Protein Binding Sites Research Articles

Structure-based Virtual Screening, and Design of Some Lead Compounds as Inhibitors of Kras-G12D of Pancreatic Cancer

Pancreatic cancer is an abnormal cell growth in the pancreas. In 2021, approximately 60,430 individuals were diagnosed in the USA, with the annual increasing incidence rates. Pancreatic cancer is anticipated to become the second leading cause of cancer mortality by 2030. This escalating challenge has prompted a search for innovative therapeutic agents. Virtual screening, a computational technique, was employed to discover novel drug-like compounds from a diverse set of 30 chemical compounds, sourced from the PubChem database. These compounds were evaluated based on some important properties, including pharmacokinetics, lipophilicity, drug-likeness, water-solubility, and physicochemical characteristics. Seventeen compounds emerged as promising candidates for pancreatic cancer treatment. Subsequent molecular docking studies focused on the Kras-G12D protein target and identified Ligand 18 as the leading candidate, exhibiting a binding energy (BE) of -10.5 kcal mol-1 and extensive interactions with the target protein. Additionally, a newly designed compound, D4, displayed an even higher BE of -10.8 kcal mol-1, fitting more effectively into the protein's binding site than existing drugs like Gemcitabine and Irinotecan. All newly designed compounds met the five scientists' rule, indicating favorable drug-likeness and bioavailability. These findings pave the way for developing a new generation of less toxic therapeutic compounds for pancreatic cancer treatment.Keywords: virtual screening, binding energy, kras-G12D, pancreatic cancer, designed compounds.

Deep Learning for Predicting Biomolecular Binding Sites of Proteins

Deep Learning for Predicting Biomolecular Binding Sites of Proteins

Reaction and interaction dynamics of azobenzene-tethered DNA with T7 RNA polymerase.

Reaction and interaction dynamics of azobenzene-tethered DNA (photoresponsive DNA) with T7 RNA polymerase (T7RNAP) were studied after photoisomerization of azobenzene from the cis- to trans-forms using the transient grating (TG) and time-resolved fluorescence polarization techniques. Two types of photoresponsive DNA were examined: AzoPBD, tethered at the protein binding site, and AzoTATA, tethered at the unwinding site. A diffusion change was observed after photoexcitation of cis-AzoPBD within 1 ms, and this change is explained in terms of a structural change from a bent to an extended conformation upon the cis-to-trans photoisomerization. The association and dissociation rates of cis- and trans-AzoPBD-T7RNAP complex were measured by the fluorescence polarization measurements, and the dissociation constants for cis- and trans-AzoPBD were respectively determined to be 3.9 μM and 21 μM. The result indicates that trans-AzoPBD is dissociated upon photoexcitation of cis-AzoPBD-T7RNAP complex. The efficient dissociation is mainly caused by a small association rate constant. The time-resolved diffusion measurement showed a conformational change with a time constant of 2.4 ms and a dissociation reaction with a slower rate, which depends on the concentration of T7RNAP. Although AzoTATA does not exhibit significant structural changes upon isomerization, a diffusion change was observed upon photoexcitation of AzoTATA-T7RNAP. The origin is attributed to changes from the unwound to closed states of AzoTATA, but AzoTATA does not dissociate from T7RNAP. These findings highlight a critical role of the azobenzene insertion position in modulating DNA-T7RNAP interaction dynamics, providing new insights into light-regulated transcription control.

QM Investigation of Rare Earth Ion Interactions with First Hydration Shell Waters and Protein-Based Coordination Models.

