Enhancing bezlotoxumab binding to C. difficile toxin B2: insights from computational simulations and mutational analyses for antibody design.

Clostridioides difficile infection (CDI) is a significant concern caused by widespread antibiotic use, resulting in diarrhea and inflammation from the gram-positive anaerobic bacterium C. difficile. Although bezlotoxumab (Bez), a monoclonal antibody (mAb), was developed to address CDI recurrences, the recurrence rate remains high, partly due to reduced neutralization efficiency against toxin B2. In this study, we aimed to enhance the binding of Bez to C. difficile toxin B2 by combining computational simulations and mutational analyses. We identified specific mutations in Bez, including S28R, S31W/K, Y32R, S56W and G103D/S in the heavy chain (Hc), and S32F/H/R/W/Y in the light chain (Lc), which significantly improved binding to toxin B2 and formed critical protein-protein interactions. Through molecular dynamics simulations, several single mutations, such as HcS28R, LcS32H, LcS32R, LcS32W and LcS32Y, exhibited superior binding affinities to toxin B2 compared to Bez wild-type (WT), primarily attributed to Coulombic interactions. Combining the HcS28R mutation with four different mutations at residue LcS32 led to even greater binding affinities in double mutants (MTs), particularly HcS28R/LcS32H, HcS28R/LcS32R and HcS28R/LcS32Y, reinforcing protein-protein binding. Analysis of per-residue decomposition free energy highlighted key residues contributing significantly to enhanced binding interactions, emphasizing the role of electrostatic interactions. These findings offer insights into rational Bez MT design for improved toxin B2 binding, providing a foundation for developing more effective antibodies to neutralize toxin B2 and combat-related infections.Communicated by Ramaswamy H. Sarma.

Corosolic Acid Induced Apoptosis via Upregulation of Bax/Bcl-2 Ratio and Caspase-3 Activation in Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA), an aggressive malignancy arising from the biliary epithelium, exhibits a high incidence in Thailand. CCA usually lacks specific symptoms and is typically diagnosed in its advanced stages, presenting significant treatment challenges. Current CCA therapeutic options, including surgery, chemotherapy, and radiation, have limited success rates and often cause side effects. Nature-derived compounds hold promise for reducing undesirable adverse effects and are an excellent source of anticancer drugs. Corosolic acid (CA), a triterpenoid found in Lagerstroemia speciosa L. leaves, exhibits anticancer properties; however, the effectiveness of CA against CCA and its molecular mechanisms remained unexplored. Herein, the anti-CCA and apoptosis-inducing effects of CA were investigated using various techniques, i.e., the MTT assay, flow cytometry with FITC-labeled Annexin V (Annexin V-FITC) and propidium iodide double staining, JC-1 staining, western blot analysis, caspase-3 activity assay, and molecular dynamics (MD) simulations. CA inhibited the proliferation of KKU-213A and KKU-213B CCA cells and triggered apoptosis through alterations in mitochondrial membrane potential (ΔΨm), and increases in the Bax/Bcl-2 expression ratio, cytochrome c release, and caspase-3 activity. As indicated by MD simulations, CA has the potential to bind to Bcl-2 through hydrogen bonds between amino acid residues R146 and N143. These findings underscore the potential of CA as a promising candidate for treatment of CCA.

Elucidation of bezlotoxumab binding specificity to toxin B in Clostridioides difficile.

C. difficile or Clostridioides difficile infection (CDI) is currently one of the major causes of epidemics worldwide. Toxin B from Clostridioides difficile toxin B (TcdB) infection is the main target protein inhibiting CDI recurrence. Clinical research suggested that bezlotoxumab's (Bez) efficiency is significantly reduced in neutralizing the B2 strain compared to the B1 strain. The monoclonal antibody (mAb) functions by binding to the epitope 1 and 2 regions in the combined repetitive oligopeptide (CROP) domain. Some binding residues are distinctively different between B1 and B2 strains. In this work, we aimed to elucidate and compare insights into the interaction of toxins B1 and B2 in complex with Bez by using all-atom molecular dynamics (MD) simulations and binding free energy calculations. The predicted ΔGbinding values suggested that the antibody (Ab) could bind to toxin B1 significantly better than B2, supported by higher salt bridge and hydrogen bonding (H-bonding) interactions, as well as the number of contact residues between the two focused proteins. The toxin B1 residues important for binding with Bez were E1878, T1901, E1902, F1905, N1941, V1946, N2031, T2032, E2033, V2076, V2077, and E2092. The lower susceptibility of Bez towards toxin B2 was primarily due to a change of residue E2033 from glutamate to alanine (A2033) and the loss of E1878 and E1902 contributions, as determined by the intermolecular interaction changes from the dynamic residue interaction network (dRIN) analysis. The obtained data strengthen our understanding of Bez/toxin B binding.

