High efficacy of the F-ATP synthase inhibitor TBAJ-5307 against nontuberculous mycobacteria in vitro and in vivo.

The F1FO-ATP synthase engine is essential for viability and growth of nontuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions. Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the FO domain of the engine and preventing rotation and proton translocation. TBAJ-5307 is active at low nanomolar concentrations against fast- and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP. As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity. Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem-avibactam pair showed attractive potentiation. Furthermore, the TBAJ-5307-tebipenem-avibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections.

Membrane platform protein PulF of the Klebsiella type II secretion system forms a trimeric ion channel essential for endopilus assembly and protein secretion.

IMPORTANCE: Type IV pili and type II secretion systems are members of the widespread type IV filament (T4F) superfamily of nanomachines that assemble dynamic and versatile surface fibers in archaea and bacteria. The assembly and retraction of T4 filaments with diverse surface properties and functions require the plasma membrane platform proteins of the GspF/PilC superfamily. Generally considered dimeric, platform proteins are thought to function as passive transmitters of the mechanical energy generated by the ATPase motor, to somehow promote insertion of pilin subunits into the nascent pilus fibers. Here, we generate and experimentally validate structural predictions that support the trimeric state of a platform protein PulF from a type II secretion system. The PulF trimers form selective proton or sodium channels which might energize pilus assembly using the membrane potential. The conservation of the channel sequence and structural features implies a common mechanism for all T4F assembly systems. We propose a model of the oligomeric PulF-PulE ATPase complex that provides an essential framework to investigate and understand the pilus assembly mechanism.

Variations of the Mycobacterium abscessus F-ATP synthase subunit a-c interface alter binding and potency of the anti-TB drug bedaquiline.

The anti-tuberculosis therapeutic bedaquiline (BDQ) is used against Mycobacterium abscessus. In M. abscessus BDQ is only bacteriostatic and less potent compared to M. tuberculosis or M. smegmatis. Here we demonstrate its reduced ATP synthesis inhibition against M. abscessus inside-out vesicles, including the F1FO-ATP synthase. Molecular dynamics simulations and binding free energy calculations highlight the differences in drug-binding of the M. abscessus and M. smegmatis FO-domain at the lagging site, where the drug deploys its mechanistic action, inhibiting ATP synthesis. These data pave the way for improved anti-M. abscessus BDQ analogs.

Bioactive Suture with Added Innate Defense Functionality for the Reduction of Bacterial Infection and Inflammation.

Surgical site infections (SSI) are a clinical and economic burden. Suture-associated SSI may develop when bacteria colonize the suture surface and form biofilms that are resistant to antibiotics. Thrombin-derived C-terminal peptide (TCP)-25 is a host defense peptide with a unique dual mode of action that can target both bacteria and the excessive inflammation induced by bacterial products. The peptide demonstrates therapeutic potential in preclinical in vivo wound infection models. In this study, the authors set out to explore whether TCP-25 can provide a new bioactive innate immune feature to hydrophilic polyglactin sutures (Vicryl). Using a combination of biochemical, biophysical, antibacterial, biofilm, and anti-inflammatory assays in vitro, in silico molecular modeling studies, along with experimental infection and inflammation models in mice, a proof-of-concept that TCP-25 can provide Vicryl sutures with a previously undisclosed host defense capacity, that enables targeting of bacteria, biofilms, and the accompanying inflammatory response, is shown.

Targeting Toll-like receptor-driven systemic inflammation by engineering an innate structural fold into drugs.

There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.

A pH-dependent cluster of charges in a conserved cryptic pocket on flaviviral envelopes.

