·1 per 1000 participant-years) over a median follow-up of 7·1 years ([IQR 4·3–9·9]; total sum of follow-up across studies was 297 837 person-years). Quartile median steps per day were 3553 for quartile 1, 5801 for quartile 2, 7842 for quartile 3, and 10 901 for quartile 4. Compared with the lowest quartile, the adjusted HR for all-cause mortality was 0·60 (95% CI 0·51–0·71) for quartile 2, 0·55 (0·49–0·62) for quartile 3, and 0·47 (0·39–0·57) for quartile 4. Restricted cubic splines showed progressively decreasing risk of mortality among adults aged 60 years and older with increasing number of steps per day until 6000–8000 steps per day and among adults younger than 60 years until 8000–10000 steps per day. Adjusting for number of steps per day, comparing quartile 1 with quartile 4, the association between higher stepping rates and mortality was attenuated but remained significant for a peak of 30 min (HR 0·67 [95% CI 0·56–0·83]) and a peak of 60 min (0·67 [0·50–0·90]), but not significant for time (min per day) spent walking at 40 steps per min or faster (1·12 [0·96–1·32]) and 100 steps per min or faster (0·86 [0·58–1·28]). Interpretation Taking more steps per day was associated with a progressively lower risk of all-cause mortality, up to a level that varied by age. The findings from this meta-analysis can be used to inform step guidelines for public health promotion of physical activity

PRMT5 is upregulated by B-cell receptor signaling and forms a positive-feedback loop with PI3K/AKT in lymphoma cells

PRMT5, which regulates gene expression by symmetric dimethylation of histones and non-histone target proteins, is overexpressed and plays a pathogenic role in many cancers. In diffuse large B cell lymphoma (DLBCL), the mechanisms of PRMT5 dysregulation and its role in lymphomagenesis remain largely unknown. Here we demonstrate that B cell receptor (BCR) signaling regulates PRMT5 expression in DLBCL cells. Immunohistochemical analysis reveals elevated levels of PRMT5 expression in DLBCL cases and in germinal center (GC) B cells when compared to naive B cells. PRMT5 can be induced in naive B cells by BCR stimulation. We discovered that BTK-NF-κB signaling induces PRMT5 transcription in activated B cell-like (ABC) DLBCL cells while BCR downstream PI3K-AKT-MYC signaling upregulates PRMT5 expression in both ABC and GCB DLBCL cells. PRMT5 inhibition inhibits the growth of DLBCL cells in vitro and patient derived xenografts. Genomic and biochemical analysis demonstrate that PRMT5 promotes cell cycle progression and activates PI3K-AKT signaling, suggesting a feedback regulatory mechanism to enhance cell survival and proliferation. Co-targeting PRMT5 and AKT by their specific inhibitors is lethal to DLBCL cell lines and primary cancer cells. Therefore, this study provides a mechanistic rationale for clinical trials to evaluate PRMT5 and AKT inhibitors for DLBCL

The π Configuration of the WWW Motif of a Short Trp-Rich Peptide Is Critical for Targeting Bacterial Membranes, Disrupting Preformed Biofilms, and Killing Methicillin-Resistant <i>Staphylococcus aureus</i>

Tryptophan-rich peptides, being short and suitable for large-scale chemical synthesis, are attractive candidates for developing a new generation of antimicrobials to combat antibiotic-resistant bacteria (superbugs). Although there are numerous pictures of the membrane-bound structure of a single tryptophan (W), how multiple Trp amino acids assemble themselves and interact with bacterial membranes is poorly understood. This communication presents the three-dimensional structure of an eight-residue Trp-rich peptide (WWW­L­R­KIW-NH₂ with 50% W) determined by the improved two-dimensional nuclear magnetic resonance method, which includes the measurements of ¹³C and ¹⁵N chemical shifts at natural abundance. This peptide forms the shortest two-turn helix with a distinct amphipathic feature. A unique structural arrangement is identified for the Trp triplet, WWW, that forms a π configuration with W2 as the horizontal bar and W1/W3 forming the two legs. An arginine scan reveals that the WWW motif is essential for killing methicillin-resistant <i>Staphylococcus aureus</i> USA300 and disrupting preformed bacterial biofilms. This unique π configuration for the WWW motif is stabilized by aromatic–aromatic interactions as evidenced by ring current shifts as well as nuclear Overhauser effects. Because the WWW motif is maintained, a change of I7 to R led to a potent antimicrobial and antibiofilm peptide with 4-fold improvement in cell selectivity. Collectively, this study elucidated the structural basis of antibiofilm activity of the peptide, identified a better peptide candidate via structure–activity relationship studies, and laid the foundation for engineering future antibiotics based on the WWW motif

Structure-guided discovery of potent and oral soluble epoxide hydrolase inhibitors for the treatment of neuropathic pain

Soluble epoxide hydrolase (sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding (88.6%) and better metabolic stability in&nbsp;vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0&nbsp;g/kg. Inhibitor A20 also displayed robust analgesic effect in&nbsp;vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor (±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain

Discovery of memantyl urea derivatives as potent soluble epoxide hydrolase inhibitors against lipopolysaccharide-induced sepsis

Sepsis, a systemic inflammatory response, caused by pathogenic factors including microorganisms, has high mortality and limited therapeutic approaches. Herein, a new soluble epoxide hydrolase (sEH) inhibitor series comprising a phenyl ring connected to a memantyl moiety via a urea or amide linkage has been designed. A preferential urea pharmacophore that improved the binding properties of the compounds was identified for those series via biochemical assay in&nbsp;vitro and in&nbsp;vivo studies. Molecular docking displayed that 3,5-dimethyl on the adamantyl group in B401 could make van der Waals interactions with residues at a hydrophobic pocket of sEH active site, which might indirectly explain the subnanomolar level activities of memantyl urea derivatives in&nbsp;vitro better than AR-9281. Among them, compound B401 significantly improved the inhibition potency with human and murine sEH IC50 values as 0.4&nbsp;nM and 0.5&nbsp;nM, respectively. Although the median survival time of C57BL/6 mice in LPS-induced sepsis model was slightly increased, the survival rate did not reach significant efficacy. Based on safety profile, metabolic stability, pharmacokinetic and in&nbsp;vivo efficacy, B401 demonstrated the proof of potential for this class of memantyl urea-based sEH inhibitors as therapeutic agents in sepsis

Two distinct amphipathic peptide antibiotics with systemic efficacy.

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibiotic-resistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-stat