Explore the vast range of titles available.

We previously reported an antibiotic discovery screening platform that identifies whole-cell active compounds with high sensitivity while simultaneously providing mechanistic insight, necessary for hit prioritization. Named PROSPECT, (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), this platform measures chemical-genetic interactions between small molecules and pooled Mycobacterium tuberculosis mutants, each depleted of a different essential protein. Here, we introduce Perturbagen CLass (PCL) analysis, a computational method that infers a compound’s mechanism-of-action (MOA) by comparing its chemical-genetic interaction profile to those of a curated reference set of 437 known molecules. In leave-one-out cross-validation, we correctly predict MOA with 70% sensitivity and 75% precision, and achieve comparable results (69% sensitivity, 87% precision) with a test set of 75 antitubercular compounds with known MOA previously reported by GlaxoSmithKline (GSK). From 98 additional GSK antitubercular compounds with unknown MOA, we predict 60 to act via a reference MOA and functionally validate 29 compounds predicted to target respiration. Finally, from a set of ~5,000 compounds from larger unbiased libraries, we identify a novel QcrB-targeting scaffold that initially lacked wild-type activity, experimentally confirming this prediction while chemically optimizing this scaffold. PCL analysis of PROSPECT data enables rapid MOA assignment and hit prioritization, streamlining antimicrobial discovery. Bond et al. predict mechanism of action of hit compounds from a pooled screen of Mycobacterium tuberculosis mutants underproducing essential proteins by comparing the strain-specific responses of screening hits to those elicited by known antimicrobials.

A convergent total synthesis platform led to the discovery of TP-2758 from a series of novel 7-methoxy-8-heterocyclyl tetracycline analogs. TP-2758 demonstrated high in vitro potency against key Gram-negative pathogens including extended spectrum β-lactamases- and carbapenemase-producing Enterobacteriaceae and Acinetobacter spp. strains. This compound was efficacious when administered either intravenously or orally in multiple murine infection models and displayed a favorable preclinical pharmacological profile supporting its advancement into clinical development.

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against and . Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of . To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in . CRISPRi-induced partial depletion of lipooligosaccharide transport genes ( - , ) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on genes in Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains.

PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) is an antimicrobial discovery platform based on chemical-genetic interaction (CGI) profiling of compounds against a pool of Mycobacterium tuberculosis (Mtb) hypomorphs, each depleted of an essential gene. From prior screening data, we have now identified a novel N-oxolan-3-yl pyrazole carboxamide inhibitor (BRD1554) that had increased, selective activity against strains depleted of polyketide synthase 13 (Pks13), an essential enzyme in mycolic acid synthesis, and Rv2581c, an uncharacterized protein similar to glyoxylase II enzymes. Perturbagen CLass (PCL) analysis, a reference-based approach to mechanism of action (MOA) assignment from PROSPECT, predicted Pks13, a polyketide synthase with five catalytic domains responsible for the terminal condensation step in mycolic acid biosynthesis, was the likely target, potentially implicating the thioesterase domain. We synthesized a more active analogue while assigning the absolute stereochemistry of the active diastereomer, resulting in 1554-06-3R,4S with an MIC90 of 3.0 uM against Mtb H37Rv. Exposure to 1554-06 led to the upregulation of the pks13 operon along with the iniBAC operon and other genes linked to mycolic acid synthesis. Isolation of mutants resistant to 1554-06 revealed single nucleotide polymorphisms in the thioesterase domain of Pks13. Finally, we biochemically confirmed that 1554-06 inhibits the activity of recombinant Pks13 thioesterase domain, with computational docking of 1554-06 steroisomers consistent with the stereospecific activity seen in whole cell assays. We found unique chemical genetic interactions between inhibitors of the different Pks13 domains and different detoxifying enzymes of Mtb, thus revealing novel gene-gene interactions. These results highlight how PROSPECT can not only immediately reveal, with domain-level resolution, the MOA of new whole-cell active chemical inhibitors of Mtb, allowing the integration of biological insight into compound triage and accelerated early development, but can also illuminate genetic interactions linked to those mechanisms that could inform predictions of synergy for antitubercular drug development.Competing Interest StatementThe authors have declared no competing interest.Funder Information DeclaredGates Foundation, https://ror.org/0456r8d26, INV-040933

