While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S-transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.

Dengue virus is a single positive-strand RNA virus that is composed of three structural proteins including capsid, envelope, and precursor membrane while seven non-structural proteins (NS1, NS2A, NS2B, NS3A, NS3B, NS4, and NS5). Dengue is a viral infection caused by the dengue virus (DENV). DENV infections are asymptomatic or produce only mild illness. However, DENV can occasionally cause more severe cases and even death. There is no specific treatment for dengue virus infections. Therapeutic peptides have several important advantages over proteins or antibodies: they are small in size, easy to synthesize, and have the ability to penetrate the cell membranes. They also have high activity, specificity, affinity, and less toxicity. Based on the known peptide inhibitor, the current study designs peptide inhibitors for dengue virus envelope protein using an alanine and residue scanning technique. By replacing I21 with Q21, L14 with H14, and V28 with K28, the binding affinity of the peptide inhibitors was increased. The newly designed peptide inhibitors with single residue mutation improved the binding affinity of the peptide inhibitors. The inhibitory capability of the new promising peptide inhibitors was further confirmed by the utilization of MD simulation and free binding energy calculations. The molecular dynamics simulation demonstrated that the newly engineered peptide inhibitors exhibited greater stability compared to the wild-type peptide inhibitors. According to the binding free energies MM(GB)SA of these developed peptides, the first peptide inhibitor was the most effective against the dengue virus envelope protein. All peptide derivatives had higher binding affinities for the envelope protein and have the potential to treat dengue virus-associated infections. In this study, new peptide inhibitors were developed for the dengue virus envelope protein based on the already reported peptide inhibitor.

Leishmaniasis is a fatal disease caused by the leishmania parasite. For the survival of the leishmania parasite, Sterol C24-Methyl Transferase (SMT) is essential which is an enzyme of the ergosterol pathway. SMT protein mutation is responsible for Amphotericin-B drug resistance in Leishmania, which is the main treatment for visceral leishmaniasis. Amphotericin-B resistance is caused by three mutated residues V131I, V321I and F72C. The underlying mechanisms and structural changes in SMT enzymes responsible for resistance due to mutation are still not well understood. In the current study, the potential mechanism of resistance due to these mutations and the structure variation of wild and mutant SMT proteins were investigated through molecular dynamics simulations and molecular docking analysis. The results showed that AmB established strong bonding interaction with wild SMT as compare to mutants SMT. The binding energy calculation showed that binding energy of AmB with mutants SMT increases as compare to the wild SMT. Further structural based virtual screening was carried out to design potential inhibitors for the mutant SMT. On the basis of structural-based virtual screening four inhibitors (SANC01057, SANC00882, SANC00414, SANC01047) were computationally identified as potential mutant SMT (F72C) inhibitors. This work provides valuable information for improved management of drug resistant Leishmaniasis.Communicated by Ramaswamy H. Sarma.

The Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) serves as a molecular switch, cycling between guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound states. KRAS modulates numerous signal transduction pathways including the conventional RAF-MEK-ERK pathway. Mutations in the RAS genes have been linked to the formation of malignant tumors. Human malignancies typically show mutations in the Ras gene including HRAS, KRAS, and NRAS. Among all the mutations in exon 12 and exon 13 of the KRAS gene, the G12D mutation is more prevalent in pancreatic and lung cancer and accounts for around 41% of all G12 mutations, making them potential anticancer therapeutic targets. The present study is aimed at repurposing the peptide inhibitor KD2 of the KRAS G12D mutant. We employed an in-silico mutagenesis approach to design novel peptide inhibitors from the experimentally reported peptide inhibitor, and it was found that substitutions (N8W, N8I, and N8Y) might enhance the peptide\'s binding affinity toward the KRAS. Molecular dynamics simulations and binding energy calculations confirmed that the newly designed peptide inhibitors are stable and that their binding affinities are stronger as compared to the wild-type peptide. The detailed analysis revealed that newly designed peptides have the potential to inhibit KRAS/Raf interaction and the oncogenic signal of the KRAS G12D mutant. Our findings strongly suggest that these peptides should be tested and clinically validated to combat the oncogenic activity of KRAS.Communicated by Ramaswamy H. Sarma.

