The Hybrid Antibiotic Thiomarinol A Overcomes Intrinsic Resistance in Escherichia coli Using a Privileged Dithiolopyrrolone Moiety.

An impermeable outer membrane and multidrug efflux pumps work in concert to provide Gram-negative bacteria with intrinsic resistance against many antibiotics. These resistance mechanisms reduce the intracellular concentrations of antibiotics and render them ineffective. The natural product thiomarinol A combines holothin, a dithiolopyrrolone antibiotic, with marinolic acid A, a close analogue of mupirocin. The hybridity of thiomarinol A converts the mupirocin scaffold from inhibiting Gram-positive bacteria to inhibiting both Gram-positive and -negative bacteria. We found that thiomarinol A accumulates significantly more than mupirocin within the Gram-negative bacterium Escherichia coli, likely contributing to its broad-spectrum activity. Antibiotic susceptibility testing of E. coli mutants reveals that thiomarinol A overcomes the intrinsic resistance mechanisms that render mupirocin inactive. Structure-activity relationship studies suggest that the dithiolopyrrolone is a privileged moiety for improving the accumulation and antibiotic activity of the mupirocin scaffold without compromising binding to isoleucyl-tRNA synthetase. These studies also highlight that accumulation is required but not sufficient for antibiotic activity. Our work reveals a role of the dithiolopyrrolone moiety in overcoming intrinsic mupirocin resistance in E. coli and provides a starting point for designing dual-acting and high-accumulating hybrid antibiotics.

Structural insight into selectivity of amylin and calcitonin receptor agonists.

Amylin receptors (AMYRs), heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins, are promising obesity targets. A hallmark of AMYR activation by Amy is the formation of a 'bypass' secondary structural motif (residues S19-P25). This study explored potential tuning of peptide selectivity through modification to residues 19-22, resulting in a selective AMYR agonist, San385, as well as nonselective dual amylin and calcitonin receptor agonists (DACRAs), with San45 being an exemplar. We determined the structure and dynamics of San385-bound AMY3R, and San45 bound to AMY3R or CTR. San45, via its conjugated lipid at position 21, was anchored at the edge of the receptor bundle, enabling a stable, alternative binding mode when bound to the CTR, in addition to the bypass mode of binding to AMY3R. Targeted lipid modification may provide a single intervention strategy for design of long-acting, nonselective, Amy-based DACRAs with potential anti-obesity effects.

A Permissive Amide N-Methyltransferase for Dithiolopyrrolones.

Amide N-methylation is important for the activity and permeability of bioactive compounds but can be challenging to perform selectively. The broad-spectrum antimicrobial natural products thiolutin and holomycin differ only by an N-methyl group at the endocyclic amide of thiolutin, but only thiolutin exhibits antifungal activity. The enzyme responsible for amide N--methylation in thiolutin biosynthesis has remained elusive. Here, we identified and characterized the amide N-methyltransferase DtpM that is encoded >400 kb outside of the thiolutin gene cluster. DtpM catalyzes efficient conversion of holomycin to thiolutin, exhibits broad substrate scope toward dithiolopyrrolones, and has high thermal stability. In addition, sequence similarity network analysis suggests DtpM is more closely related to phenol O-methyltransferases than some amide methyltransferases. This study expands the limited examples of amide N-methyltransferases and may facilitate chemoenzymatic synthesis of diverse dithiolopyrrolone compounds as potential therapeutics.

American Trauma Care: A System of Systems.

OBJECTIVE: The benefits of organized trauma systems have been well-documented during 50 years of trauma system development in the United States. Unfortunately, despite this evidence, trauma system development has occurred only sporadically in the 50 states. METHODS: The relevant literature related to trauma system design and development was reviewed based on relevance to the study. Information from these sources was summarized into a SWOT (strengths, weaknesses, opportunities, and threats) analysis. RESULTS: Strengths discovered were leadership brought forth by the American College of Surgeons Committee on Trauma and meaningful change generated from The National Academy of Sciences, Engineering, and Medicine report addressing the fractionation of the nation's trauma systems, whereas weaknesses included patient outcome disparities due to the lack of a national governing authority, undertriage, underresourced rural trauma, and underfunded trauma research. Opportunities included the creation of level IV trauma centers; telemedicine; the development of rural trauma management courses; air medical transport to bring high-intensity care to the patient, particularly in rural areas; trauma research; and trauma prevention through outreach and educational programs. The following threats were determined: mass casualty incidents, motor vehicle collisions because of the high rate of motor vehicle collision deaths in the United States relative to other developed countries, and underfunded trauma systems. CONCLUSION: Much work remains to be done in the development of an American trauma system. Recommendations include implementation of trauma care governance at the federal level; national oversight and support of emergency medical services systems, particularly in rural areas with strict reporting processes for emergency medical services programs; national organization of our mass casualty response; increased federal and state funding allocated to trauma centers; a consistent model for trauma system development; and trauma research.

