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1.
Haematologica ; 106(6): 1616-1623, 2021 06 01.
Article in English | MEDLINE | ID: mdl-32354869

ABSTRACT

Complement C3 binds fibrinogen and compromises fibrin clot lysis thereby enhancing thrombosis risk. We investigated the role of fibrinogen-C3 interaction as a novel therapeutic target to reduce thrombosis risk by analysing: i) consistency in the fibrinolytic properties of C3, ii) binding sites between fibrinogen and C3 and iii) modulation of fibrin clot lysis by manipulating fibrinogen-C3 interactions. Purified fibrinogen and C3 from the same individuals (n=24) were used to assess inter-individual variability in the anti-fibrinolytic effects of C3. Microarray screening and molecular modelling evaluated C3 and fibrinogen interaction sites. Novel synthetic conformational proteins, termed Affimers, were used to modulate C3-fibrinogen interaction and fibrinolysis. C3 purified from patients with type 1 diabetes showed enhanced prolongation of fibrinolysis compared with healthy control protein [195±105 and 522±166 seconds, respectively (p=0.04)], with consistent effects but a wider range (5-51% and 5-18% lysis prolongation, respectively). Peptide microarray screening identified 2 potential C3-fibrinogen interactions sites within fibrinogen ß chain (residues 424-433, 435-445). One fibrinogen-binding Affimer was isolated that displayed sequence identity with C3 in an exposed area of the protein. This Affimer abolished C3-induced prolongation of fibrinolysis (728±25.1 seconds to 632±23.7 seconds, p=0.005) and showed binding to fibrinogen in the same region that is involved in C3-fibrinogen interactions. Moreover, it shortened plasma clot lysis of patients with diabetes, cardiovascular disease or controls by 7-11%. C3 binds fibrinogen ß-chain and disruption of fibrinogen-C3 interaction using Affimer proteins enhances fibrinolysis, which represents a potential novel target tool to reduce thrombosis in high risk individuals.


Subject(s)
Fibrinogen , Thrombosis , Complement C3 , Fibrin , Fibrinolysis , Humans , Thrombosis/drug therapy , Thrombosis/etiology , Thrombosis/prevention & control
2.
Proc Natl Acad Sci U S A ; 115(1): E72-E81, 2018 01 02.
Article in English | MEDLINE | ID: mdl-29247053

ABSTRACT

Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.


Subject(s)
Antigen-Antibody Complex/chemistry , Immunoglobulin G/chemistry , Molecular Dynamics Simulation , Receptors, IgG/chemistry , Allosteric Regulation , Antigen-Antibody Complex/immunology , Humans , Immunoglobulin G/immunology , Receptors, IgG/immunology
3.
Bioorg Med Chem ; 27(16): 3546-3550, 2019 08 15.
Article in English | MEDLINE | ID: mdl-31257079

ABSTRACT

Previously we have reported on a series of pyridine-3-carboxamide inhibitors of DNA gyrase and DNA topoisomerase IV that were designed using a computational de novo design approach and which showed promising antibacterial properties. Herein we describe the synthesis of additional examples from this series aimed specifically at DNA gyrase, along with crystal structures confirming the predicted mode of binding and in vitro ADME data which describe the drug-likeness of these compounds.


Subject(s)
DNA Gyrase/genetics , Escherichia coli/metabolism , Topoisomerase II Inhibitors/therapeutic use , Models, Molecular , Structure-Activity Relationship , Topoisomerase II Inhibitors/pharmacology
4.
Mol Microbiol ; 106(3): 492-504, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28876489

ABSTRACT

Clavulanic acid and avibactam are clinically deployed serine ß-lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo ß-lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine ß-lactamase) but not L1 (metallo ß-lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X-ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam-like ß-lactams plus nonclassical ß-lactamase inhibitors, particularly avibactam-like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.


