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The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coliare mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1is a substrate-competitive inhibitor targeting apo LpxA, and compound 2is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2exhibited more favorable biological and physicochemical properties than compound 1and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.

Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaristogether with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgarisand 391 from Escherichia coli. Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fidePPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in western countries and typically occurs in elderly individuals. There will be an estimated 21,250 newly diagnosed cases in the United States this year and 4,320 deaths. Due to the highly variable clinical course of this disease, prognostication and risk stratification methods are necessary for guiding decisions on clinical management. Integrated prognostic models incorporating laboratory testing for multiple molecular, cytogenetic, and other biomarkers have recently been proposed by major clinical guidelines to classify patients into risk subgroups. The current NCCN Guidelines for Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia describe such an integrated prognostic model known as the Rossi model that includes TP53, NOTCH1, SF3B1, and BIRC3mutations along with the cytogenetic abnormalities detected by fluorescence in situ hybridization (FISH) to classify CLL patients into 4 distinct prognostic subgroups: high-risk (TP53and/or BIRC3abnormalities), intermediate-risk (NOTCH1and/or SF3B1mutations and/or deletion 11q), low-risk (trisomy 12 and wild-type for all genetic lesions), and very low-risk (deletion 13q only). The 10-year survival rates for these subgroups are 29%, 37%, 57%, and 69%, respectively. To assess the clinical value of an integrated biomarker testing approach, we analyzed results of 651 consecutive cases submitted to our clinical diagnostic laboratory for testing on our integrated panel of molecular and cytogenetic biomarkers for CLL. Our panel includes detection of genomic alterations by FISH (deletion 6q, 13q, 11q, 17p, trisomy 12, IGH rearrangement, and IGH/CCND1 translocation) and detection of sequence variants in BIRC3, BTK, MYD88, NOTCH1, PCLG2, SF3B1, and TP53by next-generation sequencing (NGS). In total, 472 cases had positive findings by either FISH (90%) or NGS (46%) for a detection rate of 72.5%. Using the Rossi integrated prognostic model, 17.5% of cases fell into the high-risk subgroup, 20% of cases fell into the intermediate-risk subgroup, 43.5% of cases fell into the low-risk subgroup, and 19% of cases fell into the very low-risk subgroup. Importantly, among cases with positive FISH findings, 40.1% of cases also had positive molecular findings. In approximately 84% of cases belonging to the low-risk cytogenetic subgroups by FISH assessment alone, the incorporation of molecular findings resulted in reclassification into a higher-risk subgroup. Among the FISH-negative cases, 17% were classified as high-risk or intermediate-risk based on the molecular findings. Together, these findings support the clinical value of an integrative biomarker testing approach that includes both molecular and cytogenetic biomarkers to stratify CLL patients into risk subgroups to help guide decisions on clinical management.

Prathapam: Quest Diagnostics: Current Employment. Maharjan: Quest Diagnostics: Current Employment. Powers: Quest Diagnostics: Current Employment. Freitas: Quest Diagnostics: Current Employment. Sun: Quest Diagnostics: Current Employment. Tan: Quest Diagnostics: Current Employment. Gupta: Quest Diagnostics: Current Employment. Hucthagowder: Quest Diagnostics: Current Employment. Graham: Quest Diagnostics: Current Employment. Whitman: Quest Diagnostics: Current Employment. Khadgi: Quest Diagnostics: Current Employment. Daniel: Quest Diagnostics: Current Employment. Racke: Quest Diagnostics: Current Employment. Champion: Quest Diagnostics: Current Employment.