Understanding the hydrological response of a headwater-dominated catchment by analysis of distributed surface-subsurface interactions.

We computationally explore the relationship between surface-subsurface exchange and hydrological response in a headwater-dominated high elevation, mountainous catchment in East River Watershed, Colorado, USA. In order to isolate the effect of surface-subsurface exchange on the hydrological response, we compare three model variations that differ only in soil permeability. Traditional methods of hydrograph analysis that have been developed for headwater catchments may fail to properly characterize catchments, where catchment response is tightly coupled to headwater inflow. Analyzing the spatially distributed hydrological response of such catchments gives additional information on the catchment functioning. Thus, we compute hydrographs, hydrological indices, and spatio-temporal distributions of hydrological variables. The indices and distributions are then linked to the hydrograph at the outlet of the catchment. Our results show that changes in the surface-subsurface exchange fluxes trigger different flow regimes, connectivity dynamics, and runoff generation mechanisms inside the catchment, and hence, affect the distributed hydrological response. Further, changes in surface-subsurface exchange rates lead to a nonlinear change in the degree of connectivity-quantified through the number of disconnected clusters of ponding water-in the catchment. Although the runoff formation in the catchment changes significantly, these changes do not significantly alter the aggregated streamflow hydrograph. This hints at a crucial gap in our ability to infer catchment function from aggregated signatures. We show that while these changes in distributed hydrological response may not always be observable through aggregated hydrological signatures, they can be quantified through the use of indices of connectivity.

New Insights into the Structure and Function of Class B1 GPCRs.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. Class B1 GPCRs constitute a subfamily of 15 receptors that characteristically contain large extracellular domains (ECDs) and respond to long polypeptide hormones. Class B1 GPCRs are critical regulators of homeostasis, and, as such, many are important drug targets. While most transmembrane proteins, including GPCRs, are recalcitrant to crystallization, recent advances in cryo-electron microscopy (cryo-EM) have facilitated a rapid expansion of the structural understanding of membrane proteins. As a testament to this success, structures for all the class B1 receptors bound to G proteins have been determined by cryo-EM in the past 5 years. Further advances in cryo-EM have uncovered dynamics of these receptors, ligands, and signaling partners. Here, we examine the recent structural underpinnings of the class B1 GPCRs with an emphasis on structure-function relationships.

Membranes under the Magnetic Lens: A Dive into the Diverse World of Membrane Protein Structures Using Cryo-EM.

Membrane proteins are highly diverse in both structure and function and can, therefore, present different challenges for structure determination. They are biologically important for cells and organisms as gatekeepers for information and molecule transfer across membranes, but each class of membrane proteins can present unique obstacles to structure determination. Historically, many membrane protein structures have been investigated using highly engineered constructs or using larger fusion proteins to improve solubility and/or increase particle size. Other strategies included the deconstruction of the full-length protein to target smaller soluble domains. These manipulations were often required for crystal formation to support X-ray crystallography or to circumvent lower resolution due to high noise and dynamic motions of protein subdomains. However, recent revolutions in membrane protein biochemistry and cryo-electron microscopy now provide an opportunity to solve high resolution structures of both large, >1 megadalton (MDa), and small, <100 kDa (kDa), drug targets in near-native conditions, routinely reaching resolutions around or below 3 Å. This review provides insights into how the recent advances in membrane biology and biochemistry, as well as technical advances in cryo-electron microscopy, help us to solve structures of a large variety of membrane protein groups, from small receptors to large transporters and more complex machineries.

A Structurally Characterized Staphylococcus aureus Evolutionary Escape Route from Treatment with the Antibiotic Linezolid.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment. Clonal isolates which had evolved a linezolid-resistant phenotype were characterized by whole-genome sequencing. Linezolid-resistant mutants were identified which had accumulated mutations in the ribosomal protein uL3. Multiple clones which had two mutations in uL3 exhibited resistance to linezolid, 2-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single-particle cryo-electron microscopic analysis and compared to the ribosomes from the parent strain. We found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix 90, propagating a structural change over 15 Å away. This distal change swings nucleotide U2504 into the binding site of the antibiotic, causing linezolid resistance. IMPORTANCE Antibiotic resistance poses a critical problem to human health and decreases the utility of these lifesaving drugs. Of particular concern is the "superbug" methicillin-resistant Staphylococcus aureus (MRSA), for which treatment of infection requires the use of last-line antibiotics, including linezolid. In this paper, we characterize the atomic rearrangements which the ribosome, the target of linezolid, undergoes during its evolutionary journey toward becoming drug resistant. Using cryo-electron microscopy, we describe a particular molecular mechanism which MRSA uses to become resistant to linezolid.