Flaviviruses are enveloped viruses which include human pathogens that are predominantly transmitted by mosquitoes and ticks. Some, such as dengue virus, exhibit the phenomenon of antibody-dependent enhancement (ADE) of disease, making vaccine-based routes of fighting infections problematic. The pH-dependent conformational change of the envelope (E) protein required for fusion between the viral and endosomal membranes is an attractive point of inhibition by antivirals as it has the potential to diminish the effects of ADE. We examined six flaviviruses by employing large-scale molecular dynamics (MD) simulations of raft systems that represent a substantial portion of the flaviviral envelope. We utilised a benzene-mapping approach that led to a discovery of shared hotspots and conserved cryptic sites. A cryptic pocket previously shown to bind a detergent molecule exhibited strain-specific characteristics. An alternative conserved cryptic site at the E protein domain interfaces showed a consistent dynamic behaviour across flaviviruses and contained a conserved cluster of ionisable residues. Constant-pH simulations revealed cluster and domain-interface disruption under low pH conditions. Based on this, we propose a cluster-dependent mechanism that addresses inconsistencies in the histidine-switch hypothesis and highlights the role of cluster protonation in orchestrating the domain dissociation pivotal for the formation of the fusogenic trimer.

SARS-CoV-2 spike protein aggregation is triggered by bacterial lipopolysaccharide.

SARS-CoV-2 spike (S) protein is crucial for virus invasion in COVID-19. Here, we showed that lipopolysaccharide (LPS) can trigger S protein aggregation at high doses of LPS and S protein. We demonstrated the formation of S protein aggregates by microscopy analyses, aggregation and gel shift assays. LPS at high levels boosts the formation of S protein aggregates as detected by amytracker and thioflavin T dyes that specifically bind to aggregating proteins. We validated the role of LPS by blocking the formation of aggregates by the endotoxin-scavenging thrombin-derived peptide TCP-25. Aggregation-prone sequences in S protein are predicted to be nearby LPS binding sites, while molecular simulations showed stable formation of S protein-LPS higher-order oligomers. Collectively, our results provide evidence of LPS-induced S protein aggregation.

Health effects of androgen abuse: a review of the HAARLEM study.

PURPOSE OF REVIEW: Data on the health effects of androgen abuse are mainly derived from lower level evidence, such as case series and cross-sectional studies. In the last few years a relatively large and prospective cohort initiative, the HAARLEM (health risks of anabolic androgenic steroid use by male amateur athletes) study, made an important contribution to current knowledge. RECENT FINDINGS: The HAARLEM study showed that all androgen abusers experience positive and negative effects, such as an increase in strength and acne and gynecomastia, respectively. Effects are generally reversible and acute life-threatening toxicity is rare. There is a distinct but limited impact on liver and kidney function. Gonadal function is disrupted but resumes normally after abuse is discontinued in the majority of athletes. The negative impact of androgens on cardiovascular parameters, such as blood pressure, hematocrit and lipid metabolism, as well as cardiac structure and function, seems to be the mechanism for premature atherosclerosis and cardiomyopathy, respectively, in long-term users. SUMMARY: It is beyond dispute that androgen abuse is harmful and much of the short-term toxicity is well documented. To prevent the long-term health hazards, there should be ample focus on preventive measures, both primary and secondary, and effective harm reduction strategies should be developed.

Anabolic-androgenic steroids: How do they work and what are the risks?

Anabolic-androgenic steroids (AAS) are a class of hormones that are widely abused for their muscle-building and strength-increasing properties in high, nontherapeutic, dosages. This review provides an up-to-date and comprehensive overview on how these hormones work and what side effects they might elicit. We discuss how AAS are absorbed into the circulation after intramuscular injection or oral ingestion and how they are subsequently transported to the tissues, where they will move into the extravascular compartment and diffuse into their target cells. Inside these cells, AAS can biotransform into different metabolites or bind to their cognate receptor: the androgen receptor. AAS and their metabolites can cause side effects such as acne vulgaris, hypertension, hepatotoxicity, dyslipidemia, testosterone deficiency, erectile dysfunction, gynecomastia, and cardiomyopathy. Where applicable, we mention treatment options and self-medication practices of AAS users to counteract these side effects. Clinicians may use this review as a guide for understanding how AAS use can impact health and to assist in patient education and, in some cases, the management of side effects.

