Elucidating the structural basis for differing enzyme inhibitor potency by cryo-EM

Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both Saccharomyces cerevisiae (Sc_IGPD) and Arabidopsis thaliana (At_IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies. The structure–activity relationship of potent triazole-phosphonate inhibitors of IGPD has been determined in both homologs, revealing that the lead inhibitor (C348) is an order of magnitude more potent against Sc_IGPD than At_IGPD; however, the molecular basis of this difference has not been established. Here we have used single-particle electron microscopy (EM) to study structural differences between the At and Sc_IGPD homologs, which could influence the difference in inhibitor potency. The resulting EM maps at ∼3 Å are sufficient to de novo build the protein structure and identify the inhibitor binding site, which has been validated against the crystal structure of the At_IGPD/C348 complex. The structure of Sc_IGPD reveals that a 24-amino acid insertion forms an extended loop region on the enzyme surface that lies adjacent to the active site, forming interactions with the substrate/inhibitor binding loop that may influence inhibitor potency. Overall, this study provides insights into the IGPD family and demonstrates the power of using an EM approach to study inhibitor binding

Development of a triclosan scaffold which allows for adaptations on both the A- and B-ring for transport peptides

The enoyl acyl-carrier protein reductase (ENR) enzyme is harbored within the apicoplast of apicomplexan parasites providing a significant challenge for drug delivery, which may be overcome through the addition of transductive peptides, which facilitates crossing the apicoplast membranes. The binding site of triclosan, a potent ENR inhibitor, is occluded from the solvent making the attachment of these linkers challenging. Herein, we have produced 3 new triclosan analogs with bulky A- and B-ring motifs, which protrude into the solvent allowing for the future attachment of molecular transporters for delivery

From TgO/GABA-AT, GABA, and T-263 mutant to conception of Toxoplasma

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat’s oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with “Rosetta stone”-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite’s capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease

New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections

Toxoplasma gondii, the most common parasitic infection of human brain and eye, persists across lifetimes, can progressively damage sight, and is currently incurable. New, curative medicines are needed urgently. Herein, we develop novel models to facilitate drug development: EGS strain T. gondii forms cysts in vitro that induce oocysts in cats, the gold standard criterion for cysts. These cysts highly express cytochrome b. Using these models, we envisioned, and then created, novel 4-(1H)-quinolone scaffolds that target the cytochrome bc₁ complex Qi site, of which, a substituted 5,6,7,8-tetrahydroquinolin-4-one inhibits active infection (IC₅₀, 30 nM) and cysts (IC₅₀, 4 μM) in vitro, and in vivo (25 mg/kg), and drug resistant Plasmodium falciparum (IC₅₀, <30 nM), with clinically relevant synergy. Mutant yeast and co-crystallographic studies demonstrate binding to the bc₁ complex Q[subscript]i site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites

Design, synthesis and biological evaluation of new bacterial RNAP inhibitors

Bacterial resistance to antibiotics has been well documented since the emergence of resistance to the beta-lactams during the 1930s. The World Health Organisation considers it to be one of the three greatest threats to human health. Since the late 1980s, the pharmaceutical industry has relied upon the synthesis of analogues based on established classes to combat resistance. However, resistance mechanisms for these classes quickly evolve to render these derivatives ineffective, offering only short-term solutions. There is, therefore, an urgent need for the identification of new antibacterial scaffolds, as well as novel bacterial targets, which do not share this cross-resistance. Structure-based drug design is a rational approach to drug discovery which uses protein structures and computational simulations to guide the design process and deliver lead molecules with greater efficiency. SPROUT is a de novo molecular design program for the generation of small molecule inhibitors, developed within our research group at the University of Leeds. Recent in silica efforts to design novel inhibitors of RNA polymerase (RNAP), an established antibacterial drug target, have focussed on the newly identified myxopyronin B (MyxB) binding region. In conjunction with the X-ray co-crystal structure of Thermus thermophilus RNAP-MyxB, SPROUT has been used to deSign putative ligands, and analogues of MyxB, for the MyxB region. Several series of molecules have been prepared and a number of these compounds displayed IC50 values between 5 and 10 uM against Escherichia coli RNAP. In parallel, a drug delivery approach has been explored to circumvent the problem of enzyme inhibitors that lack cell membrane penetration. Expanding our focus to include scaffolds that inhibit other antibacterial targets, we have conjugated 'warheads' to bacterial iron-transporters called siderophores to enhance their antibacterial activity against Staphylococcus aureus (64 ug/ml), compared to that displayed by the parent scaffold (256 ug/ml). Combining this delivery approach with structure based molecular design represents a new strategy for the generation of novel antibacterial scaffolds, tackling the fundamental problem of poor success in discovering new antibacterial agents over the last 30 years.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Developments in Non-Intercalating Bacterial Topoisomerase Inhibitors: Allosteric and ATPase Inhibitors of DNA Gyrase and Topoisomerase IV

