Multicenter Evaluation of the Unyvero Platform for Testing Bronchoalveolar Lavage Fluid.

Bronchoalveolar lavage (BAL) culture is a standard, though time-consuming, approach for identifying microorganisms in patients with severe lower respiratory tract (LRT) infections. The sensitivity of BAL culture is relatively low, and prior antimicrobial therapy decreases the sensitivity further, leading to overuse of empirical antibiotics. The Unyvero LRT BAL Application (Curetis GmbH, Germany) is a multiplex molecular panel that detects 19 bacteria, 10 antibiotic resistance markers, and a fungus, Pneumocystis jirovecii, in BAL fluid in ∼4.5 h. Its performance was evaluated using 1,016 prospectively collected and 392 archived specimens from 11 clinical trial sites in the United States. Overall positive and negative percent agreements with culture results for identification of bacteria that grow in routine cultures were 93.4% and 98.3%, respectively, with additional potential pathogens identified by Unyvero in 21.7% of prospectively collected specimens. For detection of P. jirovecii, the positive percent agreement with standard testing was 87.5%. Antibiotic resistance marker results were compared to standard antibiotic susceptibility test results to determine positive predictive values (PPVs). PPVs ranged from 80 to 100%, based on the microorganism and specific resistance marker(s). The Unyvero LRT BAL Application provides accurate detection of common agents of bacterial pneumonia and of P. jirovecii The sensitivity and rapidity of this panel suggest significant clinical value for choosing appropriate antibiotics and for antibiotic stewardship.

Structure-Based Design, Docking and Binding Free Energy Calculations of A366 Derivatives as Spindlin1 Inhibitors.

The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation, through which it has been linked to several types of malignant tumors. In the current work, we report on the development of novel analogs of the previously published lead inhibitor A366. In an effort to improve the activity and explore the structure-activity relationship (SAR), a series of 21 derivatives was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light on the important interactions. Our study highlighted the main structural features that are required for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184. Different protocols were tested to identify a fast in silico method that could help to discriminate between active and inactive compounds within the A366 series. Rescoring the docking poses with MM-GBSA calculations was successful in this regard. Because A366 is known to be a G9a inhibitor, the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the selectivity profile of the A366 analogs. This resulted in the discovery of diverse selective compounds, among which 1s and 1t showed Spindlin1 activity in the nanomolar range and selectivity over G9a and GLP. Finally, future design hypotheses were suggested based on our findings.

Species Identification and Antibiotic Resistance Prediction by Analysis of Whole-Genome Sequence Data by Use of ARESdb: an Analysis of Isolates from the Unyvero Lower Respiratory Tract Infection Trial.

Whole-genome sequencing (WGS) is now routinely performed in clinical microbiology laboratories to assess isolate relatedness. With appropriately developed analytics, the same data can be used for prediction of antimicrobial susceptibility. We assessed WGS data for identification using open-source tools and antibiotic susceptibility testing (AST) prediction using ARESdb compared to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification and broth microdilution phenotypic susceptibility testing on clinical isolates from a multicenter clinical trial of the FDA-cleared Unyvero lower respiratory tract infection (LRTI) application (Curetis). For the trial, more than 2,000 patient samples were collected from intensive care units across nine hospitals and tested for LRTI. The isolate subset used in this study included 620 clinical isolates originating from 455 LRTI culture-positive patient samples. Isolates were sequenced using the Illumina Nextera XT protocol and FASTQ files with raw reads uploaded to the ARESdb cloud platform (ares-genetics.cloud; released for research use in 2020). The platform combines Ares Genetics' proprietary database ARESdb with state-of-the-art bioinformatics tools and curated public data. For identification, WGS showed 99 and 93% concordance with MALDI-TOF MS at the genus and species levels, respectively. WGS-predicted susceptibility showed 89% categorical agreement with phenotypic susceptibility across a total of 129 species-compound pairs analyzed, with categorical agreement exceeding 90% in 78 species-compound pairs and reaching 100% in 32. Results of this study add to the growing body of literature showing that, with improvement of analytics, WGS data could be used to predict antimicrobial susceptibility.

Structure-guided design of a selective inhibitor of the methyltransferase KMT9 with cellular activity.

Inhibition of epigenetic regulators by small molecules is an attractive strategy for cancer treatment. Recently, we characterised the role of lysine methyltransferase 9 (KMT9) in prostate, lung, and colon cancer. Our observation that the enzymatic activity was required for tumour cell proliferation identified KMT9 as a potential therapeutic target. Here, we report the development of a potent and selective KMT9 inhibitor (compound 4, KMI169) with cellular activity through structure-based drug design. KMI169 functions as a bi-substrate inhibitor targeting the SAM and substrate binding pockets of KMT9 and exhibits high potency, selectivity, and cellular target engagement. KMT9 inhibition selectively downregulates target genes involved in cell cycle regulation and impairs proliferation of tumours cells including castration- and enzalutamide-resistant prostate cancer cells. KMI169 represents a valuable tool to probe cellular KMT9 functions and paves the way for the development of clinical candidate inhibitors as therapeutic options to treat malignancies such as therapy-resistant prostate cancer.

Discovery of a Potent and Selective Fragment-like Inhibitor of Methyllysine Reader Protein Spindlin 1 (SPIN1).

By screening an epigenetic compound library, we identified that UNC0638, a highly potent inhibitor of the histone methyltransferases G9a and GLP, was a weak inhibitor of SPIN1 (spindlin 1), a methyllysine reader protein. Our optimization of this weak hit resulted in the discovery of a potent, selective, and cell-active SPIN1 inhibitor, compound 3 (MS31). Compound 3 potently inhibited binding of trimethyllysine-containing peptides to SPIN1, displayed high binding affinity, was highly selective for SPIN1 over other epigenetic readers and writers, directly engaged SPIN1 in cells, and was not toxic to nontumorigenic cells. The crystal structure of the SPIN1-compound 3 complex indicated that it selectively binds tudor domain II of SPIN1. We also designed a structurally similar but inactive compound 4 (MS31N) as a negative control. Our results have demonstrated for the first time that potent, selective, and cell-active fragment-like inhibitors can be generated by targeting a single tudor domain.