In an era of increasing resistance, new and effective strategies are needed for antibiotic discovery. Whole-cell active screens yield candidate compounds lacking mechanism-of-action (MOA) information and thus do not provide biological insight for prioritization. We previously reported PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of Mycobacterium tuberculosis mutants, each depleted of a different essential protein target. PROSPECT facilitates efficient hit prioritization by simultaneously identifying whole-cell active compounds with high sensitivity and providing early insights into their MOA. Here, we report a reference-based approach to infer MOA from often complex PROSPECT data. For this aim, we curated a reference set of 437 compounds with published, annotated MOA and known or suspected antitubercular activity, and applied PROSPECT to it. We then developed Perturbagen CLass (PCL) analysis, a computational method that predicts MOA by comparing chemical-genetic interaction profiles of unknown compounds to those of this reference set. In leave-one-out cross-validation, PCL analysis correctly predicted MOA with 70% sensitivity and 75% precision. When applied to 75 antitubercular leads with known MOA previously reported by GlaxoSmithKline (GSK), PCL analysis similarly achieved 69% sensitivity and 87% precision. We also analyzed 98 GSK compounds lacking MOA information, predicting 60 of them to act via a reference MOA, and followed up with functional validation of 29 compounds predicted to target respiration-related MOAs. Finally, we applied PROSPECT and PCL analysis to ∼5,000 compounds from larger unbiased libraries that had not been preselected for antitubercular activity. PCL analysis identified a novel scaffold lacking wild-type activity but predicted to inhibit respiration via QcrB, and we confirmed this prediction while chemically optimizing this scaffold to achieve wild-type activity. PCL analysis of PROSPECT data thus enables rapid MOA assignment and hit prioritization, advancing the discovery of new, potent antitubercular compounds.

Proteus mirabilis is a predominant cause of catheter associated urinary tract infection (CAUTI), and a key virulence factor is its urease enzyme which can increase urine pH and form urinary stones, causing catheter blockage and facilitating bacteremia. The only FDA approved urease inhibitor, acetohydroxamic acid (AHA), has side effects that limit its clinical use, necessitating new approaches to target urease activity. We previously discovered that common urinary tract colonizers modulate P. mirabilis urease activity via secreted small molecules. In this study, we conduct a metabolomics analysis of six modulatory bacterial species to reveal urease-dampening metabolites. Of 31 candidate metabolites, seven reproducibly decrease P. mirabilis urease activity. All seven metabolites dampen urease activity in other urease-positive bacterial species, suggesting conserved targets. Six of the metabolites act via mixed inhibition of the urease enzyme. One metabolite, D-imidazole lactate, exhibits a non-competitive mechanism of urease inhibition along with antimicrobial activity and repression of the urease operon in P. mirabilis . Metabolite combinations with AHA demonstrate synergistic activity and prevent catheter encrustation in an in vitro model for CAUTI. Prophylactic use of urease dampening metabolites with AHA could improve the efficacy of antimicrobial treatment against catheter biofilms. Authors utilise a metabolomics approach to identify microbial-derived metabolites that synergistically inhibit urease activity in Proteus mirabilis , a cause of urease-induced kidney stones. They reveal that two metabolites prevented urinary catheter encrustation and improved antimicrobial efficacy against catheter biofilm.

