Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to ...enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 BRD4(1) as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.
Automatic identification and sorting of livestock organs in the meat processing industry could reduce costs and improve efficiency. Two hyperspectral sensors encompassing the visible (400-900 nm) and ...short-wave infrared (900-1700 nm) spectra were used to identify the organs by type. A total of 104 parenchymatous organs of cattle and sheep (heart, kidney, liver, and lung) were scanned in a multi-sensory system that encompassed both sensors along a conveyor belt. Spectral data were obtained and averaged following manual markup of three to eight regions of interest of each organ. Two methods were evaluated to classify organs: partial least squares discriminant analysis (PLS-DA) and random forest (RF). In addition, classification models were obtained with the smoothed reflectance and absorbance and the first and second derivatives of the spectra to assess if one was superior to the rest. The in-sample accuracy for the visible, short-wave infrared, and combination of both sensors was higher for PLS-DA compared to RF. The accuracy of the classification models was not significantly different between data pre-processing methods or between visible and short-wave infrared sensors. Hyperspectral sensors, particularly those in the visible spectrum, seem promising to identify organs from slaughtered animals which could be useful for the automation of quality and process control in the food supply chain, such as in abattoirs.
Abstract
Background
Development of human immunodeficiency virus (HIV) remission strategies requires precise information on time to HIV rebound after treatment interruption, but there is uncertainty ...regarding whether modern antiretroviral therapy (ART) regimens and timing of ART initiation may affect this outcome.
Methods
AIDS Clinical Trials Group (ACTG) A5345 enrolled individuals who initiated ART during chronic or early HIV infection and on suppressive ART for ≥2 years. Participants underwent carefully monitored antiretroviral interruption. ART was restarted upon 2 successive viral loads ≥1000 copies/mL. We compared participants of A5345 with participants of 6 historic ACTG treatment interruption studies.
Results
Thirty-three chronic-treated and 12 early-treated participants interrupted ART with evaluable time to viral rebound. Median time to viral rebound ≥1000 HIV RNA copies/mL was 22 days. Acute retroviral rebound syndrome was diagnosed in 9% of the chronic-treated and none of the early-treated individuals. All participants of the historic studies were on older protease inhibitor-based regimens, whereas 97% of A5345 participants were on integrase inhibitor-based ART. There were no differences in the timing of viral rebound comparing A5345 versus historic studies. In a combined analysis, a higher percentage of early-treated participants remained off ART at posttreatment interruption week 12 (chronic vs early: 2% vs 9%, P = .0496). One chronic-treated and one early-treated A5345 participant remained off ART for >24 weeks. All participants resuppressed after ART reinitiation.
Conclusions
Early ART initiation, using either older or newer ART regimens, was associated with a significant delay in the time to HIV rebound after ART interruption, lowering the barrier for HIV remission.
In A5345, we detected no differences in the timing of viral rebound with modern versus historic ART. Early ART was associated with a significant delay in the time to HIV rebound after ART interruption, lowering the barrier for HIV remission.
Covalent Bruton's tyrosine kinase (BTK) inhibitors are efficacious in multiple B-cell malignancies, but patients discontinue these agents due to resistance and intolerance. We evaluated the safety ...and efficacy of pirtobrutinib (working name; formerly known as LOXO-305), a highly selective, reversible BTK inhibitor, in these patients.
Patients with previously treated B-cell malignancies were enrolled in a first-in-human, multicentre, open-label, phase 1/2 trial of the BTK inhibitor pirtobrutinib. The primary endpoint was the maximum tolerated dose (phase 1) and overall response rate (ORR; phase 2). This trial is registered with ClinicalTrials.gov, NCT03740529.
323 patients were treated with pirtobrutinib across seven dose levels (25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, and 300 mg once per day) with linear dose-proportional exposures. No dose-limiting toxicities were observed and the maximum tolerated dose was not reached. The recommended phase 2 dose was 200 mg daily. Adverse events in at least 10% of 323 patients were fatigue (65 20%), diarrhoea (55 17%), and contusion (42 13%). The most common adverse event of grade 3 or higher was neutropenia (32 10%). There was no correlation between pirtobrutinib exposure and the frequency of grade 3 treatment-related adverse events. Grade 3 atrial fibrillation or flutter was not observed, and grade 3 haemorrhage was observed in one patient in the setting of mechanical trauma. Five (1%) patients discontinued treatment due to a treatment-related adverse event. In 121 efficacy evaluable patients with chronic lymphocytic leukaemia (CLL) or small lymphocytic lymphoma (SLL) treated with a previous covalent BTK inhibitor (median previous lines of treatment 4), the ORR with pirtobrutinib was 62% (95% CI 53–71). The ORR was similar in CLL patients with previous covalent BTK inhibitor resistance (53 67% of 79), covalent BTK inhibitor intolerance (22 52% of 42), BTK C481-mutant (17 71% of 24) and BTK wild-type (43 66% of 65) disease. In 52 efficacy evaluable patients with mantle cell lymphoma (MCL) previously treated with covalent BTK inhibitors, the ORR was 52% (95% CI 38–66). Of 117 patients with CLL, SLL, or MCL who responded, all but eight remain progression-free to date.
Pirtobrutinib was safe and active in multiple B-cell malignancies, including patients previously treated with covalent BTK inhibitors. Pirtobrutinib might address a growing unmet need for alternative therapies for these patients.
Loxo Oncology.
Abstract
Background
During antiretroviral treatment (ART) with plasma HIV RNA below the limit of quantification, HIV RNA can be detected in genital or rectal secretions, termed discordant shedding ...(DS). We hypothesized that proliferating cells produce virions without HIV replication.
