Exploring the potential of structural modeling and molecular docking for efficient siRNA screening: A promising approach to Combat viral mutants, with a focus on HIV-1.

RNA interference (RNAi) holds immense potential for sequence-specific downregulation of disease-related genes. Small interfering RNA (siRNA) therapy has made remarkable strides, with FDA approval for treating specific human diseases, showcasing its promising future in disease treatment. Designing highly efficient siRNAs is a critical step in this process. Previous studies have introduced various algorithms and parameters for siRNA design and scoring. However, these attempts have often fallen short of meeting all essential criteria or required modifications, resulting in variable and unclear effectiveness of screened siRNAs, particularly against viral mutants with non-conserved short sequences. In this study, we present a fully optimized siRNA screening system considering all necessary parameters. Notably, we highlight the critical role of molecular docking simulations between siRNA and two functional domains of the Argonaute protein (PAZ and PIWI) in identifying the most efficient siRNAs, since the appropriate interaction between the guide siRNA strand and the RISC complex is crucial. Through our stringent method, we designed approximately 50 potential siRNAs targeting the HIV-1 vpr gene. Evaluation through XTT, qRT-PCR, and flow cytometry analysis on RAW 264.7 macrophage stable cells revealed negligible cytotoxicity and exceptional gene-silencing efficiency at both the transcriptional and translational levels for the top-ranked screened siRNAs. Given the growing interest in siRNA-based therapeutics, we anticipate that the insights from this study will contribute to improving treatment strategies against mutant viruses, particularly HIV-1.

Characterization of recombinant pumpkin 2S albumin and mutation studies to unravel potential DNA/RNA binding site.

The native pumpkin 2S albumin, a multifunctional protein, possess a variety of potential biotechnologically exploitable properties. The present study reports the characterization of recombinant pumpkin 2S albumin (rP2SA) and unraveling of its potential DNA/RNA binding site. The purification and characterization of the rP2SA established that it retains the characteristic α-helical structure and exhibited comparable DNase, RNase, antifungal and anti-proliferative activities as native protein. In vitro studies revealed that rP2SA exhibits potent antiviral activity against chikungunya virus (CHIKV) at a non-toxic concentration with an IC50 of 114.5 µg/mL. In silico studies and site-directed mutagenesis were employed to unravel the potential DNA/RNA binding site. A strong positive charge distribution due to presence of many arginine residues in proximity of helix 5 was identified as a potential site. The two of the arginine residues, conserved in some 2S albumins, were selected for the mutation studies. The mutated forms of recombinant protein (R84A and R91A) showed a drastic reduction in DNase and RNase activities suggesting their presence at binding site and involvement in the nuclease activity. A metal binding site was also identified adjacent to DNA/RNA binding site. The present study demonstrated the structural and functional integrity of the rP2SA and reports potential antiviral activity against CHIKV. Further, potential DNA/RNA binding site was unraveled through mutation studies and bioinformatics analysis.

The analysis of subtle internal communications through mutation studies in periplasmic metal uptake protein CLas-ZnuA2.

The subtle internal communications through an intricate network of interactions play a key role in metal-binding and release in periplasmic metal uptake proteins of cluster A-I family, a component of ABC transport system. These proteins have evolved different mechanisms of metal-binding and release through sequence and thereby structure-function divergence. The CLas-ZnuA2 from Candidatus Liberibacter asiaticus (CLA), in previous studies, showed a lower metal-binding affinity. The subtle communications within and between domains from crystal structure analysis revealed that protein seems to prefer a metal-free state. The unique features of CLas-ZnuA2 included a highly restrained loop L3 and presence of a proline in linker helix. In present work, S38A and Y68F mutants were studied as they play an important role during metal-binding in CLas-ZnuA2. The mutations in linker helix could not be studied as the expressed protein was not soluble and in most cases degraded with time. The crystal structure analysis of (S38A and Y68F) mutants in metal-free and metal-bound forms showed variations in interactions, an increase in number of alternate conformations and distortions in secondary structure elements, despite a similar overall structure, suggesting alterations in internal communications. The results suggested that any change in critical residues could alter the subtle internal communications and result in disturbing the fine-tuned structure required for optimal functioning.

John W. (Jan) Drake: A Biochemical View of a Geneticist Par Excellence.

John W. Drake died 02-02-2020, a mathematical palindrome, which he would have enjoyed, given his love of "word play and logic," as stated in his obituary and echoed by his family, friends, students, and colleagues. Many aspects of Jan's career have been reviewed previously, including his early years as a Caltech graduate student, and when he was editor-in-chief, with the devoted assistance of his wife Pam, of this journal for 15 impactful years. During his editorship, he raised the profile of GENETICS as the flagship journal of the Genetics Society of America and inspired and contributed to the creation of the Perspectives column, coedited by Jim Crow and William Dove. At the same time, Jan was building from scratch the Laboratory of Molecular Genetics on the newly established Research Triangle Park campus of the National Institute of Environmental Health Science, which he headed for 30 years. This commentary offers a unique perspective on Jan's legacy; we showcase Jan's 1969 benchmark discovery of antimutagenic T4 DNA polymerases and the research by three generations (and counting) of scientists whose research stems from that groundbreaking discovery. This is followed by a brief discussion of Jan's passion: his overriding interest in analyzing mutation rates across species. Several anecdotal stories are included to bring alive one of Jan's favorite phrases, "to think like a geneticist." We feature Jan's genetical approach to mutation studies, along with the biochemistry of DNA polymerase function, our area of expertise. But in the end, we acknowledge, as Jan did, that genetics, also known as in vivo biochemistry, prevails.

Binding to large enzyme pockets: small-molecule inhibitors of trypanothione reductase.

The causative agents of the parasitic disease human African trypanosomiasis belong to the family of trypanosomatids. These parasitic protozoa exhibit a unique thiol redox metabolism that is based on the flavoenzyme trypanothione reductase (TR). TR was identified as a potential drug target and features a large active site that allows a multitude of possible ligand orientations, which renders rational structure-based inhibitor design highly challenging. Herein we describe the synthesis, binding properties, and kinetic analysis of a new series of small-molecule inhibitors of TR. The conjunction of biological activities, mutation studies, and virtual ligand docking simulations led to the prediction of a binding mode that was confirmed by crystal structure analysis. The crystal structures revealed that the ligands bind to the hydrophobic wall of the so-called "mepacrine binding site". The binding conformation and potency of the inhibitors varied for TR from Trypanosoma brucei and T. cruzi.