Fibroblast growth factor 10 functions as a paracrine mesenchymal molecule to initiate signalling pathways regarding to cellular development and health. However, the low thermal stability restricts it\'s functionality in the human body and the shelf-life of FGF10-based formulations. The current study aimed to employ rational design and bioinformatics approaches to identify some point mutations which may improve the thermal stability of FGF10. Bioinformatics analyses resulted in N105D, C106F, K144R, K153M and I156R as the potential stability conferring mutations. The identified mutants were subjected to MD simulation indicating that all mutations are both structurally and energetically favoured. Finally, the effects of the identified mutations on receptor binding of FGF10 were predicted and the results showed that K144R and K153M mutations may increase the binding affinity relative to the wild type. The findings of the current study propose potentially improved FGF10 analogues for further experimental investigations.

UNASSIGNED: Drug resistance caused by G1202R/G1202del mutation in anaplastic lymphoma kinase (ALK) represents a great challenge in the clinic. The effect of other mutation(s) at G1202 on the available tyrosine kinase inhibitors (TKIs) in the clinic remains unknown.
UNASSIGNED: A 50-year-old Chinese male non-smoker with lung adenocarcinoma progressed with spinal metastasis after receiving chest radiation together with Pemetrexed and Cisplatin as adjuvant chemotherapy. Targeted next generation sequencing (NGS) identified EML4-ALK gene fusion in the resected left lung tissue. Local radiation followed by Crizotinib were used in the following treatment and the spinal metastasis was found to shrink, but the progression free survival (PFS) only lasted for 2 months with the appearance of brain metastasis. Afterwards, the patient benefited from the therapy of Alectinib with a PFS of 8 months. Then he progressed with metastases in right lung and pleural, and did not show response to the chemotherapy with Docetaxel plus Bevacizumab. The targeted sequencing consistently identified EML4-ALK gene fusion in both plasma and pleural effusion (PE), as well as a novel ALK G1202K mutation (c.3604_3605delGGinsAA). Given the lack of established or known drug treatment for this novel mutation, we implemented molecular dynamics (MD) simulation-guided drug sensitivity prediction, which results suggested Lorlatinib remains potent against G1202K mutant ALK. Therefore, Lorlatinib was used as the fourth-line therapy, which lead to the considerable efficacy with improved performance status (PS) score and reduced lung metastases. The structural mechanism underlying G1202K-induced drug resistance to different ALK-TKIs was also discussed.
UNASSIGNED: Our case suggested the ALK-G1202K mutation may serve as a novel mechanism underlying the resistance to Alectinib, and provide direct evidence to support its sensitization to Lorlatinib. Our work represented an example of integrating in silico predictions into clinical practice.

Designing dual small molecule inhibitors against enzymes associated with cancer has turned into a new strategy in cancer chemotherapy. Targeting DNA methyltransferase (DNMT) and histone deacetylase (HDAC) enzymes, involved in epigenetic modifications, are considered as promising treatments for a wide range of cancers, due to their association with the initiation, proliferation, and survival of cancer cells. In this study, for the first time, the dual inhibitors of the histone deacetylases 8 (HDAC8) and DNA methyltransferase 1 (DNMT1) has introduced as novel potential candidates for epigenetic-based cancer therapeutics. This research has been facilitated by employing pharmacophore-based virtual screening of ZINC and Maybridge databases, as well as performing molecular docking, molecular dynamics simulations and free binding energy calculation on the top derived compound. Results have demonstrated that the suggested compounds not only adopt highly favorable conformations but also possess strong binding interaction with the HDAC8 enzyme. Additionally, the obtained results from the experimental assay confirmed the predicted behavior of inhibitors from virtual screening. These results can be used for further optimization to yield promising more effective candidates for the treatment of cancer.Communicated by Ramaswamy H. Sarma.