Somatic genetic rescue of a germline ribosome assembly defect.

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS.

The nanotube express: Delivering a stapled peptide to the cell surface.

HYPOTHESIS: Carbon nanotubes (CNTs) represent a novel platform for cellular delivery of therapeutic peptides. Chemically-functionalized CNTs may enhance peptide uptake by improving their membrane targeting properties. EXPERIMENTS: Using coarse-grained (CG) molecular dynamics (MD) simulations, we investigate membrane interactions of a peptide conjugated to pristine and chemically-modified CNTs. As proof of principle, we focus on their interactions with PM2, an amphipathic stapled peptide that inhibits the E3 ubiquitin ligase HDM2 from negatively regulating the p53 tumor suppressor. CNT interaction with both simple planar lipid bilayers as well as spherical lipid vesicles was studied, the latter as a surrogate for curved cellular membranes. FINDINGS: Membrane permeation was rapid and spontaneous for both pristine and oxidized CNTs when unconjugated. This was slowed upon addition of a noncovalently attached peptide surface "sheath", which may be an effective way to slow CNT entry and avert membrane rupture. The CNT conjugates were observed to "desheath" their peptide layer at the bilayer interface upon insertion, leaving their cargo behind in the outer leaflet. This suggests that a synergy may exist to optimize CNT safety whilst enhancing the delivery efficiency of "hitchhiking" therapeutic molecules.

Antibody affinity versus dengue morphology influences neutralization.

Different strains within a dengue serotype (DENV1-4) can have smooth, or "bumpy" surface morphologies with different antigenic characteristics at average body temperature (37°C). We determined the neutralizing properties of a serotype cross-reactive human monoclonal antibody (HMAb) 1C19 for strains with differing morphologies within the DENV1 and DENV2 serotypes. We mapped the 1C19 epitope to E protein domain II by hydrogen deuterium exchange mass spectrometry, cryoEM and molecular dynamics simulations, revealing that this epitope is likely partially hidden on the virus surface. We showed the antibody has high affinity for binding to recombinant DENV1 E proteins compared to those of DENV2, consistent with its strong neutralizing activities for all DENV1 strains tested regardless of their morphologies. This finding suggests that the antibody could out-compete E-to-E interaction for binding to its epitope. In contrast, for DENV2, HMAb 1C19 can only neutralize when the epitope becomes exposed on the bumpy-surfaced particle. Although HMAb 1C19 is not a suitable therapeutic candidate, this study with HMAb 1C19 shows the importance of choosing a high-affinity antibody that could neutralize diverse dengue virus morphologies for therapeutic purposes.

The impact of Gag non-cleavage site mutations on HIV-1 viral fitness from integrative modelling and simulations.

The high mutation rate in retroviruses is one of the leading causes of drug resistance. In human immunodeficiency virus type-1 (HIV-1), synergistic mutations in its protease and the protease substrate - the Group-specific antigen (Gag) polyprotein - work together to confer drug resistance against protease inhibitors and compensate the mutations affecting viral fitness. Some Gag mutations can restore Gag-protease binding, yet most Gag-protease correlated mutations occur outside of the Gag cleavage site. To investigate the molecular basis for this, we now report multiscale modelling approaches to investigate various sequentially cleaved Gag products in the context of clinically relevant mutations that occur outside of the cleavage sites, including simulations of the largest Gag proteolytic product in its viral membrane-bound state. We found that some mutations, such as G123E and H219Q, involve direct interaction with cleavage site residues to influence their local environment, while certain mutations in the matrix domain lead to the enrichment of lipids important for Gag targeting and assembly. Collectively, our results reveal why non-cleavage site mutations have far-reaching implications outside of Gag proteolysis, with important consequences for drugging Gag maturation intermediates and tackling protease inhibitor resistance.

