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A computational design, make and test strategy was used to identify antibacterial inhibitors of bacterial DNA gyrase and topoisomerase IV, proposed to bind at a novel allosteric site.

Antibiotic-resistant bacteria represent a serious threat to modern medicine and human life. Only a minority of antibacterial agents are active against Gram-negative bacteria. Hence, the development of novel antimicrobial agents will always be a vital need. In an effort to discover new therapeutics against Gram-negative bacteria, we previously reported a structure-activity-relationship (SAR) study on 1,2-disubstituted benzimidazole derivatives. Compound III showed a potent activity against tolC-mutant Escherichia coli with an MIC value of 2 μg/mL, representing a promising lead for further optimization. Building upon this study, herein, 49 novel benzimidazole compounds were synthesized to investigate their antibacterial activity against Gram-negative bacteria. Our design focused on three main goals, to address the low permeability of our compounds and improve their cellular accumulation, to expand the SAR study to the unexplored ring C, and to optimize the lead compound (III) by modification of the methanesulfonamide moiety. Compounds (25a-d, 25f-h, 25k, 25l, 25p, 25r, 25s, and 26b) exhibited potent activity against tolC-mutant E. coli with MIC values ranging from 0.125 to 4 μg/mL, with compound 25d displaying the highest potency among the tested compounds with an MIC value of 0.125 μg/mL. As its predecessor, III, compound 25d exhibited an excellent safety profile without any significant cytotoxicity to mammalian cells. Time-kill kinetics assay indicated that 25d exhibited a bacteriostatic activity and significantly reduced E. coli JW55031 burden as compared to DMSO. Additionally, combination of 25d with colistin partially restored its antibacterial activity against Gram-negative bacterial strains (MIC values ranging from 4 to 16 μg/mL against E. coli BW25113, K. pneumoniae, A. baumannii, and P. aeruginosa). Furthermore, formulation of III and 25d as lipidic nanoparticles (nanocapsules) resulted in moderate enhancement of their antibacterial activity against Gram-negative bacterial strains (A. Baumannii, N. gonorrhoeae) and compound 25d demonstrated superior activity to the lead compound III. These findings establish compound 25d as a promising candidate for treatment of Gram-negative bacterial infections and emphasize the potential of nano-formulations in overcoming poor cellular accumulation in Gram-negative bacteria where further optimization and investigation are warranted to improve the potency and broaden the spectrum of our compounds.

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.

The insulin receptor and insulin like growth factor-1 receptor are heterodimers consisting of two extracellular α-subunits and two transmembrane β-subunits. IR αβ and IGF1R αβ hemi-receptors can heterodimerize to form hybrids composed of one IR αβ and one IGF1R αβ. Widely distributed in mammalian tissues, in contrast to IR and IGF1R the physiological function of hybrids is unclear. To identify tool compounds that inhibit hybrid formation we performed a high-throughput small molecule screen based on a homology model of hybrid structure. Our studies unveil a first in class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells with no effect on IR or IGF1R expression. Downstream of IR and IGF1R our small molecule led to reduced expression of the negative regulatory p85α subunit of phosphatidylinositol 3-kinase, an increase in phosphorylation of its downstream target Akt and enhanced insulin and shear-induced phosphorylation of Akt. We show that hybrids have a role in human endothelial cell physiology distinct from IR and IGF1R.

FMS-like tyrosine kinase 3 (FLT3) enzyme overexpression and mutations are the most common molecular abnormalities associated with acute myeloid leukemia (AML). In addition, recent studies investigated the role of tropomyosin receptor kinase A (TrKA) enzyme fusions in promoting AML growth and survival. Based on these premises, targeting both kinases using dual inhibitors would constitute a promising therapeutic approach to target resistant AML. Guided by ligand-based design and structure simplification of the FLT3 inhibitor, quizartinib, we developed a benzimidazole-based small molecule, 4ACP, that exhibited nanomolar activity against wild-type FLT3, FLT3-Internal tandem duplications (FLT3-ITD), and FLT3-D835Y (FLT3-TKD) mutation (IC

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.

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., 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 γ-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 γ-aminobutyric acid both in vitro and when overexpressed in E. coli. Additionally, transport was greatly reduced in the presence of β-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that γ-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that γ-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 γ-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 β-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.

Aurora B is a pivotal cell cycle regulator where errors in its function results in polyploidy, genetic instability, and tumorigenesis. It is overexpressed in many cancers, consequently, targeting Aurora B with small molecule inhibitors constitutes a promising approach for anticancer therapy. Guided by structure-based design and molecular hybridization approach we developed a series of fifteen indolin-2-one derivatives based on a previously reported indolin-2-one-based multikinase inhibitor (1). Seven derivatives, 5g, 6a, 6c-e, 7, and 8a showed preferential antiproliferative activity in NCI-60 cell line screening and out of these, carbamate 6e and cyclopropylurea 8a derivatives showed optimum activity against Aurora B (IC50 = 16.2 and 10.5 nM respectively) and MDA-MB-468 cells (IC50 = 32.6 ± 9.9 and 29.1 ± 7.3 nM respectively). Furthermore, 6e and 8a impaired the clonogenic potential of MDA-MB-468 cells. Mechanistic investigations indicated that 6e and 8a induced G2/M cell cycle arrest, apoptosis, and necrosis of MDA-MB-468 cells and western blot analysis of 8a effect on MDA-MB-468 cells revealed 8a‘s ability to reduce Aurora B and its downstream target, Histone H3 phosphorylation. 6e and 8a displayed better safety profiles than multikinase inhibitors such as sunitinib, showing no cytotoxic effects on normal rat cardiomyoblasts and murine hepatocytes. Finally, 8a demonstrated a more selective profile than 1 when screened against ten related kinases. Based on these findings, 8a represents a promising candidate for further development to target breast cancer via Aurora B selective inhibition.

Recent developments in electron microscopy have provided new opportunities in the field of structure-based drug design. This review looks at the challenges that remain and the future prospects for the use of electron microscopy in this area., Recent developments in electron microscopy (EM) have led to a step change in our ability to solve the structures of previously intractable systems, especially membrane proteins and large protein complexes. This has provided new opportunities in the field of structure-based drug design, with a number of high-profile publications resolving the binding sites of small molecules and peptide inhibitors. There are a number of advantages of EM over the more traditional X-ray crystallographic approach, such as resolving different conformational states and permitting the dynamics of a system to be better resolved when not constrained by a crystal lattice. There are still significant challenges to be overcome using an EM approach, not least the speed of structure determination, difficulties with low-occupancy ligands and the modest resolution that is available. However, with the anticipated developments in the field of EM, the potential of EM to become a key tool for structure-based drug design, often complementing X-ray and NMR studies, seems promising.

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 Qi site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites.

Made available in DSpace on 2020-12-10T17:35:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Royal Society - Newton Advanced Fellowship Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Wellcome Trust Chikungunya virus (CHIKV) is a mosquito-transmitted virus of special concern as it causes Chikungunya fever, characterized by an acute febrile illness, rash, and arthralgia that can progress to chronic and debilitating arthritic symptoms. The effects of climate change on the geographic distribution of the mosquito vector has the potential to expose more of the globe to this virus. No antiviral agents or vaccines are currently available against CHIKV infection and the development of novel therapies that may lead to a future treatment is therefore necessary. In this context, the ADP-ribose binding site of the CHIKV nsP3 macro domain has been reported as a potential target for the development of antivirals. Mutations in the ADP-ribose binding site demonstrated decreased viral replication in cell culture and reduced virulence. In this study, 48,750 small molecules were screened in silico for their ability to bind to the ADP-ribose binding site of the CHIKV nsP3 macro domain. From this in silico analysis, 12 molecules were selected for in vitro analysis using a CHIKV subgenomic replicon in Huh-7 cells. Cell viability and CHIKV replication were evaluated and molecules C5 and C13 demonstrated 53 and 66% inhibition of CHIKV replication, respectively. By using a CHIKV-Dual luciferase replicon contain two reporter genes, we also demonstrated that the treatment with either compounds are probably interfering in the early replication rather than after RNA replication has occurred. Sao Paulo State Univ, IBILCE, Sao Jose Do Rio Preto, SP, Brazil Univ Fed Uberlandia, Inst Biomed Sci, Lab Virol, ICBIM, Uberlandia, MG, Brazil Univ Leeds, Fac Engn & Phys Sci, Sch Chem, Leeds LS2 9JT, W Yorkshire, England Univ Leeds, Astbury Ctr Struct Mol Biol, Leeds LS2 9JT, W Yorkshire, England Univ Leeds, Fac Biol Sci, Sch Mol & Cellular Biol, Leeds LS2 9JT, W Yorkshire, England Univ Tartu, Inst Technol, Nooruse 1, EE-50411 Tartu, Estonia Sao Paulo State Univ, IBILCE, Sao Jose Do Rio Preto, SP, Brazil Royal Society - Newton Advanced Fellowship: NA 150195 CNPq: 445021/2014-4 FAPEMIG: APQ-00587-14 FAPEMIG: SICONV 793988/2013 CAPES: 001 Wellcome Trust: 096670

The transient receptor potential ankyrin 1 (TRPA1) channel is a non-selective cation channel, which detects noxious stimuli leading to pain, itch and cough. However, the mechanism(s) of channel modulation by many of the known, non-reactive modulators has not been fully elucidated. N-cinnamoylanthranilic acid derivatives (CADs) contain structural elements from the TRPA1 modulators cinnamaldehyde and flufenamic acid, so it was hypothesized that specific modulators could be found amongst them and more could be learnt about modulation of TRPA1 with these compounds. A series of CADs was therefore screened for agonism and antagonism in HEK293 cells stably transfected with WT-human (h)TRPA1, or C621A, F909A or F944A mutant hTRPA1. Derivatives with electron-withdrawing and/or electron-donating substituents were found to possess different activities. CADs with inductive electron-withdrawing groups were agonists with desensitizing effects, and CADs with electron-donating groups were either partial agonists or antagonists. Site-directed mutagenesis revealed the CADs do not undergo conjugate addition reaction with TRPA1 and reveal that F944 is a key residue involved in the non-covalent modulation of TRPA1 by CADs, as well as many other structurally distinct non-reactive TRPA1 ligands already reported.

Twenty-two compounds belonging to several classes of polyamine analogs have been examined for their ability to inhibit the growth of the human malaria parasite Plasmodium falciparum in vitro and in vivo. Four lead compounds from the thiourea sub-series and one compound from the urea-based analogs were found to be potent inhibitors of both chloroquine-resistant (Dd2) and chloroquine-sensitive (3D7) strains of Plasmodium with IC50 values ranging from 150 to 460 nM. In addition, the compound RHW, N1,N7-bis (3-(cyclohexylmethylamino) propyl) heptane-1,7-diamine tetrabromide was found to inhibit Dd2 with an IC50 of 200 nM. When RHW was administered to P. yoelii-infected mice at 35 mg/kg for 4 days, it significantly reduced parasitemia. RHW was also assayed in combination with the ornithine decarboxylase inhibitor difluoromethylornithine, and the two drugs were found not to have synergistic antimalarial activity. Furthermore, these inhibitors led to decreased cellular spermidine and spermine levels in P. falciparum, suggesting that they exert their antimalarial activities by inhibition of spermidine synthase.

Background: Parasite diseases are a huge burden on human health causing significant morbidity and mortality. However, parasite structure based drug discovery programmes have been hindered by a lack of high resolution structural information from parasite derived proteins and have often relied upon homology models from mammalian systems. The recent renaissance in electron microscopy (EM) has caused a dramatic rise in the number of structures being determined at high resolution and subsequently enabled it to be thought of as a tool in drug discovery. Results: In this review, we discuss the challenges associated with the structural determination of parasite proteins including the difficulties in obtaining sufficient quantities of protein. We then discuss the reasons behind the resurgence in EM, how it may overcome some of these challenges and provide examples of EM derived parasite protein structures. Finally, we discuss the challenges which EM needs to overcome before it is used as a mainstream technique in anti-parasite drug discovery. Conclusions: This review reports the progress that has been made in obtaining sufficient quantities of proteins for structural studies and the role EM may play in future structure based drug design programs. The outlook for future structure based drug design programs against some of the most devastating parasite diseases looks promising.