A structural basis for amylin receptor phenotype.

Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), AMY1R, AMY2R, and AMY3R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY1R with salmon CT (sCT), AMY2R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics.

Cryo-EM structure of the dual incretin receptor agonist, peptide-19, in complex with the glucagon-like peptide-1 receptor.

Dual agonists that can activate both the glucagon-like peptide-1 receptor (GLP-1R) and the gastric inhibitory polypeptide receptor (GIPR) have demonstrated high efficacy for the treatment of metabolic disease. Peptide-19 is a prototypical dual agonist that has high potency at both GLP-1R and GIPR but has a distinct signalling profile relative to the native peptides at the cognate receptors. In this study, we solved the structure of peptide-19 bound to the GLP-1R in complex with Gs protein, and compared the structure and dynamics of this complex to that of published structures of GLP-1R:Gs in complex with other receptor agonists. Unlike other peptide-bound receptor complexes, peptide-19:GLP-1R:Gs demonstrated a more open binding pocket where transmembrane domain (TM) 6, TM7 and the interconnecting extracellular loop 3 (ECL3) were located away from the peptide, with no interactions between peptide-19 and TM6/ECL3. Analysis of conformational variance of the complex revealed that peptide-19 was highly dynamic and underwent binding and unbinding motions facilitated by the more open TM binding pocket. Both the consensus structure of the GLP-1R complex with peptide-19 and the dynamics of this complex were distinct from previously described GLP-1R structures providing unique insights into the mode of GLP-1R activation by this dual agonist.

Evolving cryo-EM structural approaches for GPCR drug discovery.

G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in stabilization of GPCR:transducer complexes, together with improvements in cryoelectron microscopy (cryo-EM) have recently been applied to structure-assisted drug design for GPCR agonists. Nonetheless, limitations in the commercial application of these approaches, including the use of nanobody 35 (Nb35) to aid complex stabilization and the high cost of 300 kV imaging, have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated the formation of stable complexes with a modified Gs protein in the absence of Nb35. In parallel, we compare 200 versus 300 kV image acquisition using a Falcon 4 or K3 direct electron detector. Moreover, the 200 kV Glacios-Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.

Human TRPC5 structures reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site.

TRPC1/4/5 channels are non-specific cation channels implicated in a wide variety of diseases, and TRPC1/4/5 inhibitors have recently entered clinical trials. However, fundamental and translational studies require a better understanding of TRPC1/4/5 channel regulation by endogenous and exogenous factors. Although several potent and selective TRPC1/4/5 modulators have been reported, the paucity of mechanistic insights into their modes-of-action remains a barrier to the development of new chemical probes and drug candidates. Xanthine-based modulators include the most potent and selective TRPC1/4/5 inhibitors described to date, as well as TRPC5 activators. Our previous studies suggest that xanthines interact with a, so far, elusive pocket of TRPC1/4/5 channels that is essential to channel gating. Here we report the structure of a small-molecule-bound TRPC1/4/5 channel-human TRPC5 in complex with the xanthine Pico145-to 3.0 Å. We found that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a bound phospholipid. Our findings explain the mode-of-action of xanthine-based TRPC1/4/5 modulators, and suggest a structural basis for TRPC1/4/5 modulation by endogenous factors such as (phospho)lipids and Zn2+ ions. These studies lay the foundations for the structure-based design of new generations of TRPC1/4/5 modulators.

Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites.

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Cryo-EM Structure and Molecular Dynamics Analysis of the Fluoroquinolone Resistant Mutant of the AcrB Transporter from Salmonella.

Salmonella is an important genus of Gram-negative pathogens, treatment of which has become problematic due to increases in antimicrobial resistance. This is partly attributable to the overexpression of tripartite efflux pumps, particularly the constitutively expressed AcrAB-TolC. Despite its clinical importance, the structure of the Salmonella AcrB transporter remained unknown to-date, with much of our structural understanding coming from the Escherichia coli orthologue. Here, by taking advantage of the styrene maleic acid (SMA) technology to isolate membrane proteins with closely associated lipids, we report the very first experimental structure of Salmonella AcrB transporter. Furthermore, this novel structure provides additional insight into mechanisms of drug efflux as it bears the mutation (G288D), originating from a clinical isolate of Salmonella Typhimurium presenting an increased resistance to fluoroquinolones. Experimental data are complemented by state-of-the-art molecular dynamics (MD) simulations on both the wild type and G288D variant of Salmonella AcrB. Together, these reveal several important differences with respect to the E. coli protein, providing insights into the role of the G288D mutation in increasing drug efflux and extending our understanding of the mechanisms underlying antibiotic resistance.