Conventional methods for extracting rare earth metals (REMs) from mined mineral ores are inefficient, expensive, and environmentally damaging. Recent discovery of lanmodulin (LanM), a protein that coordinates REMs with high-affinity and selectivity over competing ions, provides inspiration for new REM refinement methods. Here, we used quantum mechanical (QM) methods to investigate trivalent lanthanide cation (Ln3+) interactions with coordination systems representing bulk solvent water and protein binding sites. Energy decomposition analysis (EDA) showed differences in the energetic components of Ln3+ interaction with representatives of solvent (water, H2O) and protein binding sites (acetate, CH3COO-), highlighting the importance of accurate description of electrostatics and polarization in computational modeling of REM interactions with biological and bioinspired molecules. Relative binding free energies were obtained for Ln3+ with coordination complexes originating from binding sites in PDB structures of a lanthanum binding peptide (PDB entry 7CCO) and LanM, with explicit consideration of the first hydration shell waters, according to quasi-chemical theory (QCT). Beyond the first shell, the bulk solvent environment was represented with an implicit continuum model. Ln3+ interactions with (H2O)9 and both binding site models became more favorable, moving down the periodic series. This trend was more pronounced with the protein binding site models than with water, resulting in affinity increasing with periodic number, except for the last REM, Lu3+, which bound less favorably than the preceding element, Yb3+. Using the truncated 7CCO binding site model, the magnitude and trend of the experimental Ln3+ relative binding free energies for the whole 7CCO peptide were reproduced. Conversely, the previously reported experimental data for LanM show a preference for the earlier lanthanides; this is likely due to longer-range interactions and cooperative effects, which are not represented by the reduced models. Using the truncated 7CCO binding site model, the magnitude and trend of the experimental Ln3+ relative binding free energies for the whole 7CCO peptide were reproduced. In contrast to the previously reported experimental data for LanM, the peptide preferentially binds the earlier lanthanides. This difference likely arises due to longer-range interactions and cooperative effects not represented by the peptide. Further investigation of Ln3+ interactions with whole proteins using polarizable molecular mechanics models with explicit solvent is warranted to understand the influence of longer-ranged interactions, cooperativity, and bulk solvent. Nevertheless, the present work provides new insights into Ln3+ interactions with biomolecules and presents an effective computational platform for designing specific single-site REM binding peptides more efficiently.

Assessing the efficacy of iso-mukaadial acetate and betulinic acid against selected Plasmodium falciparum glycolytic pathway proteins: in silico and in vitro studies

Malaria is the extensive health concern in sub-Saharan Africa, with Plasmodium falciparum being the most lethal strain. The continued emergence of drug-resistant P. falciparum advocates for the development of new antimalarials. Our current study aimed to effectively explore the interaction capabilities of iso-mukaadial acetate (IMA) and betulinic acid (BA) against two essential P. falciparum glycolytic pathway proteins, PfLDH and PfHk. Recombinant PfLDH and PfHk were independently expressed in E. coli BL21 (DE3) cells and subsequently purified using affinity chromatography. Protein–ligand interaction studies probed in silico and in vitro approaches. Parasite inhibition studies confirmed potent antimalarial activity against the P. falciparum NF54 strains, with BA and IMA showing IC50 values of 1.27 µg/ml and 1.03 µg/ml against the asexual stage of P. falciparum, respectively. FTIR experiments confirmed interactions between the compounds and the secondary structure of the proteins. Direct protein–ligand interaction studies analysis using microscale thermophoresis (MST) showed a KD value of 0.1036 ± 0.6001 µM for the PfLDH-BA complex and 0.7473 ± 0.3554 µM KD value for PfLDH-IMA. Meanwhile, PfHk-IMA had 0.39701 ± 0.16298 µM KD value, while the PfHk-BA complex had no interaction detected. Molecular docking and molecular dynamics simulation studies were used to measure and confirm the interactive strength of complexes. Molecular docking reported a binding score of − 1.155 kcal/mol for the PfLDH-BA complex and a binding score of − 3.200 kcal/mol for PfLDH-IMA. The PfHk-BA complex had − 2.871 kcal/mol and PfHk-IMA complex had − 4.225 kcal/mol binding score. In conclusion, BA and IMA compounds had better interactions and remained bound within the binding sites of the glycolytic pathway proteins (PfLDH and PfHk).

Active Learning to Select the Most Suitable Reagents and One-Step Organic Chemistry Reactions for Prioritizing Target-Specific Hits from Ultralarge Chemical Spaces.

Designing chemically novel and synthesizable ligands from the largest possible chemical space is a major issue in modern drug discovery to identify early hits that are easily amenable to medicinal chemistry optimization. Starting from the sole three-dimensional structure of a protein binding site, we herewith describe a fully automated active learning protocol to propose the commercial chemical reagents and one-step organic chemistry reactions necessary to enumerate target-specific primary hits from ultralarge chemical spaces. When applied in different scenarios (single transform and multiple transforms) addressing chemical spaces of various sizes (from 670 million to 4.5 billion compounds), the method was able to recover up to 98% of virtual hits discovered by an exhaustive docking-based approach while scanning only 5% of the full chemical space. It is therefore applicable to the structure-based screening of trillion-sized chemical spaces at a very high throughput with minimal computational resources.