Subject(s)
Stenotrophomonas maltophilia/metabolism , beta-Lactamase Inhibitors/metabolism , Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/metabolism , Azabicyclo Compounds/pharmacology , Aztreonam , Bacterial Proteins/metabolism , Ceftazidime , Crystallography, X-Ray/methods , Drug Resistance, Bacterial , Microbial Sensitivity Tests , beta-Lactamase Inhibitors/chemistry , beta-Lactamases/genetics , beta-Lactamases/metabolism
5.
EMBO J ; 33(16): 1831-44, 2014 Aug 18.
Article in English | MEDLINE | ID: mdl-24952894

ABSTRACT

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Binding Sites , Biological Transport , Crystallography, X-Ray , Hydantoins/metabolism , Hydrogen Bonding , Ligands , Micrococcaceae/chemistry , Models, Molecular , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Mutation , Protein Conformation , Structure-Activity Relationship
6.
Article in English | MEDLINE | ID: mdl-28115348

ABSTRACT

ß-Lactamase-mediated resistance is a growing threat to the continued use of ß-lactam antibiotics. The use of the ß-lactam-based serine-ß-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-ß-lactam inhibitor avibactam has extended the utility of ß-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-ß-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-ß-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of "dual-action" inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-ß-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of ß-lactamase, including the class A extended spectrum ß-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of ß-lactams against Gram-negative clinical isolates expressing a variety of ß-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other ß-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-ß-lactamase catalysis.


Subject(s)
Anti-Bacterial Agents/pharmacology , Boronic Acids/pharmacology , Enterobacteriaceae/drug effects , beta-Lactam Resistance/drug effects , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/chemistry , Amino Acid Motifs , Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Boronic Acids/chemical synthesis , Cloning, Molecular , Crystallography, X-Ray , Cyclization , Enterobacteriaceae/enzymology , Enterobacteriaceae/genetics , Enterobacteriaceae/growth & development , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Kinetics , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Thermodynamics , beta-Lactam Resistance/genetics , beta-Lactamase Inhibitors/chemical synthesis , beta-Lactamases/genetics , beta-Lactamases/metabolism , beta-Lactams/pharmacology
7.
Bioorg Med Chem Lett ; 27(16): 3878-3882, 2017 08 15.
Article in English | MEDLINE | ID: mdl-28669445

ABSTRACT

Two new tricyclic ß-aminoacrylate derivatives (2e and 3e) have been found to be inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) with Ki 0.037 and 0.15µM respectively. 1H and 13C NMR spectroscopic data show that these compounds undergo ready cis-trans isomerisation at room temperature in polar solvents. In silico docking studies indicate that for both molecules there is neither conformation nor double bond configuration which bind preferentially to PfDHODH. This flexibility is favourable for inhibitors of this channel that require extensive positioning to reach their binding site.


Subject(s)
Acrylates/pharmacology , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Plasmodium falciparum/enzymology , Acrylates/chemical synthesis , Acrylates/chemistry , Dihydroorotate Dehydrogenase , Dose-Response Relationship, Drug , Molecular Docking Simulation , Molecular Structure , Oxidoreductases Acting on CH-CH Group Donors/genetics , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Structure-Activity Relationship
8.
Nat Chem Biol ; 10(6): 457-62, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24776929

ABSTRACT

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder ß-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.


Subject(s)
Cyclic AMP/metabolism , Cyclic GMP/analogs & derivatives , Dinucleoside Phosphates/metabolism , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Ion Channel Gating/physiology , Muscle Proteins/metabolism , Potassium Channels/metabolism , Animals , Binding Sites , Blotting, Western , Crystallography, X-Ray , Cyclic GMP/chemistry , Cyclic GMP/metabolism , Dinucleoside Phosphates/chemistry , HEK293 Cells , High-Throughput Screening Assays , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics , Ion Channel Gating/drug effects , Ligands , Mice , Mice, Inbred C57BL , Molecular Docking Simulation , Molecular Structure , Muscle Proteins/genetics , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Potassium Channels/genetics , Sinoatrial Node/cytology , Sinoatrial Node/drug effects , Sinoatrial Node/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Transfection
9.
Orig Life Evol Biosph ; 46(4): 425-434, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27220497

ABSTRACT

We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H2P2O52-; PPi(III)], to the phosphate group of 5'-adenosine mono phosphate (5'-AMP). We show further that this P-transfer process is accelerated both by divalent metal ions (M2+) and by organic co-factors such as acetate (AcO-). In this specific case of P-transfer from PPi(III) to 5'-AMP, we show a synergistic enhancement of transfer in the combined presence of M2+ & AcO-. Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5'-AMP, [P(III)P(V)-5'-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus.