A second shell residue modulates a conserved ATP-binding site with radically different affinities for ATP.

BACKGROUND: Prediction of ligand binding and design of new function in enzymes is a time-consuming and expensive process. Crystallography gives the impression that proteins adopt a fixed shape, yet enzymes are functionally dynamic. Molecular dynamics offers the possibility of probing protein movement while predicting ligand binding. Accordingly, we choose the bacterial F1Fo ATP synthase ε subunit to unravel why ATP affinity by ε subunits from Bacillus subtilis and Bacillus PS3 differs ~500-fold, despite sharing identical sequences at the ATP-binding site. METHODS: We first used the Bacillus PS3 ε subunit structure to model the B. subtilis ε subunit structure and used this to explore the utility of molecular dynamics (MD) simulations to predict the influence of residues outside the ATP binding site. To verify the MD predictions, point mutants were made and ATP binding studies were employed. RESULTS: MD simulations predicted that E102 in the B. subtilis ε subunit, outside of the ATP binding site, influences ATP binding affinity. Engineering E102 to alanine or arginine revealed a ~10 or ~54 fold increase in ATP binding, respectively, confirming the MD prediction that E102 drastically influences ATP binding affinity. CONCLUSIONS: These findings reveal how MD can predict how changes in the "second shell" residues around substrate binding sites influence affinity in simple protein structures. Our results reveal why seemingly identical ε subunits in different ATP synthases have radically different ATP binding affinities. GENERAL SIGNIFICANCE: This study may lead to greater utility of molecular dynamics as a tool for protein design and exploration of protein design and function.

An Alternative HIV-1 Non-Nucleoside Reverse Transcriptase Inhibition Mechanism: Targeting the p51 Subunit.

The ongoing development of drug resistance in HIV continues to push for the need of alternative drug targets in inhibiting HIV. One such target is the Reverse transcriptase (RT) enzyme which is unique and critical in the viral life cycle-a rational target that is likely to have less off-target effects in humans. Serendipitously, we found two chemical scaffolds from the National Cancer Institute (NCI) Diversity Set V that inhibited HIV-1 RT catalytic activity. Computational structural analyses and subsequent experimental testing demonstrated that one of the two chemical scaffolds binds to a novel location in the HIV-1 RT p51 subunit, interacting with residue Y183, which has no known association with previously reported drug resistance. This finding supports the possibility of a novel druggable site on p51 for a new class of non-nucleoside RT inhibitors that may inhibit HIV-1 RT allosterically. Although inhibitory activity was shown experimentally to only be in the micromolar range, the scaffolds serve as a proof-of-concept of targeting the HIV RT p51 subunit, with the possibility of medical chemistry methods being applied to improve inhibitory activity towards more effective drugs.

A Benzene-Mapping Approach for Uncovering Cryptic Pockets in Membrane-Bound Proteins.

Molecular dynamics (MD) simulations in combination with small organic probes present in the solvent have previously been used as a method to reveal cryptic pockets that may not have been identified in experimental structures. We report such a method implemented within the CHARMM force field using the GROMACS simulation package to effectively explore cryptic pockets on the surfaces of membrane-embedded proteins using benzene as a probe molecule. This method, for which we have made implementation files freely available, relies on modified nonbonded parameters in addition to repulsive potentials between membrane lipids and benzene molecules. The method was tested on part of the outer shell of the dengue virus (DENV), for which research into a safe and effective neutralizing antibody or drug molecule is still ongoing. In particular, the envelope (E) protein, associated with the membrane (M) protein, is a lipid membrane-embedded complex which forms a dimer in the mature viral envelope. Solvent mapping was performed for the full, membrane-embedded EM protein complex and compared with similar calculations performed for the isolated, soluble E protein ectodomain dimer in the solvent. Ectodomain-only simulations with benzene exhibited unfolding effects not observed in the more physiologically relevant membrane-associated systems. A cryptic pocket which has been experimentally shown to bind n-octyl-ß-d-glucoside detergent was consistently revealed in all benzene-containing simulations. The addition of benzene also enhanced the flexibility and hydrophobic exposure of cryptic pockets at a key, functional interface in the E protein and revealed a novel, potentially druggable pocket that may be targeted to prevent conformational changes associated with viral entry into the cell.