Discovery of Novel Naphthoquinone-Chalcone Hybrids as Potent FGFR1 Tyrosine Kinase Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling.

This work presents a flexible synthesis of 10 novel naphthoquinone-chalcone derivatives (1-10) by nucleophilic substitution of readily accessible aminochalcones and 2,3-dichloro-1,4-naphthoquinone. All compounds displayed broad-spectrum cytotoxic activities against all the tested cancer cell lines (i.e., HuCCA-1, HepG2, A549, MOLT-3, T47D, and MDA-MB-231) with IC50 values in the range of 0.81-62.06 µM, especially the four most potent compounds 1, 3, 8, and 9. The in vitro investigation on the fibroblast growth factor receptor 1 (FGFR1) inhibitory effect indicated that eight derivatives (1-2, 4-5, and 7-10) were active FGFR1 inhibitors (IC50 = 0.33-3.13 nM) with more potency than that of the known FGFR1 inhibitor, AZD4547 (IC50 = 12.17 nM). Promisingly, compounds 5 (IC50 = 0.33 ± 0.01 nM), 9 (IC50 = 0.50 ± 0.04 nM), and 7 (IC50 = 0.85 ± 0.08 nM) were the three most potent FGFR1 inhibitors. Molecular docking, molecular dynamics simulations, and MM/GBSA-based free energy calculation revealed that the key amino acid residues involved in the binding of the compounds 5, 7, and 9 and the target FGFR1 protein were similar with those of the AZD4547 (i.e., Val492, Lys514, Ile545, Val561, Ala640, and Asp641). These findings revealed that the newly synthesized naphthoquinone-chalcone scaffold is a promising structural feature for an efficient inhibition of FGFR1.

BI6727 and GSK461364A, potent PLK1 inhibitors induce G2/M arrest and apoptosis against cholangiocarcinoma cell lines.

Polo-like kinase 1 (PLK1) is an essential mitotic checkpoint protein that plays a key role in cell cycle division. Overexpression of PLK1 has been associated with poor prognosis in various cancers. Cholangiocarcinoma (CCA) is a lethal bile duct cancer and the current treatments in inoperable patients have not been satisfactory. In order to develop novel targeted therapies, we investigated the efficacy of BI6727 (volasertib) and GSK461364A, polo-like kinase 1 (PLK1) inhibitors in KKU-100 and KKU-213A CCA cell lines. PLK1 expression was significantly up-regulated in CCA cases compared with normal tissues based on the results derived from GEPIA. Western blot results exhibited PLK1 protein expression in both CCA cell lines. Molecular dynamics simulations and free energy calculations based on MM/GBSA method revealed that BI6727-PLK1 and GSK461364A-PLK1 complexes were stable in an aqueous environment, and their complexation was mainly driven by Van der Waals interaction. BI6727 and GSK461364A clearly suppressed CCA cell proliferation and induced G2/M arrest, accompanied with upregulation of cyclin B1 and phosphorylated Histone H3 at Ser10 (pS10H3), specific markers of mitosis. Furthermore, both compounds triggered mitotic catastrophe followed by cell apoptosis via activation of PARP and Caspase 3, as well as downregulation of Mcl-1 anti-apoptotic protein in both CCA cell lines. In conclusion, pharmacologic PLK1 inhibition by BI6727 and GSK461364A blocked survival of CCA cells by several mechanisms. Our study provides evidence that BI6727 and GSK461364A could be alternative drugs and have potential implications at the clinical level for CCA therapy.