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Abstract: This study describes the affinity maturation, molecular engineering, and preclinical assessment of depemokimab, an enhanced anti-interleukin-5 antagonist antibody. The molecular design objective for depemokimab was to generate a therapeutic antibody enabling a less frequent dosing regimen of once every 6 months compared with every 4 weeks for mepolizumab. Mepolizumab is a marketed monoclonal antibody used as an add-on prescription maintenance treatment for patients with severe asthma with an eosinophilic phenotype and other eosinophilic-associated disorders. A complementarity-determining region restricted affinity maturation strategy was used where affinity improved interleukin-5 binding antibody variants were subject to affinity driven selective pressure and identified using the Adimab yeast-based platform. Improved complementarity-determining region variants were combined with serum half-life extending amino acid mutations introduced into the fragment crystallizable region of the antibody. When compared with mepolizumab, depemokimab demonstrated improved in vitro interleukin-5 neutralization in a TF-1 (human erythroleukemia) functional cell assay. In vivo, depemokimab displayed significantly extended pharmacokinetic performance and pharmacodynamic duration determined via eosinophil suppression in cynomolgus monkey (Macaca fascicularis). These data provide compelling evidence that a less frequent dosing regimen for depemokimab in humans is possible and supported the advancement of depemokimab into a Phase I study in patients with asthma.

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Abstract: The trematode liver fluke Fasciola hepatica causes the neglected tropical disease fascioliasis in humans and is associated with significant losses in agricultural industry due to reduced animal productivity. Triosephosphate isomerase (TPI) is a glycolytic enzyme that has been researched as a drug target for various parasites, including F. hepatica. The high-resolution crystal structure of F. hepatica TPI (FhTPI) has been solved at 1.51 Å resolution in its monoclinic form. The structure has been used to perform molecular-docking studies with the most successful fasciolocide triclabendazole (TCBZ), which has recently been suggested to target FhTPI. Two FhTPI residues, Lys50 and Asp51, are located at the dimer interface and are found in close proximity to the docked TCBZ. These residues are not conserved in mammalian hosts.

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Abstract: Enzymes from salt-in halophiles are stable in conditions of low water activity with applications in chiral synthesis requiring organic solvents, yet the origins of such stability remains poorly understood. Here we describe the molecular basis of the reaction mechanism and dual NADH/NADPH-specificity of D2HDH, a 2-hydroxyacid dehydrogenase from the extreme halophile Haloferax mediterranei, an organism whose proteins have to remain active in high intracellular concentrations of KCl. Halophilic adaptations of D2HDH include the expected acidic surface and a reduction in hydrophobic surface resulting from a lower lysine content. Structure determination of crystals of D2HDH grown with KCl showed that bound K+ ions were coordinated predominantly by clusters of main chain protein carbonyl ligands, with no involvement of the numerous exposed surface carboxyls. Structural comparisons identified similar sites in other halophilic proteins suggesting that the generic use of carbonyl clusters to coordinate K+ ions may also contribute in a carboxylate-independent way to the stabilisation of the folded state of the protein in its high salt environment.

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Abstract: Enterobacterales, a large order of Gram-negative bacteria, including Escherichia coli and Klebsiella pneumoniae, are major causes of urinary tract and gastrointestinal infections, pneumonia, and other diseases in healthcare settings and communities. ESBL-producing Enterobacterales and carbapenem-resistant Enterobacterales can break down commonly used antibiotics, with some strains being resistant to all available antibiotics. This public health threat necessitates the development of novel antibiotics, ideally targeting new pathways in these bacteria. Gram-negative bacteria possess an outer membrane enriched with lipid A, a saccharolipid that serves as the membrane anchor of lipopolysaccharides and the active component of the bacterial endotoxin, causing septic shock. The biosynthesis of lipid A is crucial for the viability of Gram-negative bacteria, and as an essential enzyme in this process, LpxH has emerged as a promising target for developing novel antibiotics against multidrug-resistant Gram-negative pathogens. Here, we report the development of pyridinyl sulfonyl piperazine LpxH inhibitors. Among them, ortho-substituted pyridinyl compounds significantly boost LpxH inhibition and antibiotic activity over the original phenyl series. Structural and QM/MM analyses reveal that these improved activities are primarily due to the enhanced interaction between F141 of the LpxH insertion lid and the pyridinyl group. Incorporation of the N-methyl-N-phenyl-methanesulfonamide moiety into the pyridinyl sulfonyl piperazine backbone results in JH-LPH-106 and JH-LPH-107, both of which exhibit potent antibiotic activity against wild-type Enterobacterales such as K. pneumoniae and E. coli. JH-LPH-107 exhibits a low rate of spontaneous resistance and a high safety window in vitro, rendering it an excellent lead for further clinical development.

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Abstract: Herein, we report the identification and optimization of a series of potent inhibitors of EGFR Exon20 insertions with significant selectivity over wild-type EGFR. A strategically designed HTS campaign, multiple iterations of structure-based drug design (SBDD), and tactical linker replacement led to a potent and wild-type selective series of molecules and ultimately the discovery of 36. Compound 36 is a potent and selective inhibitor of EGFR Exon20 insertions and has demonstrated encouraging efficacy in NSCLC EGFR CRISPR-engineered H2073 xenografts that carry an SVD Exon20 insertion and reduced efficacy in a H2073 wild-type EGFR xenograft model compared to CLN-081 (5), indicating that 36 may have lower EGFR wild-type associated toxicity.