The use of virtual screening and differential scanning fluorimetry for the rapid identification of fragments active against MEK1.

We report the analysis of an in-house fragment screening campaign for the oncology target MEK1. The application of virtual screening (VS) as a primary fragment screening approach, followed by biophysical validation using differential screening fluorimetry (DSF), with resultant binding mode determination by X-ray crystallography (X-ray), is presented as the most time and cost-effective combination of in silico and in vitro methods to identify fragments. We demonstrate the effectiveness of the VS-DSF workflow for the early identification of fragments to both 'jump-start' the drug discovery project and to complement biochemical screening data.

KRAS G12C fragment screening renders new binding pockets.

KRAS genes belong to the most frequently mutated family of oncogenes in cancer. The G12C mutation, found in a third of lung, half of colorectal and pancreatic cancer cases, is believed to be responsible for a substantial number of cancer deaths. For 30 years, KRAS has been the subject of extensive drug-targeting efforts aimed at targeting KRAS protein itself, but also its post-translational modifications, membrane localization, protein-protein interactions and downstream signalling pathways. So far, most KRAS targeting strategies have failed, and there are no KRAS-specific drugs available. However, clinical candidates targeting the KRAS G12C protein have recently been developed. MRTX849 and recently approved Sotorasib are covalent binders targeting the mutated cysteine 12, occupying Switch II pocket.Herein, we describe two fragment screening drug discovery campaigns that led to the identification of binding pockets on the KRAS G12C surface that have not previously been described. One screen focused on non-covalent binders to KRAS G12C, the other on covalent binders.

Genetically detoxified pertussis toxin displays near identical structure to its wild-type and exhibits robust immunogenicity.

The mutant gdPT R9K/E129G is a genetically detoxified variant of the pertussis toxin (PTx) and represents an attractive candidate for the development of improved pertussis vaccines. The impact of the mutations on the overall protein structure and its immunogenicity has remained elusive. Here we present the crystal structure of gdPT and show that it is nearly identical to that of PTx. Hydrogen-deuterium exchange mass spectrometry revealed dynamic changes in the catalytic domain that directly impacted NAD+ binding which was confirmed by biolayer interferometry. Distal changes in dynamics were also detected in S2-S5 subunit interactions resulting in tighter packing of B-oligomer corresponding to increased thermal stability. Finally, antigen stimulation of human whole blood, analyzed by a previously unreported mass cytometry assay, indicated broader immunogenicity of gdPT compared to pertussis toxoid. These findings establish a direct link between the conserved structure of gdPT and its ability to generate a robust immune response.

Discovery of 6-(2,4-Dichlorophenyl)-5-[4-[(3S)-1-(3-fluoropropyl)pyrrolidin-3-yl]oxyphenyl]-8,9-dihydro-7H-benzo[7]annulene-2-carboxylic acid (SAR439859), a Potent and Selective Estrogen Receptor Degrader (SERD) for the Treatment of Estrogen-Receptor-Positive Breast Cancer.

More than 75% of breast cancers are estrogen receptor alpha (ERα) positive (ER+), and resistance to current hormone therapies occurs in one-third of ER+ patients. Tumor resistance is still ERα-dependent, but mutations usually confer constitutive activation to the hormone receptor, rendering ERα modulator drugs such as tamoxifen and aromatase inhibitors ineffective. Fulvestrant is a potent selective estrogen receptor degrader (SERD), which degrades the ERα receptor in drug-resistant tumors and has been approved for the treatment of hormone-receptor-positive metastatic breast cancer following antiestrogen therapy. However, fulvestrant shows poor pharmacokinetic properties in human, low solubility, weak permeation, and high metabolism, limiting its administration to inconvenient intramuscular injections. This Drug Annotation describes the identification and optimization of a new series of potent orally available SERDs, which led to the discovery of 6-(2,4-dichlorophenyl)-5-[4-[(3S)-1-(3-fluoropropyl)pyrrolidin-3-yl]oxyphenyl]-8,9-dihydro-7H-benzo[7]annulene-2-carboxylic acid (43d), showing promising antitumor activity in breast cancer mice xenograft models and whose properties warranted clinical evaluation.

An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery.

Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns.

In vitro activity of AVE1330A, an innovative broad-spectrum non-beta-lactam beta-lactamase inhibitor.

OBJECTIVES: Production of beta-lactamases is the main mechanism of beta-lactam resistance in Gram-negative bacteria. Despite the current use of clavulanic acid, sulbactam and tazobactam, the prevalence of class A and class C enzymes is increasing worldwide, demanding new beta-lactamase inhibitors. Here we report the antimicrobial properties of AVE1330A, a representative of a novel class of bridged bicyclico[3.2.1]diazabicyclo-octanones in combination with ceftazidime. MATERIALS AND METHODS: IC(50) and kinetic parameters of the hydrolysis reaction were used to characterize beta-lactamase inhibition by AVE1330A. MICs for >600 strains were determined with the combination ceftazidime/AVE1330A at a fixed ratio of 4:1. RESULTS: IC(50)s of AVE1330A for TEM-1 and P99 enzymes were 0.0023 mg/L (8 nM) and 0.023 mg/L (80 nM), compared with 0.027 mg/L (130 nM) and 205.1 mg/L (1 x 10(6) nM) of clavulanic acid and 0.013 mg/L (40 nM) and 1.6 mg/L (5000 nM) of tazobactam. A highly stable covalent complex led to a low turnover of AVE1330A. MICs of ceftazidime/AVE1330A for Enterobacteriaceae were at least eight-fold lower than those of ceftazidime alone. All of the Escherichia coli, Klebsiella pneumoniae, Citrobacter and Proteus mirabilis strains, including ceftazidime-resistant isolates, were inhibited at 4-8 mg/L. Only 2 mg/L were required to inhibit other Proteeae, Enterobacter, Salmonella and Serratia. CONCLUSION: The combination of ceftazidime with AVE1330A exhibited broad-spectrum activity against Ambler class A- and class C-producing Enterobacteriaceae.