PPDock: Pocket Prediction-Based Protein-Ligand Blind Docking.

Predicting the docking conformation of a ligand in the protein binding site (pocket), i.e., protein-ligand docking, is crucial for drug discovery. Traditional docking methods have a long inference time and low accuracy in blind docking (when the pocket is unknown). Recently, blind docking techniques based on deep learning have significantly improved inference efficiency and achieved good docking results. However, these methods often use the entire protein for docking, which makes it difficult to identify the correct pocket and results in poor generalization performance. In this study, we propose a two-stage docking paradigm, where pocket prediction is followed by pocket-based docking. Following this paradigm, we design a new blind docking method based on pocket prediction (PPDock). Through extensive experiments on benchmark data sets, our proposed PPDock outperforms existing methods in nearly all evaluation metrics, demonstrating strong docking accuracy, generalization ability, and efficiency.

Selective Novel Metal‐Coordinated Biomedical Agents Encompassing Tetradentate Salen Ligand: Structural Elucidation, DFT Calculation, Cytotoxic, and Antioxidant Activities Supported by Molecular Docking Approach

ABSTRACTSeveral physicochemical and analytical methods were employed to elucidate the structural analysis of some novel complexes derived from the {3,4‐bis‐[(3‐ethoxy‐2‐hydroxy‐benzylidene)‐amino]‐phenyl}‐phenyl‐methanone (ESAB ligand). Decomposition point determination, elemental analysis (CHN), spectroscopy (IR, NMR, and mass spectrometry), conductivity, magnetic susceptibility, UV–Vis spectrum study, and theoretical investigations were among these methods. With conductance values ranging from 7.5 to 15.12 Ω−1 cm2 mol−1, molar conductance values showed that the Zn (II), Cu (II), and Ru (III) complexes are nonelectrolytes in fresh DMSO solutions, with the exception of the ESABRu complex, which is a monoelectrolyte. According to IR spectra, the ligand uses the (N & O) donor sites from the (C=N & C‐O) groups in the ligand moiety to coordinate through the metal ions in a tetradentate form. A 1:1 (metal:ligand) molar ratio was proposed by Job's approach based on analytical data from solution complexation. According to the stability constant (Kf) values, the complexes' stability order was found to be ESABRu > ESABCu > ESABZn. The pH profile showed that the complexes under study are stable throughout a broad pH range, usually between pH = 4 and pH = 10. The complexes' geometric structures and ligand coordination capabilities were inferred with the use of magnetic and electronic spectrum studies. The DFT method uses quantum chemical simulations to evaluate the electronic structures of the ligand under investigation and its complexes. The unbound ligand's B3LYP level, B3LYP/6/311G* degree, and the complexes' B3LYP/6/311G**/LANL2DZ functional categories were employed in the computations of the density function concept (DFT). The findings revealed the consistency between the experimental and DFT computations. Natural bond orbital (NBO) analysis was used to investigate bond strength between molecules' charge exchange, hyperconjugative connections, and molecular equilibrium. By computing the hyperpolarizability (β) and molecular polarizability (α) parameters, the ensuing nonlinear optical properties were investigated, leading to a number of surprising optical properties for the produced compounds. The antipathogenic activity of the generated materials was experimentally verified against a subset of gram (+) and gram (−) bacteria as well as some fungi using the agar well diffusion method. Additionally, hepatic cellular carcinomas, cells from colon and breast cancer, were utilized to test the cytotoxic activity of the ESAB ligand and its metal chelates. Furthermore, the examined compounds' ability to suppress the DPPH radical was examined. Additionally, simulations of molecular docking were performed to ascertain how the produced compounds attached to the specific protein binding sites.

Discovery of Diverse CRISPR Leader Motifs, Putative Functions, and Applications for Enhanced CRISPR Detection and Subtype Annotation.