Subject(s)
Adenosine Monophosphate/chemistry , Cations, Divalent/chemistry , Origin of Life , Phosphorus Compounds/chemistry , Metals
10.
Blood ; 121(11): 2117-26, 2013 Mar 14.
Article in English | MEDLINE | ID: mdl-23303819

ABSTRACT

Formation of a stable fibrin clot is dependent on interactions between factor XIII and fibrin. We have previously identified a key residue on the αC of fibrin(ogen) (Glu396) involved in binding activated factor XIII-A(2) (FXIII-A(2)*); however, the functional role of this interaction and binding site(s) on FXIII-A(2)* remains unknown. Here we (1) characterized the functional implications of this interaction; (2) identified by liquid-chromatography-tandem mass spectrometry the interacting residues on FXIII-A(2)* following chemical cross-linking of fibrin(ogen) αC389-402 peptides to FXIII-A(2)*; and (3) carried out molecular modeling of the FXIII-A(2)*/peptide complex to identify contact site(s) involved. Results demonstrated that inhibition of the FXIII-A(2)*/αC interaction using αC389-402 peptide (Pep1) significantly decreased incorporation of biotinamido-pentylamine and α2-antiplasmin to fibrin, and fibrin cross-linking, in contrast to Pep1-E396A and scrambled peptide controls. Pep1 did not inhibit transglutaminase-2 activity, and incorporation of biotinyl-TVQQEL to fibrin was only weakly inhibited. Molecular modeling predicted that Pep1 binds the activation peptide cleft (AP-cleft) within the ß-sandwich domain of FXIII-A(2)* localizing αC cross-linking Q366 to the FXIII-A(2)* active site. Our findings demonstrate that binding of fibrin αC389-402 to the AP-cleft is fundamental to clot stabilization and presents this region of FXIII-A(2)* as a potential site involved in glutamine-donor substrate recognition.


Subject(s)
Catalytic Domain , Factor XIII/chemistry , Factor XIII/metabolism , Fibrinogen/metabolism , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Thrombin/metabolism , Animals , CHO Cells , Catalytic Domain/genetics , Cricetinae , Cricetulus , Fibrinogen/chemistry , Models, Molecular , Peptide Fragments/chemistry , Protein Binding/physiology , Protein Interaction Domains and Motifs/physiology , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Structure, Quaternary
11.
Org Biomol Chem ; 12(3): 486-94, 2014 Jan 21.
Article in English | MEDLINE | ID: mdl-24276506

ABSTRACT

An approach for designing bioactive small molecules has been developed in which de novo structure-based ligand design (SBLD) was focused on regions of chemical space accessible using a diversity-oriented synthetic approach. The approach was exploited in the design and synthesis of a focused library of platensimycin analogues in which the complex bridged ring system was replaced with a series of alternative ring systems. The affinity of the resulting compounds for the C163Q mutant of FabF was determined using a WaterLOGSY competition binding assay. Several compounds had significantly improved affinity for the protein relative to a reference ligand. The integration of synthetic accessibility with ligand design enabled focus to be placed on synthetically-accessible regions of chemical space that were relevant to the target protein under investigation.