Methods
ART-naive Peruvians initiating ART were observed for DS over 2 years. HIV env and pol genomes were amplified from DS. Antiretrovirals and cytokines/chemokines concentrations were compared at DS and control time points.
Results
Eighty-two participants had ART suppression. DS was detected in 24/82 (29%) participants: 13/253 (5%) cervicovaginal lavages, 20/322 (6%) seminal plasmas, and 6/85 (7%) rectal secretions. HIV RNA in DS specimens was near the limit of quantification and not reproducible. HIV DNA was detected in 6/13 (46%) DS cervicovaginal lavages at low levels. Following DNase treatment, 5/39 DS specimens yielded HIV sequences, all without increased genetic distances. Women with and without DS had similar plasma antiretroviral levels and DS in 1 woman was associated with inflammation.
Conclusions
HIV RNA and DNA sequences and therapeutic antiretroviral plasma levels did not support HIV replication as the cause of DS from the genital tract. Rather, our findings infer that HIV RNA is shed due to proliferation of infected cells with virion production.
Findings suggest that, during ART suppression, discordant shedding of HIV in genital secretions arises from HIV-infected cells producing virions near the limit of quantification in association with infected cell proliferation. HIV replication isolated to the genital tract was not detected.
The post-mortem inspection process of livestock viscera at abattoirs is expensive and laborious, but it is essential for the detection and condemnation of defective edible organs and carcases due to ...food safety issues. Lesions in hearts, kidneys, livers, lungs, and their associated lymph nodes are amongst the most common offal defects found in abattoirs. Visible (VIS) and short-wave infrared (SWIR) hyperspectral imaging implemented in a multisensory platform were used to differentiate between sheep parenchymatous organs passed as fit (Healthy, n = 42) or not fit (Diseased, n = 47) for human consumption. Partial least squares discriminant analysis (PLS-DA) and random forest (RF) were used to classify organs as healthy or diseased in heart (n = 28), kidney (n = 15), liver (n = 24), and lung (n = 22). PLS-DA produced equal or greater classification accuracy and sensitivity than RF for all organs except for lung when the VIS sensor was used (means 84.4% and 78.3%, respectively). Livers and hearts (86.9%) showed higher accuracy than lungs and kidneys (75.9%). Limited differences occurred between VIS and SWIR sensors, although a single sensor tended to be more accurate than a combination of both. SWIR outperformed VIS in accuracy across all organs (84.8% vs. 76.3%), and the combination of VIS and SWIR was also accurate (83.0%). The use of hyperspectral imaging is an attractive proposition for the meat processing industry as a non-invasive imaging technology to detect defects in offal, and it can also provide automatic detection, saving time and labour costs.
•ST612-MRSA in Australia form distinct equine/veterinarian-associated and human-associated clades.•Bacteraemia-causing ST612-MRSA isolate in Western Australia is of the equine and ...veterinarian-associated ST612 clade.•ST612-MRSA is closely related to ST8 USA500 and carries conserved insertion sequences in virulence-modulating genes.•Rifampicin use in equine veterinary practice may encourage colonisation of horses by ST612-MRSA.
Staphylococcus aureus is a serious human and animal pathogen. Multilocus sequence type 612 (ST612) is the dominant methicillin-resistant S. aureus (MRSA) clone in certain South African hospitals and is sporadically isolated from horses and horse-associated veterinarians in Australia. Colonisation and infection by ST612-MRSA is increasing in Western Australia. Whole-genome sequencing was performed for 51 isolates of ST612-MRSA from Western Australian patients and healthcare workers, South African hospital patients, Australian veterinarians and New South Wales horses. Core genome phylogenies suggested that Australian equine and veterinarian-associated ST612-MRSA were monophyletic. Individual Western Australian isolates grouped either with this equine-associated lineage or more diverse lineages related to those in South African hospitals. Bioinformatic analyses of the complete ST612-MRSA reference genome SVH7513 confirmed that ST612-MRSA was closely related to ST8 USA500 MRSA. Common use of rifampicin in South Africa and equine veterinarian practice may favour ST612-MRSA in these settings. Humans and horses colonised with ST612-MRSA are potential reservoirs for MRSA in Australia.
The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drug-resistant bacteria that are adapted to thrive in hospitalized patients. Of particular ...concern are globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clones that cause outbreaks and epidemics associated with health care. The most rapidly spreading and tenacious health-care-associated clone in Europe currently is EMRSA-15, which was first detected in the UK in the early 1990s and subsequently spread throughout Europe and beyond. Using phylogenomic methods to analyze the genome sequences for 193 S. aureus isolates, we were able to show that the current pandemic population of EMRSA-15 descends from a health-care-associated MRSA epidemic that spread throughout England in the 1980s, which had itself previously emerged from a primarily community-associated methicillin-sensitive population. The emergence of fluoroquinolone resistance in this EMRSA-15 subclone in the English Midlands during the mid-1980s appears to have played a key role in triggering pandemic spread, and occurred shortly after the first clinical trials of this drug. Genome-based coalescence analysis estimated that the population of this subclone over the last 20 yr has grown four times faster than its progenitor. Using comparative genomic analysis we identified the molecular genetic basis of 99.8% of the antimicrobial resistance phenotypes of the isolates, highlighting the potential of pathogen genome sequencing as a diagnostic tool. We document the genetic changes associated with adaptation to the hospital environment and with increasing drug resistance over time, and how MRSA evolution likely has been influenced by country-specific drug use regimens.
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