Bacteria and archaea acquire resistance to genetic parasites by preferentially integrating short fragments of foreign DNA at one end of a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). "Leader" DNA upstream of CRISPR loci regulates transcription and foreign DNA integration into the CRISPR. Here, we analyze 37,477 CRISPRs from 39,277 bacterial and 556 archaeal genomes to identify conserved sequence motifs in CRISPR leaders. A global analysis of all leader sequences fails to identify universally conserved motifs. However, an analysis of leader sequences that have been grouped by 16S rRNA-based taxonomy and CRISPR subtype reveals 87 specific motifs in type I, II, III, and V CRISPR leaders. Fourteen of these leader motifs have biochemically demonstrated roles in CRISPR biology including integration, transcription, and CRISPR RNA processing. Another 28 motifs are related to DNA binding sites for proteins with functions that are consistent with regulating CRISPR activity. In addition, we show that these leader motifs can be used to improve existing CRISPR detection methods and enhance the accuracy of CRISPR classification.

Cyclodextrin increases the effectiveness of albumin’s interaction with antibacterial drugs and their combinations

Fluorescence studies indicate that a two-drug combination demonstrates the synergism in binding to human serum albumin if drugs prefer different protein binding sites. The formation of a drug complex with methylated β-cyclodextrin leads to an increase in albumin’s quenching and drug binding affinity, while the fluorescence quenching mechanism remains unchanged.

Unveiling the potential anticancer activity of new dihydropyrimidines through dual inhibition of EGFR and TrkA: Design, synthesis, and in silico study.

A series of designed scaffold of dihydropyrimidine was synthesized as dual tyrosine kinase targets inhibitors using a multicomponent Biginelli reaction which provided a high atom economy in a single pot reaction. Several 1,4-DHPM hybrids were obtained via alkylation with different chloroacetylamine derivatives. All the synthesized derivatives were screened for their antiproliferative efficacy towards various cancer cell lines (HCT-116, PC-3, and MCF-7) and normal cell line WI-38 using MTT assay. The results indicated that compounds 8h and 8i have the most significant inhibitory effect on all evaluated cancer cell lines, displaying IC50 of 3.94-15.78µM. Also, they demonstrated favorable selectivity towards normal cell lines. Moreover, the most active hybrids 8h and 8i were evaluated for their EGFR and TrkA inhibitory activity. The findings indicated that compound 8h had superior inhibitory activity compared to compound 8i on the targeted kinases, effectively stopping the G1 phase of the MCF-7 cell cycle and encouraging apoptosis. Additionally, the molecular docking studies declared that the most active compounds exhibited a notable binding interaction with the binding site of the target proteins. Furthermore, their physicochemical properties, ADMET profiles, and bioavailability radar plots were predicted and analyzed.

Antioxidant taurine inhibits chondrocyte ferroptosis through upregulation of OGT/Gpx4 signaling in osteoarthritis induced by anterior cruciate ligament transection.

The aim of this study was to investigate the potential molecular mechanisms by which taurine protects against cartilage degeneration. The anterior cruciate ligament transection (ACLT) surgery was used to construct an animal model of osteoarthritis (OA). Metabolomics was used to identify characteristic metabolites in osteoarthritic chondrocytes. Transcriptomics and metabolomics were used to explore potential mechanisms by which the small molecule metabolite taurine protects against inflammatory chondrocyte damage. Cell transfection and small molecule inhibitors/agonists were used to validate the molecular mechanisms by which taurine protects inflammatory chondrocytes in vitro. Finally, adeno-associated virus and small molecule inhibitors/agonists were used to validate the molecular mechanisms by which taurine protects against cartilage degeneration in vivo. Metabolomic assays identified taurine as a possible key metabolic molecule in the progression of OA. Transcriptomics and metabolomics revealed that O-GlcNAc transferase (OGT)-dependent O-GlcNAcylation and Gpx4-dependent ferroptosis may mediate the inflammatory protective effects of taurine on chondrocytes, which was further confirmed by gain and loss of function in vitro. Subsequently, further experiments indicated that the possible existence of a direct binding site for Gpx4 and OGT proteins, which provides evidence for the presence of O-GlcNAc modification of Gpx4 protein. Finnaly, we demonstrated that Gpx4-dependent ferroptosis and OGT-dependent O-GlcNAcylation may be potential mechanisms by which taurine protects against cartilage degeneration in vivo. Antioxidant taurine inhibits chondrocyte ferroptosis through upregulation of OGT/Gpx4 signaling. Supplementation with taurine, a safe nonessential amino acid, may be a potential therapeutic strategy for OA.

