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1. Molecular and Clinical Epidemiology of Carbapenem-Resistant Enterobacteriaceae in the United States: a Prospective Cohort Study

   David van Duin M.D.; Arias C.A.; Komarow L.; Chen L.; Hanson B.M.; Weston G.; .... Multi-Drug Resistant Organism Network Investigators (June 2020, Lancet infections disease)

   Background: Carbapenem-resistant Enterobacteriaceae (CRE) are a global threat. Here, we describe the clinical and molecular characteristics of Centers for Disease Control and Prevention (CDC)-defined CRE in the US. Methods: The second Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE-2, ClinicalTrials.gov: NCT03646227) is a prospective, multicenter, cohort study. Patients hospitalized in 49 US hospitals, with clinical cultures positive for CDC-defined CRE between 30 April 2016 and 31 August 2017 were included. Primary outcome was desirability of outcome ranking (DOOR) at 30 days. Clinical data and bacteria were collected, and whole genome sequencing (WGS) was performed. Findings: 1,040 patients with unique isolates were included; 449 (43%) with infection and 591 (57%) with colonization. CDC-defined CRE admission rate was 57 CDC-defined CRE admissions/100,000 admissions (95% CI: 45–71). Three subsets of CDC-defined CRE were identified: carbapenemase-producing Enterobacteriaceae (618/1,040, 59%); non-carbapenemase-producing CRE (194/1,040, 19%); and unconfirmed CRE (228/1,040, 22%; initially reported as CRE, but susceptible to carbapenems in two central laboratories). Klebsiella pneumoniae carbapenemase (KPC)-producing clonal group 258 K. pneumoniae was the most common carbapenemase-producing Enterobacteriaceae. In 449 patients with CDC-defined CRE infections, DOOR outcomes were not significantly different in patients with carbapenemase-producing Enterobacteriaceae, non-carbapenemase-producing CRE, and unconfirmed CRE. At 30 days 107/449 (24%, 95% CI 20–28%) patients had died. Interpretation: Among patients with CDC-defined CRE, similar outcomes were observed among three subgroups, including the novel unconfirmed CRE group. CDC-defined CRE represent diverse bacteria, whose spread may not respond to interventions directed to carbapenemase-producing Enterobacteriaceae. 

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2. Chemical inhibition of a CBASS anti-bacteriophage defense

   https://doi.org/10.1101/2025.09.19.677395  

   Zhang, Chengqian; Rechkoblit, Olga; Gerdt, Joseph P (September 2025, bioRxiv)

   ABSTRACT Multidrug-resistant (MDR) bacteria pose a significant public health challenge, underscoring the urgent need for innovative antibacterial strategies. Bacteriophages (phages), viruses that specifically target bacteria, offer a promising alternative; however, bacterial immune defenses often limit their effectiveness. Developing small-molecule inhibitors of these defenses can facilitate mechanistic studies and serve as adjuvants to enhance phage therapy. Here, we identify novel inhibitors targeting the bacterial cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector Cap5. Cap5 is an HNH endonuclease activated by a cyclic nucleotide to degrade genomic DNA in virally infected cells, leading to cell death through abortive infection. Guided by the crystal structure of the Cap5 SAVED domain bound to its activating ligand, we performed structure-guided virtual screening to identify candidate inhibitors. Biochemical assays revealed that approximately 16% of the top docking hits exhibited inhibitory activity. Further cellular assays demonstrated that one potent compound could enterE. colicells and inhibit Cap5 activity. Our integrated approach—combining structure-based virtual screening with biochemical validation—provides a robust framework for discovering small-molecule inhibitors of bacterial immune defenses to advance adjunctive therapies and deepen our understanding of phage-bacteria interactions. 

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3. Sodium Malonate Inhibits the AcrAB-TolC Multidrug Efflux Pump of Escherichia coli and Increases Antibiotic Efficacy

   https://doi.org/10.3390/pathogens11121409  

   Cauilan, Allea; Ruiz, Cristian (December 2022, Pathogens)

   There is an urgent need to find novel treatments for combating multidrug-resistant bacteria. Multidrug efflux pumps that expel antibiotics out of cells are major contributors to this problem. Therefore, using efflux pump inhibitors (EPIs) is a promising strategy to increase antibiotic efficacy. However, there are no EPIs currently approved for clinical use especially because of their toxicity. This study investigates sodium malonate, a natural, non-hazardous, small molecule, for its use as a novel EPI of AcrAB-TolC, the main multidrug efflux pump of the Enterobacteriaceae family. Using ethidium bromide accumulation experiments, we found that 25 mM sodium malonate inhibited efflux by the AcrAB-TolC and other MDR pumps of Escherichia coli to a similar degree than 50 μΜ phenylalanine-arginine-β-naphthylamide, a well-known EPI. Using minimum inhibitory concentration assays and molecular docking to study AcrB-ligand interactions, we found that sodium malonate increased the efficacy of ethidium bromide and the antibiotics minocycline, chloramphenicol, and ciprofloxacin, possibly via binding to multiple AcrB locations, including the AcrB proximal binding pocket. In conclusion, sodium malonate is a newly discovered EPI that increases antibiotic efficacy. Our findings support the development of malonic acid/sodium malonate and its derivatives as promising EPIs for augmenting antibiotic efficacy when treating multidrug-resistant bacterial infections. 