Subject(s)
Acetyltransferases/antagonists & inhibitors , Adamantane/pharmacology , Aminobenzoates/pharmacology , Anilides/pharmacology , Drug Discovery , Enzyme Inhibitors/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Acetyltransferases/genetics , Acetyltransferases/metabolism , Adamantane/chemical synthesis , Adamantane/chemistry , Aminobenzoates/chemical synthesis , Aminobenzoates/chemistry , Anilides/chemical synthesis , Anilides/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Fatty Acid Synthase, Type II/antagonists & inhibitors , Fatty Acid Synthase, Type II/genetics , Fatty Acid Synthase, Type II/metabolism , Ligands , Models, Molecular , Molecular Conformation , Structure-Activity Relationship
12.
Parasitology ; 141(1): 17-27, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23768800

ABSTRACT

In light of the low success rate of target-based genomics and HTS (High Throughput Screening) approaches in anti-infective drug discovery, in silico structure-based drug design (SBDD) is becoming increasingly prominent at the forefront of drug discovery. In silico SBDD can be used to identify novel enzyme inhibitors rapidly, where the strength of this approach lies with its ability to model and predict the outcome of protein-ligand binding. Over the past 10 years, our group have applied this approach to a diverse number of anti-infective drug targets ranging from bacterial D-ala-D-ala ligase to Plasmodium falciparum DHODH. Our search for new inhibitors has produced lead compounds with both enzyme and whole-cell activity with established on-target mode of action. This has been achieved with greater speed and efficiency compared with the more traditional HTS initiatives and at significantly reduced cost and manpower.


Subject(s)
Anti-Infective Agents/chemistry , Drug Discovery , Enzyme Inhibitors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Peptide Synthases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Anti-Infective Agents/pharmacology , Bacterial Infections/drug therapy , Bacterial Infections/microbiology , Dihydroorotate Dehydrogenase , Drug Design , Enzyme Inhibitors/pharmacology , High-Throughput Screening Assays/economics , High-Throughput Screening Assays/statistics & numerical data , Humans , Ligands , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Molecular Docking Simulation , Oxidoreductases Acting on CH-CH Group Donors/genetics , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Peptide Synthases/genetics , Peptide Synthases/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , User-Computer Interface
13.
Bioorg Chem ; 55: 69-76, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24962384

ABSTRACT

In recent years bacterial resistance has been observed against many of our current antibiotics, for instance most worryingly against the cephalosporins which are typically the last line of defence against many bacterial infections. Additionally the failure of high throughput screening in the discovery of new antibacterial drug leads has led to a decline in the number of antibacterial agents reaching the market. Alternative methods of drug discovery including structure based drug design are needed to meet the threats caused by the emergence of resistance. In this review we explore the latest advancements in the identification of new antibacterial agents through the use of a number of structure based drug design programs.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Bacterial Infections/drug therapy , Drug Design , Drug Discovery , Animals , Humans , Structure-Activity Relationship
14.
Mol Membr Biol ; 30(2): 184-94, 2013 Mar.
Article in English | MEDLINE | ID: mdl-22908980

ABSTRACT

Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the elucidation of over 250 unique membrane protein crystal structures. The aim of the European Drug Initiative for Channels and Transporter (EDICT) project is to use the structures of clinically significant membrane proteins for the development of lead molecules. One of the approaches used to achieve this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family.


Subject(s)
Amino Acid Transport Systems/antagonists & inhibitors , Bacterial Proteins/antagonists & inhibitors , High-Throughput Screening Assays/methods , Leucine/metabolism , Membrane Transport Proteins/metabolism , Amino Acid Transport Systems/metabolism , Bacterial Proteins/metabolism , Binding Sites , Biological Transport , Crystallography, X-Ray , Plasma Membrane Neurotransmitter Transport Proteins/metabolism
15.
Growth Horm IGF Res ; 77: 101607, 2024 Jul 18.
Article in English | MEDLINE | ID: mdl-39033666

ABSTRACT

Type 2 diabetes is characterised by the disruption of insulin and insulin-like growth factor (IGF) signalling. The key hubs of these signalling cascades - the Insulin receptor (IR) and Insulin-like growth factor 1 receptor (IGF1R) - are known to form functional IR-IGF1R hybrid receptors which are insulin resistant. However, the mechanisms underpinning IR-IGF1R hybrid formation are not fully understood, hindering the ability to modulate this for future therapies targeting this receptor. To pinpoint suitable sites for intervention, computational hotspot prediction was utilised to identify promising epitopes for targeting with point mutagenesis. Specific IGF1R point mutations F450A, R391A and D555A show reduced affinity of the hybrid receptor in a BRET based donor-saturation assay, confirming hybrid formation could be modulated at this interface. These data provide the basis for rational design of more effective hybrid receptor modulators, supporting the prospect of identifying a small molecule that specifically interacts with this target.