Nature's defense against emerging neurodegenerative threats: Dynamic simulation, PCA, DCCM identified potential plant-based antiviral lead targeting borna disease virus nucleoprotein.

The rare zoonotic Borna disease virus (BDV) causes fatal neurological disease in various animals, with a high mortality rate exceeding 90% in central Europe. However, unlike most viruses, it establishes persistent infections within the host cell nucleus, hindering treatment. As successful BDV treatments remain elusive, the researchers turned to a computational approach, utilizing molecular docking, ADME/T, post-docking MMGBSA, MD simulation, DCCM, and PCA to identify promising phytochemical drug candidates targeting the BDV Nucleoprotein (PDB ID: 1N93). From IMPPAT 1940 unique phytochemical compounds of a total of 8617 compounds from 36 Indian medicinal plants were retrieved. Three compounds were chosen as leads with higher binding affinity of -6.244, -6.116, and -6.07 kcal/mol with CID 163114683 (IMPHY000668) Nimbochalcin, CID 20871246 (IMPHY007896) 3,4-Dihydroxy-5-oxocyclohex-3-ene-1-carboxylic acid, and CID 243 (IMPHY002962) Benzoic acid. The three top compounds coordinated with the protein's common amino acid residues at GLN 161, ARG 165, ILE 145, ILE 162, ILE 149, and VAL 229 during molecular docking, which implies that both lead compounds and the control ligand interact within the protein's shared active site. Afterwards, negative binding free energies of Nimbochalcin, 3,4-Dihydroxy-5-oxocyclohex-3-ene-1-carboxylic acid, and Benzoic acid were -51.21, -13.94, and -22.95 kcal/mol, accordingly. Favorable Pk and toxicological characteristics are shared by all of the chosen drugs, indicating their efficacy and safety. Using MD simulation, these three compounds were further assessed, and their stability in binding to the target protein was confirmed and subsequently, DCCM and PCA analyses were carried out from MD trajectory. MD simulations found that the protein binding site is highly stable when complexed with CID 20871246 and has a higher negative binding free energy value, indicating a strong interaction between the compound and the protein. Principal component analysis (PCA) identified three main components (PC1, PC2, and PC3) that accounted for 53.43%, 12.31%, and 5.97% of the variance, respectively. These findings provide intriguing evidence that the CID 20871246-1N93 complex is more stable than the other complexes. The BDV nucleoprotein was the target of this study's investigation where CID 20871246 (3,4-dihydroxy-5-oxocyclohex-3-ene-1-carboxylic acid) exhibited tremendous antiviral activity which is found in the flower of the plant Mangifera indica revealing as a possible therapeutic candidate.

Direct Vascular Effects of Angiotensin II (A Systematic Short Review).

The octapeptide angiotensin II (Ang II) is a circulating hormone as well as a locally formed agonist synthesized by the angiotensin-converting enzyme (ACE) of endothelial cells. It forms a powerful mechanism to control the amount and pressure of body fluids. All main effects are directed to save body salt and water and ensure blood pressure under basic conditions and in emergencies. All blood vessels respond to stimulation by Ang II; the immediate response is smooth muscle contraction, increasing vascular resistance, and elevating blood pressure. Such effects are conveyed by type 1 angiotensin receptors (AT1Rs) located in the plasma membrane of both endothelial and vascular smooth muscle cells. AT1Rs are heterotrimeric G protein-coupled receptors (GPCRs), but their signal pathways are much more complicated than other GPCRs. In addition to Gq/11, the G12/13, JAK/STAT, Jnk, MAPK, and ERK 1/2, and arrestin-dependent and -independent pathways are activated because of the promiscuous attachment of different signal proteins to the intracellular G protein binding site and to the intracellular C terminal loop. Substantial changes in protein expression follow, including the intracellular inflammation signal protein NF-κB, endothelial contact proteins, cytokines, matrix metalloproteinases (MMPs), and type I protocollagen, eliciting the inflammatory transformation of endothelial and vascular smooth muscle cells and fibrosis. Ang II is an important contributor to vascular pathologies in hypertensive, atherosclerotic, and aneurysmal vascular wall remodeling. Such direct vascular effects are reviewed. In addition to reducing blood pressure, AT1R antagonists and ACE inhibitors have a beneficial effect on the vascular wall by inhibiting pathological wall remodeling.