Characterizing the Hydration Properties of Proton Binding Sites in the ATP Synthase c-Rings of Bacillus Species.

The membrane-embedded domain of ATP synthases contains the c-ring, which translocates ions across the membrane, and its resultant rotation is coupled to ATP synthesis in the extramembranous domain. During rotation, the c-ring becomes accessible on both sides of the lipid bilayer to solvent via channels connected to the other membrane-embedded component, the a subunit, and thereby allows the ion to be released into the solvent environment. In recent times, many experimental structures of c-rings from different species have been solved. In some of these, a water molecule with a proposed "structural role" has been identified within the c-ring ion binding site, but in general, the requirement for high resolution to resolve specific water densities complicates their interpretation. In the present study, we use molecular dynamics (MD) simulations and rigorous free energy calculations to characterize the dynamics and energetics of a water molecule within the ion binding site of the c-ring from Bacillus pseudofirmus OF4, in its wild type (WT) and P51A mutant forms, along with the c-ring from thermophilic Bacillus PS3. Our data suggest that a water molecule stably binds to the P51A mutant, as well as helping to identify a bound water molecule in Bacillus PS3 whose presence was previously overlooked due to the limited resolution of the structural data. Sequence analysis further identifies a novel conserved sequence motif that is likely required to harbor a water molecule for stable ion coordination in the binding site of such proteins.

The Molecular Basis for Purine Binding Selectivity in the Bacterial ATP Synthase ϵ Subunit.

The ϵ subunit of ATP synthases has been proposed to regulate ATP hydrolysis in bacteria. Prevailing evidence supports the notion that when the ATP concentration falls below a certain threshold, the ϵ subunit changes its conformation from a non-inhibitory down-state to an extended up-state that then inhibits enzymatic ATP hydrolysis by binding to the catalytic domain. It has been demonstrated that the ϵ subunit from Bacillus PS3 is selective for ATP over other nucleotides, including GTP. In this study, the purine triphosphate selectivity is rationalized by using results from MD simulations and free energy calculations for the R103A/R115A mutant of the ϵ subunit from Bacillus PS3, which binds ATP more strongly than the wild-type protein. Our results are in good agreement with experimental data, and the elucidated molecular basis for selectivity could help to guide the design of novel GTP sensors.

Not all therapeutic antibody isotypes are equal: the case of IgM versus IgG in Pertuzumab and Trastuzumab.

The therapeutic potential of immunoglobulin M (IgM) is of considerable interest in immunotherapy due to its complement-activating and cell-agglutinating abilities. Pertuzumab and Trastuzumab are monoclonal antibodies used to treat human epidermal growth factor receptor 2 (HER2)-positive breast cancer but exhibit significantly different binding affinities as IgM when compared to its IgG isotype. Using integrative multiscale modelling and simulations of complete antibody assemblies, we show that Pertuzumab IgM is able to utilize all of its V-regions to bind multiple HER2 receptors simultaneously, while similar binding in Trastuzumab IgM is prohibited by steric clashes caused by the large globular domain of HER2. This is subsequently validated by confirming that Pertuzumab IgM inhibits proliferation in HER2 over-expressing live cells more effectively than its IgG counterpart and Trastuzumab IgM. Our study highlights the importance of understanding the molecular details of antibody-antigen interactions for the design and isotype selection of therapeutic antibodies.

Molecular basis of dengue virus serotype 2 morphological switch from 29°C to 37°C.

The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to "bumpy" surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become "bumpy". These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus.