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© 2015 Khare et al.Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms.

Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in β-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.

Mechanobiology is a rapidly advancing field, with growing evidence that mechanical signaling plays key roles in health and disease. To accelerate mechanobiology-based drug discovery, novel in vitro systems are needed that enable mechanical perturbation of cells in a format amenable to high throughput screening. Here, both a mechanical stretch device and 192-well silicone flexible linear stretch plate were designed and fabricated to meet high throughput technology needs for cell stretch-based applications. To demonstrate the utility of the stretch plate in automation and screening, cell dispensing, liquid handling, high content imaging, and high throughput sequencing platforms were employed. Using this system, an assay was developed as a biological validation and proof-of-concept readout for screening. A mechano-transcriptional stretch response was characterized using focused gene expression profiling measured by RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Gen sequencing. Using articular chondrocytes, a gene expression signature containing stretch responsive genes relevant to cartilage homeostasis and disease was identified. The possibility for integration of other stretch sensitive cell types (e.g., cardiovascular, airway, bladder, gut, and musculoskeletal), in combination with alternative phenotypic readouts (e.g., protein expression, proliferation, or spatial alignment), broadens the scope of high throughput stretch and allows for wider adoption by the research community. This high throughput mechanical stress device fills an unmet need in phenotypic screening technology to support drug discovery in mechanobiology-based disease areas., Competing Interests: All authors are current or prior employees of Novartis., (© 2024 Author(s).)

Cigarette smoking (CS) is the leading cause of COPD, and identifying the pathways that are driving pathogenesis in the airway due to CS exposure can aid in the discovery of novel therapies for COPD. An additional barrier to the identification of key pathways that are involved in the CS-induced pathogenesis is the difficulty in building relevant and high throughput models that can recapitulate the phenotypic and transcriptomic changes associated with CS exposure. To identify these drivers, we have developed a cigarette smoke extract (CSE)-treated bronchosphere assay in 384-well plate format that exhibits CSE-induced decreases in size and increase in luminal secretion of MUC5AC. Transcriptomic changes in CSE-treated bronchospheres resemble changes that occur in human smokers both with and without COPD compared to healthy groups, indicating that this model can capture human smoking signature. To identify new targets, we ran a small molecule compound deck screening with diversity in target mechanisms of action and identified hit compounds that attenuated CSE induced changes, either decreasing spheroid size or increasing secreted mucus. This work provides insight into the utility of this bronchopshere model to examine human respiratory disease impacted by CSE exposure and the ability to screen for therapeutics to reverse the pathogenic changes caused by CSE., Competing Interests: All authors are current or prior employees (were employees at the time of their contributions to the paper) of Novartis. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Beri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Asthma is often characterized by tissue-level mechanical phenotypes that include remodeling of the airway and an increase in airway tightening, driven by the underlying smooth muscle. Existing therapies only provide symptom relief and do not improve the baseline narrowing of the airway or halt progression of the disease. To investigate such targeted therapeutics, there is a need for models that can recapitulate the 3D environment present in this tissue, provide phenotypic readouts of contractility, and be easily integrated into existing assay plate designs and laboratory automation used in drug discovery campaigns. To address this, we have developed DEFLCT, a high-throughput plate insert that can be paired with standard labware to easily generate high quantities of microscale tissues in vitro for screening applications. Using this platform, we exposed primary human airway smooth muscle cell-derived microtissues to a panel of six inflammatory cytokines present in the asthmatic niche, identifying TGF-β1 and IL-13 as inducers of a hypercontractile phenotype. RNAseq analysis further demonstrated enrichment of contractile and remodeling-relevant pathways in TGF-β1 and IL-13 treated tissues as well as pathways generally associated with asthma. Screening of 78 kinase inhibitors on TGF-β1 treated tissues suggests that inhibition of protein kinase C and mTOR/Akt signaling can prevent this hypercontractile phenotype from emerging, while direct inhibition of myosin light chain kinase does not. Taken together, these data establish a disease-relevant 3D tissue model for the asthmatic airway, which combines niche specific inflammatory cues and complex mechanical readouts that can be utilized in drug discovery efforts., Competing Interests: Yes, all authors are current or prior employees (were employees at the time of their contributions to the paper) of Novartis., (© 2023 Author(s).)

The importance of human cell-based in vitro tools to drug development that are robust, accurate, and predictive cannot be understated. There has been significant effort in recent years to develop such platforms, with increased interest in 3D models that can recapitulate key aspects of biology that 2D models might not be able to deliver. We describe the development of a 3D human cell-based in vitro assay for the investigation of nephrotoxicity, using RPTEC-TERT1 cells. These RPTEC-TERT1 proximal tubule organoids 'tubuloids' demonstrate marked differences in physiologically relevant morphology compared to 2D monolayer cells, increased sensitivity to nephrotoxins observable via secreted protein, and with a higher degree of similarity to native human kidney tissue. Finally, tubuloids incubated with nephrotoxins demonstrate altered Na+/K+-ATPase signal intensity, a potential avenue for a high-throughput, translatable nephrotoxicity assay., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Vidal Yucha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Respiratory syncytial virus (RSV) infection can cause mucus overproduction and bronchiolitis in infants leading to severe disease and hospitalization. As a therapeutic strategy, immune modulatory agents may help prevent RSV-driven immune responses that cause severe airway disease. We developed a high throughput screen to identify compounds that reduced RSV-driven mucin 5AC (Muc5AC) expression and identified dexamethasone. Despite leading to a pronounced reduction in RSV-driven Muc5AC, dexamethasone increased RSV infection in vitro and delayed viral clearance in mice. This correlated with reduced expression of a subset of immune response genes and reduced lymphocyte infiltration in vivo. Interestingly, dexamethasone increased RSV infection levels without altering antiviral interferon signaling. In summary, the immunosuppressive activities of dexamethasone had favorable inhibitory effects on RSV-driven mucus production yet prevented immune defense activities that limit RSV infection in vitro and in vivo. These findings offer an explanation for the lack of efficacy of glucocorticoids in RSV-infected patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Upregulated expression of efflux pumps, lpxC target mutations, LpxC protein overexpression, and mutations in fabG were previously shown to mediate single-step resistance to the LpxC inhibitor CHIR-090 in P. aeruginosa Single-step selection experiments using three recently described LpxC inhibitors (compounds 2, 3, and 4) and mutant characterization showed that these mechanisms affect susceptibility to additional novel LpxC inhibitors. Serial passaging of P. aeruginosa wild-type and efflux pump-defective strains using the LpxC inhibitor CHIR-090 or compound 1 generated substantial shifts in susceptibility and underscored the interplay of efflux and nonefflux mechanisms. Whole-genome sequencing of CHIR-090 passage mutants identified efflux pump overexpression, fabG mutations, and novel mutations in fabF1 and in PA4465 as determinants of reduced susceptibility. Two new lpxC mutations, encoding A214V and G208S, that reduce susceptibility to certain LpxC inhibitors were identified in these studies, and we show that these and other target mutations differentially affect different LpxC inhibitor scaffolds. Lastly, the combination of target alteration (LpxC A214V ) and upregulated expression of LpxC was shown to be tolerated in P. aeruginosa and could mediate significant decreases in susceptibility., (Copyright © 2019 American Society for Microbiology.)

Antibiotic hypersensitive bacterial mutants (e.g., Escherichia coli imp ) are used to investigate intrinsic resistance and are exploited in antibacterial discovery to track weak antibacterial activity of novel inhibitor compounds. Pseudomonas aeruginosa Z61 is one such drug-hypersusceptible strain generated by chemical mutagenesis, although the genetic basis for hypersusceptibility is not fully understood. Genome sequencing of Z61 revealed nonsynonymous single-nucleotide polymorphisms in 153 genes relative to its parent strain, and three candidate mutations (in oprM , ampC , and lptE ) predicted to mediate hypersusceptibility were characterized. The contribution of these mutations was confirmed by genomic restoration of the wild-type sequences, individually or in combination, in the Z61 background. Introduction of the lptE mutation or genetic inactivation of oprM and ampC genes alone or together in the parent strain recapitulated drug sensitivities. This showed that disruption of oprM (which encodes a major outer membrane efflux pump channel) increased susceptibility to pump substrate antibiotics, that inactivation of the inducible β-lactamase gene ampC contributed to β-lactam susceptibility, and that mutation of the lipopolysaccharide transporter gene lptE strongly altered the outer membrane permeability barrier, causing susceptibility to large antibiotics such as rifampin and also to β-lactams., (Copyright © 2019 American Society for Microbiology.)

Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438 L172P alteration mediated a >150-fold upregulation of MexMN pump gene expression and a >50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for M ex MN r egulator and M ex MN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several β-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnS L172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD , encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnS L172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance., (Copyright © 2019 American Society for Microbiology.)

CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4 + T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34 + hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases., Competing Interests: Patents related to this work has been filed (WO 2015/095477 A1, WO 2014/151784 A1, WO 2014/151729). Several authors own shares of Novartis.

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

New chemotherapeutic agents are urgently required to combat the global spread of multidrug-resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase InhA is one of the few clinically validated targets in tuberculosis drug discovery. We report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH (reduced form of nicotinamide adenine dinucleotide)-dependent manner and blocked the enoyl substrate-binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of Mycobacterium tuberculosis infection. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB., (Copyright © 2015, American Association for the Advancement of Science.)

Renewed global efforts toward malaria eradication have highlighted the need for novel antimalarial agents with activity against multiple stages of the parasite life cycle. We have previously reported the discovery of a novel class of antimalarial compounds in the imidazolopiperazine series that have activity in the prevention and treatment of blood stage infection in a mouse model of malaria. Consistent with the previously reported activity profile of this series, the clinical candidate KAF156 shows blood schizonticidal activity with 50% inhibitory concentrations of 6 to 17.4 nM against P. falciparum drug-sensitive and drug-resistant strains, as well as potent therapeutic activity in a mouse models of malaria with 50, 90, and 99% effective doses of 0.6, 0.9, and 1.4 mg/kg, respectively. When administered prophylactically in a sporozoite challenge mouse model, KAF156 is completely protective as a single oral dose of 10 mg/kg. Finally, KAF156 displays potent Plasmodium transmission blocking activities both in vitro and in vivo. Collectively, our data suggest that KAF156, currently under evaluation in clinical trials, has the potential to treat, prevent, and block the transmission of malaria., (Copyright © 2014 Kuhen et al.)

Rifampicin resistance, a defining attribute of multidrug-resistant tuberculosis, is conferred by mutations in the β subunit of RNA polymerase. Sequencing of rifampicin-resistant (RIF-R) clinical isolates of Mycobacterium tuberculosis revealed, in addition to RIF-R mutations, enrichment of potential compensatory mutations around the double-psi β-barrel domain of the β' subunit comprising the catalytic site and the exit tunnel for newly synthesized RNA. Sequential introduction of the resistance allele followed by the compensatory allele in isogenic Mycobacterium smegmatis showed that these mutations respectively caused and compensated a starvation enhanced growth defect by altering RNA polymerase activity. While specific combinations of resistance and compensatory alleles converged in divergent lineages, other combinations recurred among related isolates suggesting transmission of compensated RIF-R strains. These findings suggest nutrient poor growth conditions impose larger selective pressure on RIF-R organisms that results in the selection of compensatory mutations in a domain involved in catalysis and starvation control of RNA polymerase transcription., (© 2014 John Wiley & Sons Ltd.)

Objectives: Current treatments for Clostridium difficile infection include vancomycin, metronidazole and fidaxomicin. LFF571 is an experimental agent undergoing evaluation in humans for the treatment of moderate C. difficile infection. Reduced susceptibility of C. difficile to fidaxomicin or LFF571 in vitro can be mediated by single point mutations in genes encoding the targets, whereas the mechanism(s) mediating reduced susceptibility to vancomycin in vitro remains elusive. To further characterize mechanisms reducing susceptibility of C. difficile to vancomycin, fidaxomicin or LFF571 in vitro, selections via serial passage at low cell density were performed, followed by whole-genome sequencing., Methods: C. difficile strain ATCC 43255 and three clinical isolates were subjected to 10 passages on medium containing a range of concentrations of fidaxomicin, LFF571 or vancomycin. Genomic DNA from isolates with reduced susceptibility was sequenced using Illumina Whole Genome Sequencing., Results: Clones exhibiting decreased susceptibility to fidaxomicin harboured mutations in rpoB and CD22120 (marR homologue). Clones exhibiting decreased susceptibility to vancomycin harboured mutations in rpoC and also in CD2725, CD3659 and sdaB, which encode a putative N-acetylglucosamine transferase, exonuclease and l-serine deaminase, respectively. All mutations resulted in non-synonymous substitutions. No clones with reduced susceptibility to LFF571 were selected in this study., Conclusions: Reduced susceptibility to fidaxomicin and vancomycin was associated with mutations mediating target modifications (RNA polymerase and cell wall, respectively), as well as with mutations that may contribute to reduced susceptibility via other mechanisms. The MIC of LFF571 was unaffected for those mutants with reduced susceptibility to fidaxomicin or vancomycin.

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1(Anaef), with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1(Anaef) mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44(hi) Helios(+) PD-1(+) CD4(+) T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1(Anaef) is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1(Anaef) naïve CD4(+) T cells. CD44 expression, CD4(+) T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1(Anaef)Mtor(chino) double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1(Anaef) T cell dysregulation. DOI: http://dx.doi.org/10.7554/eLife.01020.001.

Background: One of the main causes of mortality from severe malaria in Plasmodium falciparum infections is cerebral malaria (CM). An important host genetic component determines the susceptibility of an individual to develop CM or to clear the infection and become semi-immune. As such, the identification of genetic loci associated with susceptibility or resistance may serve to modulate disease severity., Methodology: The Plasmodium berghei mouse model for experimental cerebral malaria (ECM) reproduces several disease symptoms seen in human CM, and two different phenotypes, a susceptible (FVB/NJ) and a resistant mouse strain (DBA/2J), were examined., Results: FVB/NJ mice died from infection within ten days, whereas DBA/2J mice showed a gender bias: males survived on average nineteen days and females either died early with signs of ECM or survived for up to three weeks. A comparison of brain pathology between FVB/NJ and DBA/2J showed no major differences with regard to brain haemorrhages or the number of parasites and CD3+ cells in the microvasculature. However, significant differences were found in the peripheral blood of infected mice: For example resistant DBA/2J mice had significantly higher numbers of circulating basophils than did FVB/NJ mice on day seven. Analysis of the F2 offspring from a cross of DBA/2J and FVB/NJ mice mapped the genetic locus of the underlying survival trait to chromosome 9 with a Lod score of 4.9. This locus overlaps with two previously identified resistance loci (char1 and pymr) from a blood stage malaria model., Conclusions: Survival best distinguishes malaria infections between FVB/NJ and DBA/2J mice. The importance of char1 and pymr on chromosome 9 in malaria resistance to P. berghei was confirmed. In addition there was an association of basophil numbers with survival.

We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis by the previously uncharacterized mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser55 site in Pks13 conferred TP resistance in M. tuberculosis. Overexpression of wild-type Pks13 resulted in TP resistance, and overexpression of the Pks13(F79S) mutant conferred high resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type M. tuberculosis, but it did so to a much lesser extent in TP-resistant M. tuberculosis. TP treatment was bactericidal and equivalent to treatment with the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment resulted in sterilizing activity. Computational docking identified a possible TP-binding groove within the Pks13 acyl carrier protein domain. This study confirms that M. tuberculosis Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway.

A cell-based phenotypic screen for inhibitors of biofilm formation in mycobacteria identified the small molecule TCA1, which has bactericidal activity against both drug-susceptible and -resistant Mycobacterium tuberculosis (Mtb) and sterilizes Mtb in vitro combined with rifampicin or isoniazid. In addition, TCA1 has bactericidal activity against nonreplicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models both alone and combined with rifampicin or isoniazid. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb persistence. Genetic and affinity-based methods identified decaprenyl-phosphoryl-β-D-ribofuranose oxidoreductase DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as targets responsible for the activity of TCA1. These in vitro and in vivo results indicate that this compound functions by a unique mechanism and suggest that TCA1 may lead to the development of a class of antituberculosis agents.

SQ109, a 1,2-diamine related to ethambutol, is currently in clinical trials for the treatment of tuberculosis, but its mode of action remains unclear. Here, we demonstrate that SQ109 disrupts cell wall assembly, as evidenced by macromolecular incorporation assays and ultrastructural analyses. SQ109 interferes with the assembly of mycolic acids into the cell wall core of Mycobacterium tuberculosis, as bacilli exposed to SQ109 show immediate inhibition of trehalose dimycolate (TDM) production and fail to attach mycolates to the cell wall arabinogalactan. These effects were not due to inhibition of mycolate synthesis, since total mycolate levels were unaffected, but instead resulted in the accumulation of trehalose monomycolate (TMM), the precursor of TDM and cell wall mycolates. In vitro assays using purified enzymes showed that this was not due to inhibition of the secreted Ag85 mycolyltransferases. We were unable to achieve spontaneous generation of SQ109-resistant mutants; however, analogs of this compound that resulted in similar shutdown of TDM synthesis with concomitant TMM accumulation were used to spontaneously generate resistant mutants that were also cross-resistant to SQ109. Whole-genome sequencing of these mutants showed that these all had mutations in the essential mmpL3 gene, which encodes a transmembrane transporter. Our results suggest that MmpL3 is the target of SQ109 and that MmpL3 is a transporter of mycobacterial TMM.

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.

Testing P. aeruginosa efflux pump mutants showed that the LpxC inhibitor CHIR-090 is a substrate for MexAB-OprM, MexCD-OprJ, and MexEF-OprN. Utilizing P. aeruginosa PAO1 with a chromosomal mexC::luxCDABE fusion, luminescent mutants arose on medium containing 4 μg/ml CHIR-090, indicating upregulation of MexCD-OprJ. These mutants were less susceptible to CHIR-090 (MIC, 4 μg/ml) and had mutations in the mexCD-oprJ repressor gene nfxB. Nonluminescent mutants (MIC, 4 μg/ml) that had mutations in the mexAB-oprM regulator gene mexR were also observed. Plating the clinical isolate K2153 on 4 μg/ml CHIR-090 selected mutants with alterations in mexS (immediately upstream of mexT), which upregulates MexEF-OprN. A mutant altered in the putative1ribosomal binding site (RBS) upstream of lpxC and overexpressing LpxC was selected on a related LpxC inhibitor and exhibited reduced susceptibility to CHIR-090. Overexpression of LpxC from a plasmid reduced susceptibility to CHIR-090, and introduction of the altered RBS in this construct further increased expression of LpxC and decreased susceptibility to CHIR-090. Using a mutS (hypermutator) strain, a mutant with an altered lpxC target gene (LpxC L18V) was also selected. Purified LpxC L18V had activity similar to that of wild-type LpxC in an in vitro assay but had reduced inhibition by CHIR-090. Finally, an additional class of mutant, typified by an extreme growth defect, was identified. These mutants had mutations in fabG, indicating that alteration in fatty acid synthesis conferred resistance to LpxC inhibitors. Passaging experiments showed progressive decreases in susceptibility to CHIR-090. Therefore, P. aeruginosa can employ several strategies to reduce susceptibility to CHIR-090 in vitro.

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

A search to identify new mechanisms of isoniazid resistance in Mycobacterium bovis led to the isolation of mutants defective in mycothiol biosynthesis due to mutations in genes coding for the glycosyltransferase (mshA) or the cysteine ligase (mshC). These mutants showed low-level resistance to isoniazid but were highly resistant to ethionamide. This study further illustrates that mutations in mycothiol biosynthesis genes may contribute to isoniazid or ethionamide resistance across mycobacterial species.

Plasmodium vivax causes 25-40% of malaria cases worldwide, yet research on this human malaria parasite has been neglected. Nevertheless, the recent publication of the P. vivax reference genome now allows genomics and systems biology approaches to be applied to this pathogen. We show here that whole-genome analysis of the parasite can be achieved directly from ex vivo-isolated parasites, without the need for in vitro propagation. A single isolate of P. vivax obtained from a febrile patient with clinical malaria from Peru was subjected to whole-genome sequencing (30× coverage). This analysis revealed over 18,261 single-nucleotide polymorphisms (SNPs), 6,257 of which were further validated using a tiling microarray. Within core chromosomal genes we find that one SNP per every 985 bases of coding sequence distinguishes this recent Peruvian isolate, designated IQ07, from the reference Salvador I strain obtained in 1972. This full-genome sequence of an uncultured P. vivax isolate shows that the same regions with low numbers of aligned sequencing reads are also highly variable by genomic microarray analysis. Finally, we show that the genes containing the largest ratio of nonsynonymous-to-synonymous SNPs include two AP2 transcription factors and the P. vivax multidrug resistance-associated protein (PvMRP1), an ABC transporter shown to be associated with quinoline and antifolate tolerance in Plasmodium falciparum. This analysis provides a data set for comparative analysis with important potential for identifying markers for global parasite diversity and drug resistance mapping studies.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

An attenuated Mycobacterium bovisRD1 deletion (DeltaRD1) mutant of the Ravenel strain was constructed, characterized, and sequenced. This M. bovis DeltaRD1 vaccine strain administered to calves at 2 weeks of age provided similar efficacy as M. bovis bacillus Calmette Guerin (BCG) against low dose, aerosol challenge with virulent M. bovis at 3.5 months of age. Approximately 4.5 months after challenge, both DeltaRD1- and BCG-vaccinates had reduced tuberculosis (TB)-associated pathology in lungs and lung-associated lymph nodes and M. bovis colonization of tracheobronchial lymph nodes as compared to non-vaccinates. Mean central memory responses elicited by either DeltaRD1 or BCG prior to challenge correlated with reduced pathology and bacterial colonization. Neither DeltaRD1 or BCG elicited IFN-gamma responses to rESAT-6:CFP-10 prior to challenge, an emerging tool for modern TB surveillance programs. The DeltaRD1 strain may prove useful for bovine TB vaccine programs, particularly if additional mutations are included to improve safety and immunogenicity.

Rapid expansion of available data, both phenotypic and genotypic, for multiple strains of mice has enabled the development of new methods to interrogate the mouse genome for functional genetic perturbations. In silico mapping provides an expedient way to associate the natural diversity of phenotypic traits with ancestrally inherited polymorphisms for the purpose of dissecting genetic traits. In mouse, the current single nucleotide polymorphism (SNP) data have lacked the density across the genome and coverage of enough strains to properly achieve this goal. To remedy this, 470,407 allele calls were produced for 10,990 evenly spaced SNP loci across 48 inbred mouse strains. Use of the SNP set with statistical models that considered unique patterns within blocks of three SNPs as an inferred haplotype could successfully map known single gene traits and a cloned quantitative trait gene. Application of this method to high-density lipoprotein and gallstone phenotypes reproduced previously characterized quantitative trait loci (QTL). The inferred haplotype data also facilitates the refinement of QTL regions such that candidate genes can be more easily identified and characterized as shown for adenylate cyclase 7., Competing Interests: The authors have declared that no conflicts of interest exist.

The nature and organization of polymorphisms, or differences, between genomes of individuals are of great interest, because these variations can be associated with or even underlie phenotypic traits, including disease susceptibility. To gain insight into the genetic and evolutionary factors influencing such biological variation, we have examined the arrangement (haplotype) of single-nucleotide polymorphisms across the genomes of eight inbred strains of mice. These analyses define blocks of high or low diversity, often extending across tens of megabases that are delineated by abrupt transitions. These observations provide a striking contrast to the haplotype structure of the human genome.

In the hamster, serum total lactogenic activity increases during the latter half of gestation (Days 8-16). On Days 10 and 12 a substantial amount of lactogenic activity cannot be attributed to prolactin (PRL) and hamster placental lactogen-II (haPL-II); therefore, the presence of a molecule similar to placental lactogen-I (PL-I), as found in the rat and mouse at midpregnancy, has been hypothesized for the hamster. The objectives of this study were to identify PRL-like molecules synthesized by the hamster placenta and to determine the temporal and cellular synthesis of identified molecules. Oligonucleotides (20-23 bp) corresponding to regions of nucleotide homology between mouse PL-I (mPL-I) and rat PL-I (rPL-I) along with midgestation hamster placental RNA were used in 3' rapid amplification of cDNA ends (RACE) methodology to generate PRL-like cDNA. A 444-bp cDNA fragment that had nucleotide sequence similarity with members of the prolactin-growth hormone (PRL-GH) gene family was generated. This cDNA fragment was utilized to screen a Day 16 hamster placental bacteriophage cDNA library, and a clone containing the entire coding region was identified and sequenced. The molecule had 77% nucleotide sequence homology with mouse proliferin-related protein (mPRP) and somewhat less homology (approximately 60%) with hamster, rat, and mouse PRL or placental lactogens (PL). The derived amino acid sequence of the identified molecule contained a 15-residue signal sequence and a 219-residue peptide with a calculated molecular weight of 25477. The peptide shared 58% amino acid sequence identity with mPRP. Placental expression of the PRL-like molecule during the latter half of gestation was evaluated by Northern and slot-blot analyses using the 444-bp cDNA fragment as a hybridization probe. A 1-kb transcript was detected on Days 9-15 with peak expression on Days 10 and 11. Messenger RNA for the PRL-like molecule was localized to cytotrophoblast but not giant trophoblast cells of the placental trophospongium region. In addition, specific immunostaining using an antibody to mPRP was confined to cytotrophoblast cells.