Protein Binding Site Representation in Latent Space.

Interpretability and reliability of deep learning models are important for computer-based drug discovery. Aiming to understand feature perception by such a model, we investigatea graph neural network for affinity prediction of protein-ligand complexes. We assess a latent representation of ligand binding sites and investigate underlying geometric structure in this latent space and its relation to protein function. We introduce an automated computational pipeline for dimensionality reduction, clustering, hypothesis testing, and visualization of latent space. The results indicate that the learned protein latent space is inherently structured and not randomly distributed. Several of the identified protein binding site clusters in latent space correspond to functional protein families. Ligand size was found to be a determinant of cluster geometry. The computational pipeline proved applicable to latent space analysis and interpretation and can be adapted to work for different datasets and deep learning models.

Leveraging a Separation of States Method for Relative Binding Free Energy Calculations in Systems with Trapped Waters.

Methods for calculating the relative binding free energy (RBFE) between ligands to a target protein are gaining importance in the structure-based drug discovery domain, especially as methodological advances and automation improve accuracy and ease of use. In an RBFE calculation, the difference between the binding affinities of two ligands to a protein is calculated by transforming one ligand into another, in the protein-ligand complex, and in solvent. Alchemical binding free energy calculations are often used for such ligand transformations. Such calculations are not without challenges, however; for example, it can be challenging to handle interfacial waters when these play a crucial role in mediating protein-ligand binding. In some cases, the exchange of the interfacial waters with solvent water might be very infrequent in the course of typical molecular simulations, and such interfacial waters can be considered trapped on the simulation time scale. In these cases, RBFE calculation between two ligands, where one ligand binds with a trapped water while the other ligand displaces it, can result in inaccuracies if the surrounding water structure is not sampled adequately for both ligands. So far, a popular choice for treating the trapped waters in RBFE calculations is to combine free energy calculations with enhanced sampling methods that insert/delete waters in the binding site. Despite recent developments in the enhanced sampling methods, they can result in hysteresis in the RBFE estimate, depending on whether the simulations were started with or without the trapped waters. In this study, we introduce an alternative method, separation of states, to calculate the RBFE between ligand pairs where the ligands bind to the protein with different numbers/positions of trapped waters. The separation of states approach treats the sampling of the trapped waters separately from the free energy calculation of the ligand transformation. In our method, a trapped water in protein's binding site is decoupled from the system first, and the cavity created by its decoupling is stabilized. We then grow a larger ligand into this cavity- a ligand that is known to displace the trapped water. In this study, we show that our method results in precise and accurate estimates of RBFEs for ligand pairs involving the rearrangement of trapped water via RBFE calculations for five such ligand pairs. We have optimized our simulation protocol to be suited for large distributed computational resources and have automated our RBFE calculation workflow.

Effect of Gold Nanoparticles on the Conformation of Bovine Serum Albumin: Insights from CD Spectroscopic Analysis and Molecular Dynamics Simulations.

With the development of nanotechnology, there is growing interest in using nanoparticles (NPs) for biomedical applications, such as diagnostics, drug delivery, imaging, and nanomedicine. The protein's structural stability plays a pivotal role in its functionality, and any alteration in this structure can have significant implications, including disease progression. Herein, we performed a combined experimental and computational study of the effect of gold NPs with a diameter of 5 nm (5 nm Au-NPs) on the structural stability of bovine serum albumin (BSA) protein in the absence and presence of NaCl salt. Circular dichroism spectroscopy showed a loss in the secondary structure of BSA due to the synergistic effect of Au-NPs and NaCl, and Thioflavin T fluorescence assays showed suppressed β-sheet formation in the presence of Au-NPs in PBS, emphasizing the intricate interplay between NPs and physiological conditions. Additionally, molecular dynamics (MD) simulations revealed that 5 nm Au-NP induced changes in the secondary structure of the BSA monomer in the presence of NaCl, highlighting the initial binding mechanism between BSA and Au-NP. Furthermore, MD simulations explored the effect of smaller Au-NP (3 nm) and nanocluster (Au-NC with the size of 1 nm) on the binding sites of the BSA monomer. Although the formation of stable BSA-Au conjugates was revealed in the presence of NPs of different sizes, no specific protein binding sites were observed. Moreover, due to its small size, 1 nm Au-NC decreased helical content and hydrogen bonds in the BSA monomer, promoting protein unfolding more significantly. In summary, this combined experimental and computational study provides comprehensive insights into the interactions among Au nanosized substances, BSA, and physiological conditions that are essential for developing tailored nanomaterials with enhanced biocompatibility and efficacy.

In Silico Tools to Score and Predict Cholesterol-Protein Interactions.

Cholesterol is structurally distinct from other lipids, which confers it with singular roles in membrane organization and protein function. As a signaling molecule, cholesterol engages in discrete interactions with transmembrane, peripheral, and certain soluble proteins to control cellular responses. Accordingly, the cholesterol-protein interface is central to cholesterol-related diseases and is an essential consideration in drug design. However, cholesterol's hydrophobic, un-drug-like nature presents a unique challenge to traditional in silico analyses. In this Perspective, we survey a collection of tools designed to predict and evaluate cholesterol binding sites in proteins, including classical sequence motifs, molecular docking, template-based strategies, molecular dynamics simulations, and recent artificial intelligence approaches. We then comment on contemporary tools to evaluate ligand-protein interactions, their applicability to cholesterol, and the yet-untapped potential of cholesterol-protein interactions in human health and disease.

Specific radiation damage to halogenated inhibitors and ligands in protein-ligand crystal structures.

Protein-inhibitor crystal structures aid medicinal chemists in efficiently improving the potency and selectivity of small-molecule inhibitors. It is estimated that a quarter of lead molecules in drug discovery projects are halogenated. Protein-inhibitor crystal structures have shed light on the role of halogen atoms in ligand binding. They form halogen bonds with protein atoms and improve shape complementarity of inhibitors with protein binding sites. However, specific radiation damage (SRD) can cause cleavage of carbon-halogen (C-X) bonds during X-ray diffraction data collection. This study shows significant C-X bond cleavage in protein-ligand structures of the therapeutic cancer targets B-cell lymphoma 6 (BCL6) and heat shock protein 72 (HSP72) complexed with halogenated ligands, which is dependent on the type of halogen and chemical structure of the ligand. The study found that metrics used to evaluate the fit of the ligand to the electron density deteriorated with increasing X-ray dose, and that SRD eliminated the anomalous signal from brominated ligands. A point of diminishing returns is identified, where collecting highly redundant data reduces the anomalous signal that may be used to identify binding sites of low-affinity ligands or for experimental phasing. Straightforward steps are proposed to mitigate the effects of C-X bond cleavage on structures of proteins bound to halogenated ligands and to improve the success of anomalous scattering experiments.

Generation of Transcript Length Variants and Reprogramming of mRNA Splicing During Atherosclerosis Progression in ApoE-Deficient Mice.

Background. Variant 3'UTRs provide mRNAs with different binding sites for miRNAs or RNA-binding proteins (RBPs) allowing the establishment of new regulatory environments. Regulation of 3'UTR length impacts on the control of gene expression by regulating accessibility of miRNAs or RBPs to homologous sequences in mRNAs. Objective. Studying the dynamics of mRNA length variations in atherosclerosis (ATS) progression and reversion in ApoE-deficient mice exposed to a high-fat diet and treated with an αCD40-specific siRNA or with a sequence-scrambled siRNA as control. Methods. We gathered microarray mRNA expression data from the aortas of mice after 2 or 16 weeks of treatments, and used these data in a Bioinformatics analysis. Results. Here, we report the lengthening of the 5'UTR/3'UTRs and the shortening of the CDS in downregulated mRNAs during ATS progression. Furthermore, treatment with the αCD40-specific siRNA resulted in the partial reversion of the 3'UTR lengthening. Exon analysis showed that these length variations were actually due to changes in the number of exons embedded in mRNAs, and the further examination of transcripts co-expressed at weeks 2 and 16 in mice treated with the control siRNA revealed a process of mRNA isoform switching in which transcript variants differed in the patterns of alternative splicing or activated latent/cryptic splice sites. Conclusion. We document length variations in the 5'UTR/3'UTR and CDS of mRNAs downregulated during atherosclerosis progression and suggest a role for mRNA splicing reprogramming and transcript isoform switching in the generation of disease-related mRNA sequence diversity and variability.