The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors targeting the NAD+ bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD+ and its availability in the natural environment makes the NADH complex form of ENR an attractive target. Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella ENR, but despite good efficacy against Toxoplasma gondii, the IC50 for T. gondii ENR is poor, with no inhibitory activity at 1μM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the ENR enzyme and as such we believe that there may be significant off target effects for this family of inhibitors.

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, bc1 complex Qi site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites.

Bacterial RNA polymerase (RNAP) is essential for transcription and is an antibacterial target for small molecule inhibitors. The binding region of myxopyronin B (MyxB), a bacterial RNAP inhibitor, offers the possibility of new inhibitor design. The molecular design program SPROUT has been used in conjunction with the X-ray cocrystal structure of Thermus thermophilus RNAP with MyxB to design novel inhibitors based on a substituted pyridyl-benzamide scaffold. A series of molecules, with molecular masses

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.

Since the recent renaissance of phenotypic screening in the field of protozoan drug discovery, is there still an opportunity for the structure-based design of new anti-protozoan agents? Target-based approaches should be used in parallel to phenotypic screening to strengthen the pipeline of anti-protozoan agents. We give an overview of the protozoan drug discovery landscape highlighting four protein targets of interest: cytochrome bc1, dihydroorotate dehydrogenase, dihydrofolate reductase and calcium-dependent protein kinase 1. We discuss recent structurebased design efforts to inhibit these targets, reviewing their crystal structures and their ability to accommodate potent and selective compounds. Finally, we discuss future opportunities to apply structure-based methods to promising molecular targets within protozoan parasites discovered using chemical genomics.

The development of resistance to all current antibiotics in the clinic means there is an urgent unmet need for novel antibacterial agents with new modes of action. One of the best ways of finding these is to identify new essential bacterial enzymes to target. The advent of a number of in silico tools has aided classical methods of discovering new antibacterial targets, and these programs are the subject of this review. Many of these tools apply a cheminformatic approach, utilizing the structural information of either ligand or protein, chemogenomic databases, and docking algorithms to identify putative antibacterial targets. Considering the wealth of potential drug targets identified from genomic research, these approaches are perfectly placed to mine this rich resource and complement drug discovery programs.

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.

Many microbial pathogens rely on a type II fatty acid synthesis (FASII) pathway that is distinct from the type I pathway found in humans. Enoyl-acyl carrier protein reductase (ENR) is an essential FASII pathway enzyme and the target of a number of antimicrobial drug discovery efforts. The biocide triclosan is established as a potent inhibitor of ENR and has been the starting point for medicinal chemistry studies. We evaluated a series of triclosan analogues for their ability to inhibit the growth of Toxoplasma gondii, a pervasive human pathogen, and its ENR enzyme (TgENR). Several compounds that inhibited TgENR at low nanomolar concentrations were identified but could not be further differentiated because of the limited dynamic range of the TgENR activity assay. Thus, we adapted a thermal shift assay (TSA) to directly measure the dissociation constant (Kd) of the most potent inhibitors identified in this study as well as inhibitors from previous studies. Furthermore, the TSA allowed us to determine the mode of action of these compounds in the presence of the reduced nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide (NAD⁺) cofactor. We found that all of the inhibitors bind to a TgENR-NAD⁺ complex but that they differed in their dependence on NAD⁺ concentration. Ultimately, we were able to identify compounds that bind to the TgENR-NAD⁺ complex in the low femtomolar range. This shows how TSA data combined with enzyme inhibition, parasite growth inhibition data, and ADMET predictions allow for better discrimination between potent ENR inhibitors for the future development of medicine.

SUMMARYIn light of the low success rate of target-based genomics and HTS (High Throughput Screening) approaches in anti-infective drug discovery,in silicostructure-based drug design (SBDD) is becoming increasingly prominent at the forefront of drug discovery.In silicoSBDD 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 toPlasmodium falciparumDHODH. 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.

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.