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4. Desorption Electrospray Ionization Mass Spectrometry Assay for Label‐Free Characterization of SULT2B1b Enzyme Kinetics

   https://doi.org/10.1002/cmdc.202200043  

   Kulathunga, Samadhi_C; Morato, Nicolás_M; Zhou, Qing; Cooks, R_Graham; Mesecar, Andrew_D (February 2022, ChemMedChem)

   Abstract The sulfotransferase (SULT) 2B1b, which catalyzes the sulfonation of 3β‐hydroxysteroids, has been identified as a potential target for prostate cancer treatment. However, a major limitation for SULT2B1b‐targeted drug discovery is the lack of robust assays compatible with high‐throughput screening and inconsistency in reported kinetic data. For this reason, we developed a novel label‐free assay based on high‐throughput (>1 Hz) desorption electrospray ionization mass spectrometry (DESI‐MS) for the direct quantitation of the sulfoconjugated product (CV<10 %; <1 ng analyte). The performance of this DESI‐based assay was compared against a new fluorometric coupled‐enzyme method that we also developed. Both methodologies provided consistent kinetic data for the reaction of SULT2B1b with its major substrates, indicating the affinity trend pregnenolone>DHEA>cholesterol, for both the phospho‐mimetic and wild‐type SULT2B1b forms. The novel DESI‐MS assay developed here is likely generalizable to other drug discovery efforts and is particularly promising for identification of SULT2B1b inhibitors with potential as prostate cancer therapeutics. 

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5. Development of Natural‐Product‐Inspired ABCB1 Inhibitors Through Regioselective Tryptophan C3‐Benzylation

   https://doi.org/10.1002/chem.202401782  

   Mariya_Vincent, Dona; Mostafa, Habib; Suneer, Anza; Radha_Krishnan, Sabhashina; Ong, Mingmin; Itahana, Yoko; Itahana, Koji; Viswanathan, Rajesh (October 2024, Chemistry – A European Journal)

   Abstract The emergence of drug resistance in cancer cells eventually causing relapse is a serious threat that demands new advances. Upregulation of the ATP‐dependent binding cassette (ABC) transporters, such as ABCB1, significantly contributes to the emergence of drug resistance in cancer. Despite more than 30 years of therapeutic discovery, and several generations of inhibitors against P‐gp, the search for effective agents that minimize toxicity to human cells, while maintaining efflux pump inhibition is still underway. Leads derived from natural product scaffolds are well‐known to be effective in various therapeutic approaches. Inspired by the biosynthetic pathway to Nocardioazine A, a marine alkaloid known to inhibit the P‐gp efflux pump in cancer cells, we devised a regioselective pathway to create structurally unique indole‐C3‐benzylcyclo‐L‐Trp‐L‐Trp diketopiperazines (DKPs). Using bat cells as a model to derive effective ABCB1 inhibitors for targeting human P‐gp efflux pumps, we have recently identifiedexo‐C3‐N‐Dbn‐Trp2 (13)as a lead ABCB1 inhibitor. This C3‐benzylated lead inhibited ABCB1 better than Verapamil.^[21]Additionally,C3‐N‐Dbn‐Trp2restored chemotherapy sensitivity in drug‐resistant human cancer cells and had no adverse effect on cell proliferation in cell cultures. For a clearer structure‐activity relationship, we developed a broader screen to test C3‐functionalized pyrroloindolines as ABCB1 inhibitors and observed that C3‐benzylation is outperforming respective isoprenylated derivatives. Results arising from the molecular docking studies indicate that the interactions at the access tunnel between ABCB1 and the inhibitor result in a powerful predictor for the efficacy of the inhibitor. Based on fluorescence‐based assays, we conclude that the most efficacious inhibitor is thep‐cyano‐derivedexo‐C3‐N‐Dbn‐Trp2 (33 a), closely followed by thep‐nitro substituted analogue. By combining assay results with molecular docking studies, we further correlate that the predictions based on the inhibitor interactions at the access tunnel provide clues about the design of improved ABCB1 inhibitors. As it has been well documented that ABCB1 itself is powerfully engaged in multi‐drug resistance, this work lays the foundation for the design of a new class of inhibitors based on the endogenous amino acid‐derivedcyclo‐L‐Trp‐L‐Trp DKP scaffold. 

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