Comprehensive genome annotation is essential to understand the impact of clinically relevant variants. However, the absence of a standard for clinical reporting and browser display complicates the process of consistent interpretation and reporting. To address these challenges, Ensembl/GENCODE 1 and RefSeq 2 launched a joint initiative, the Matched Annotation from NCBI and EMBL-EBI (MANE) collaboration, to converge on human gene and transcript annotation and to jointly define a high-value set of transcripts and corresponding proteins. Here, we describe the MANE transcript sets for use as universal standards for variant reporting and browser display. The MANE Select set identifies a representative transcript for each human protein-coding gene, whereas the MANE Plus Clinical set provides additional transcripts at loci where the Select transcripts alone are not sufficient to report all currently known clinical variants. Each MANE transcript represents an exact match between the exonic sequences of an Ensembl/GENCODE transcript and its counterpart in RefSeq such that the identifiers can be used synonymously. We have now released MANE Select transcripts for 97% of human protein-coding genes, including all American College of Medical Genetics and Genomics Secondary Findings list v3.0 (ref. 3 ) genes. MANE transcripts are accessible from major genome browsers and key resources. Widespread adoption of these transcript sets will increase the consistency of reporting, facilitate the exchange of data regardless of the annotation source and help to streamline clinical interpretation. Matched Annotation from NCBI and EMBL-EBI (MANE) delivers joint transcript sets from Ensembl/GENCODE and RefSeq for standardizing variant reporting in clinical genomics and research.

BackgroundNonpalpable breast lesions require precise preoperative localization to facilitate negative margins with breast-conserving therapy. The traditional use of wires has several challenges including patient discomfort, wire migration, and coordination of schedules between radiology and the operating room. Radioactive seed localization overcomes some of these challenges, but radiation safety requirements have limited adoption of this technology. The authors examined their institutional experience with Magseed as an alternative technology for localization and compared outcomes with those of wire and radioactive seed localization.MethodsAn institutional review board (IRB)-approved retrospective study was performed to evaluate patients who underwent excisional biopsy or segmental mastectomy after wire-guided localization (WGL), radioactive seed localization (RSL), or Magseed localization (ML). The clinical and pathologic factors of the three groups were assessed with a negative margin rate as the primary outcome measure.ResultsOf the 1835 patients in the study, 825 underwent WGL, 449 underwent RSL, and 561 underwent ML. For the patients with either multiple lesions or a large lesion that required bracketing, multiple localization devices were placed in 31% of the WGL patients, 28% of the RSL patients, and 23% of the ML patients (p = 0.006). Negative margins were achieved in 91% of the WGL patients, 89% of the RSL patients, and 89% of the ML patients (p = 0.4).ConclusionLocalization of non-palpable breast lesions using Magseed is a safe and effective alternative to WGL and RSL that overcomes radiation safety limitations and increases radiology and surgery scheduling efficiency.