16.
iScience ; 27(1): 108477, 2024 Jan 19.
Article in English | MEDLINE | ID: mdl-38205261

ABSTRACT

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.

17.
Pharmaceuticals (Basel) ; 16(2)2023 Feb 08.
Article in English | MEDLINE | ID: mdl-37259406

ABSTRACT

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.

18.
RSC Med Chem ; 13(4): 360-374, 2022 Apr 20.
Article in English | MEDLINE | ID: mdl-35647546

ABSTRACT

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are dimeric disulfide-linked receptor tyrosine kinases, whose actions regulate metabolic and mitogenic signalling pathways inside the cell. It is well documented that in tissues co-expressing the IR and IGF1R, their respective monomers can heterodimerise to form IR-IGF1R hybrid receptors. Increased populations of the IR-IGF1R hybrid receptors are associated with several disease states, including type 2 diabetes and cancer. Recently, progress in the structural biology of IR and IGF1R has given insights into their structure-function relationships and mechanism of action. However, challenges in isolating IR-IGF1R hybrid receptors mean that their structural properties remain relatively unexplored. This review discusses the advances in the structural understanding of the IR and IGF1R, and how these discoveries can inform the design of small-molecule modulators of the IR-IGF1R hybrid receptors to understand their role in cell biology.

19.
J Med Chem ; 65(2): 1481-1504, 2022 01 27.
Article in English | MEDLINE | ID: mdl-34780700

ABSTRACT

Fibroblast growth factor receptors (FGFRs) are implicated in a range of cancers with several pan-kinase and selective-FGFR inhibitors currently being evaluated in clinical trials. Pan-FGFR inhibitors often cause toxic side effects and few examples of subtype-selective inhibitors exist. Herein, we describe a structure-guided approach toward the development of a selective FGFR2 inhibitor. De novo design was carried out on an existing fragment series to yield compounds predicted to improve potency against the FGFRs. Subsequent iterative rounds of synthesis and biological evaluation led to an inhibitor with nanomolar potency that exhibited moderate selectivity for FGFR2 over FGFR1/3. Subtle changes to the lead inhibitor resulted in a complete loss of selectivity for FGFR2. X-ray crystallographic studies revealed inhibitor-specific morphological differences in the P-loop which were posited to be fundamental to the selectivity of these compounds. Additional docking studies have predicted an FGFR2-selective H-bond which could be utilized to design more selective FGFR2 inhibitors.


Subject(s)
Drug Design , Drug Development , Protein Kinase Inhibitors/pharmacology , Receptor, Fibroblast Growth Factor, Type 2/antagonists & inhibitors , Urinary Bladder Neoplasms/drug therapy , Cell Proliferation , Humans , Phosphorylation , Structure-Activity Relationship , Tumor Cells, Cultured , Urinary Bladder Neoplasms/enzymology
20.
RSC Med Chem ; 13(7): 831-839, 2022 Jul 20.
Article in English | MEDLINE | ID: mdl-35919336

ABSTRACT

By 2050, it is predicted that antimicrobial resistance will be responsible for 10 million global deaths annually, more deaths than cancer, costing the world economy $100 trillion. Clearly, strategies to address this problem are essential 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 binding site, resistance is not yet documented. Using in silico molecular design methods, we have designed and synthesised a novel series of biphenyl-based inhibitors inspired by a published thiophene-based allosteric inhibitor. This series was evaluated in vitro against Escherichia coli DNA gyrase and E. coli topoisomerase IV with the most potent compounds exhibiting IC50 values towards the low micromolar range for DNA gyrase and only ∼2-fold less active against topoisomerase IV. The structure-activity relationships reported herein suggest insights to further exploit this allosteric site, offering a pathway to overcome developing fluoroquinolone resistance.

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