Structure-Based Identification and Characterization of Inhibitors of the Epilepsy-Associated KNa1.1 (KCNT1) Potassium Channel.

Drug-resistant epileptic encephalopathies of infancy have been associated with KCNT1 gain-of-function mutations, which increase the activity of KNa1.1 sodium-activated potassium channels. Pharmacological inhibition of hyperactive KNa1.1 channels by quinidine has been proposed as a stratified treatment, but mostly this has not been successful, being linked to the low potency and lack of specificity of the drug. Here we describe the use of a previously determined cryo-electron microscopy-derived KNa1.1 structure and mutational analysis to identify how quinidine binds to the channel pore and, using computational methods, screened for compounds predicated to bind to this site. We describe six compounds that inhibited KNa1.1 channels with low- and sub-micromolar potencies, likely also through binding in the intracellular pore vestibule. In hERG inhibition and cytotoxicity assays, two compounds were ineffective. These may provide starting points for the development of new pharmacophores and could become tool compounds to study this channel further.

The active form of quinol-dependent nitric oxide reductase from Neisseria meningitidis is a dimer.

Neisseria meningitidis is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from N. meningitidis (NmqNOR) reported here at 3.15 Šresolution, show monomeric assemblies, despite the more active dimeric sample being used for crystallization. Cryo-electron microscopic analysis of the same chromatographic fraction of NmqNOR, however, revealed a dimeric assembly at 3.06 Šresolution. It is shown that zinc (which is used in crystallization) binding near the dimer-stabilizing TMII region contributes to the disruption of the dimer. A similar destabilization is observed in the monomeric (∼85 kDa) cryo-EM structure of a mutant (Glu494Ala) qNOR from the opportunistic pathogen Alcaligenes (Achromobacter) xylosoxidans, which primarily migrates as a monomer. The monomer-dimer transition of qNORs seen in the cryo-EM and crystallographic structures has wider implications for structural studies of multimeric membrane proteins. X-ray crystallographic and cryo-EM structural analyses have been performed on the same chromatographic fraction of NmqNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly in crystallo and a dimeric assembly in vitrified cryo-EM grids. A number of factors have been identified that may trigger the destabilization of helices that are necessary to preserve the integrity of the dimer. These include zinc binding near the entry of the putative proton-transfer channel and the preservation of the conformational integrity of the active site. The mutation near the active site results in disruption of the active site, causing an additional destabilization of helices (TMIX and TMX) that flank the proton-transfer channel helices, creating an inert monomeric enzyme.

Metallotexaphyrins as MRI-Active Catalytic Antioxidants for Neurodegenerative Disease: A Study on Alzheimer's Disease.

The complex etiology of neurodegeneration continues to stifle efforts to develop effective therapeutics. New agents elucidating key pathways causing neurodegeneration might serve to increase our understanding and potentially lead to improved treatments. Here, we demonstrate that a water-soluble manganese(II) texaphyrin (MMn) is a suitable magnetic resonance imaging (MRI) contrast agent for detecting larger amyloid beta constructs. The imaging potential of MMn was inferred on the basis of in vitro studies and in vivo detection in Alzheimer's disease C. elegans models via MRI and ICP-MS. In vitro antioxidant- and cellular-based assays provide support for the notion that this porphyrin analog shows promise as a therapeutic agent able to mitigate the oxidative and nitrative toxic effects considered causal in neurodegeneration. The present report marks the first elaboration of an MRI-active metalloantioxidant that confers diagnostic and therapeutic benefit in Alzheimer's disease models without conjugation of a radioisotope, targeting moiety, or therapeutic payload.

Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo-electron microscopy.

Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone destroying gas nitrous oxide (N2O). Cryo-electron microscopy structures of active qNOR from Alcaligenes xylosoxidans and an activity-enhancing mutant have been determined to be at local resolutions of 3.7 and 3.2 Å, respectively. They unexpectedly reveal a dimeric conformation (also confirmed for qNOR from Neisseria meningitidis) and define the active-site configuration, with a clear water channel from the cytoplasm. Structure-based mutagenesis has identified key residues involved in proton transport and substrate delivery to the active site of qNORs. The proton supply direction differs from cytochrome c-dependent NOR (cNOR), where water molecules from the cytoplasm serve as a proton source similar to those from cytochrome c oxidase.