Pregnant women with type 1 diabetes are a high-risk population who are recommended to strive for optimal glucose control, but neonatal outcomes attributed to maternal hyperglycaemia remain suboptimal. Our aim was to examine the effectiveness of continuous glucose monitoring (CGM) on maternal glucose control and obstetric and neonatal health outcomes. In this multicentre, open-label, randomised controlled trial, we recruited women aged 18–40 years with type 1 diabetes for a minimum of 12 months who were receiving intensive insulin therapy. Participants were pregnant (≤13 weeks and 6 days' gestation) or planning pregnancy from 31 hospitals in Canada, England, Scotland, Spain, Italy, Ireland, and the USA. We ran two trials in parallel for pregnant participants and for participants planning pregnancy. In both trials, participants were randomly assigned to either CGM in addition to capillary glucose monitoring or capillary glucose monitoring alone. Randomisation was stratified by insulin delivery (pump or injections) and baseline glycated haemoglobin (HbA1c). The primary outcome was change in HbA1c from randomisation to 34 weeks' gestation in pregnant women and to 24 weeks or conception in women planning pregnancy, and was assessed in all randomised participants with baseline assessments. Secondary outcomes included obstetric and neonatal health outcomes, assessed with all available data without imputation. This trial is registered with ClinicalTrials.gov, number NCT01788527. Between March 25, 2013, and March 22, 2016, we randomly assigned 325 women (215 pregnant, 110 planning pregnancy) to capillary glucose monitoring with CGM (108 pregnant and 53 planning pregnancy) or without (107 pregnant and 57 planning pregnancy). We found a small difference in HbA1c in pregnant women using CGM (mean difference −0·19%; 95% CI −0·34 to −0·03; p=0·0207). Pregnant CGM users spent more time in target (68% vs 61%; p=0·0034) and less time hyperglycaemic (27% vs 32%; p=0·0279) than did pregnant control participants, with comparable severe hypoglycaemia episodes (18 CGM and 21 control) and time spent hypoglycaemic (3% vs 4%; p=0·10). Neonatal health outcomes were significantly improved, with lower incidence of large for gestational age (odds ratio 0·51, 95% CI 0·28 to 0·90; p=0·0210), fewer neonatal intensive care admissions lasting more than 24 h (0·48; 0·26 to 0·86; p=0·0157), fewer incidences of neonatal hypoglycaemia (0·45; 0·22 to 0·89; p=0·0250), and 1-day shorter length of hospital stay (p=0·0091). We found no apparent benefit of CGM in women planning pregnancy. Adverse events occurred in 51 (48%) of CGM participants and 43 (40%) of control participants in the pregnancy trial, and in 12 (27%) of CGM participants and 21 (37%) of control participants in the planning pregnancy trial. Serious adverse events occurred in 13 (6%) participants in the pregnancy trial (eight [7%] CGM, five [5%] control) and in three (3%) participants in the planning pregnancy trial (two [4%] CGM and one [2%] control). The most common adverse events were skin reactions occurring in 49 (48%) of 103 CGM participants and eight (8%) of 104 control participants during pregnancy and in 23 (44%) of 52 CGM participants and five (9%) of 57 control participants in the planning pregnancy trial. The most common serious adverse events were gastrointestinal (nausea and vomiting in four participants during pregnancy and three participants planning pregnancy). Use of CGM during pregnancy in patients with type 1 diabetes is associated with improved neonatal outcomes, which are likely to be attributed to reduced exposure to maternal hyperglycaemia. CGM should be offered to all pregnant women with type 1 diabetes using intensive insulin therapy. This study is the first to indicate potential for improvements in non-glycaemic health outcomes from CGM use. Juvenile Diabetes Research Foundation, Canadian Clinical Trials Network, and National Institute for Health Research.

BackgroundMost minorities receive cancer care at minority-serving hospitals (MSHs) that have been associated with disparate treatment between Black and White patients.ObjectiveOur aim was to examine the uptake of clinical trials that have changed axillary management in breast cancer patients at MSH and non-MSH cancer centers.MethodsThe National Cancer Database was used to identify patients eligible for the American College of Surgeons Oncology Group Z0011 and Z1071 trials, and mastectomy patients fulfilling the European AMAROS trial. Uptake of trial results (omission of axillary lymph node dissection) was analyzed between patients treated at MSHs and non-MSHs and adjusted for patient, tumor, and facility factors. MSHs were defined as the top decile of hospitals according to the proportion of Black and Hispanic patients treated.ResultsOf 7167 patients eligible for Z0011, 4546 for Z0171, and 9433 for AMAROS from 2015 to 2016, clinical trial uptake was seen in 1195 (74.6%) MSH and 4056 (72.9%) non-MSH patients (p = 0.173) for Z0011, 588 (41.9%) MSH and 1366 (43.5%) non-MSH patients for Z1071 (p = 0.302), and 272 (11.7%) MSH and 996 (14.0%) non-MSH patients (p = 0.005) for AMAROS. On adjusted analyses, MSH status was not significant for uptake of any of the three trials. Black race, socioeconomic status, and insurance were not associated with clinical trial uptake.ConclusionThe uptake of three landmark clinical trials of axillary management in breast cancer was not different at MSH and non-MSH centers despite adjustment for social determinants of health. At the Commission on Cancer-accredited centers in this analysis, MSH status did not affect the uptake of evidence-based care.