Increases in antibiotic usage and antimicrobial resistance occurrence have caused a dramatic reduction in the effectiveness of many frontline antimicrobial treatments. Topoisomerase inhibitors including fluoroquinolones are broad-spectrum antibiotics used to treat a range of infections, which stabilise a topoisomerase-DNA cleavage complex via intercalation of the bound DNA. However, these are subject to bacterial resistance, predominantly in the form of single-nucleotide polymorphisms in the active site. Significant research has been undertaken searching for novel bioactive molecules capable of inhibiting bacterial topoisomerases at sites distal to the fluoroquinolone binding site. Notably, researchers have undertaken searches for anti-infective agents that can inhibit topoisomerases through alternate mechanisms. This review summarises work looking at the inhibition of topoisomerases predominantly through non-intercalating agents, including those acting at a novel allosteric site, ATPase domain inhibitors, and those offering unique binding modes and mechanisms of action

Functional Characterization of the γ-Aminobutyric Acid Transporter from Mycobacterium smegmatis MC2 155 Reveals Sodium-Driven GABA Transport

Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential gamma-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis. The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported gamma-aminobutyric acid both in vitro and when overexpressed in E. coli. Additionally, transport was greatly reduced in the presence of beta-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that gamma-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that gamma-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active gamma-aminobutyric acid transport protein from mycobacteria. IMPORTANCE The spread of multidrug-resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species. Identifying new mycobacterial transporters will enhance our understanding of mycobacterial physiology and, furthermore, provides new drug targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates it is a sodium-dependent GABA transporter that may also transport beta-alanine. As GABA may well be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive target for the development of new drugs against tuberculosis.Peer reviewe

De Novo Design of Type II Topoisomerase Inhibitors as Potential Antimicrobial Agents Targeting a Novel Binding Region

By 2050 it is predicted that antimicrobial resistance will be responsible for 10 million global deaths annually, costing the world economy $100 trillion. Clearly, strategies to address this problem are required as bacterial evolution is rendering our current antibiotics ineffective. The discovery of an allosteric binding site on the established antibacterial target DNA gyrase offers a new medicinal chemistry strategy, as this site is distinct from the fluoroquinolone-DNA site binding site. Using in silico molecular design methods, we have designed and synthesised a novel series of biphenyl-based inhibitors inspired by the published thiophene allosteric inhibitor. This series was evaluated in vitro against E. coli DNA gyrase, exhibiting IC50 values in the low micromolar range. The structure-activity relationship reported herein suggests insights to further exploit this allosteric site, offering a pathway to overcome fluoroquinolone resistance

Activity of and Development of Resistance to Corallopyronin A, an Inhibitor of RNA Polymerase▿

We explored the properties of corallopyronin A (CorA), a poorly characterized inhibitor of bacterial RNA polymerase (RNAP). It displayed a 50% inhibitory concentration of 0.73 μM against RNAP, compared with 11.5 nM for rifampin. The antibacterial activity of CorA was also inferior to rifampin, and resistant mutants of Staphylococcus aureus were easily selected. The mutations conferring resistance resided in the rpoB and rpoC subunits of RNAP. We conclude that CorA is not a promising antibacterial drug candidate