Combinatorial Effects of 5-Fluorouracil and Menadione on Wnt/ß-Catenin Pathway in Human Colorectal Cancer Cells.

The incidence and mortality rates of colorectal cancer (CRC) are alarmingly high, and the scientific community is consistently engaged in developing newer therapeutic options for cancer cure or prevention. The fluoropyrimidine drug, 5-fluorouracil (5FU), remains the first line of treatment against CRC; nevertheless, relapses frequently occur since the cells gain resistance over time through various mechanisms. Studies have highlighted the significance of combinatorial treatment of a Wnt signaling inhibitor and 5FU as a better treatment strategy to overcome 5FU resistance. Small molecules that specifically target and disrupt ß-catenin-TCF interaction, a crucial step of the Wnt signaling, are promising in CRC treatment. In this study, we investigated the synergistic cytotoxic activity of menadione with 5FU as the former has previously been shown to downregulate Wnt signaling in CRC cells. Docking and experimental results suggest that the drug combination interfered with key protein-protein interactions in the ß-catenin-TCF complex, exerted synergistic anti-cancerous effects in CRC cells, and downregulated the expression of Wnt signaling proteins. Taken together, our data suggest that the simultaneous binding of 5FU and menadione to ß-catenin can block Wnt signaling by disrupting ß-catenin-TCF interaction and inhibit the proliferation of CRC cells.

rAbDesFlow: a novel workflow for computational recombinant antibody design for healthcare engineering.

Recombinant antibodies (rAbs) have emerged as a promising solution to tackle antigen specificity, enhancement of immunogenic potential and versatile functionalization to treat human diseases. The development of single chain variable fragments has helped accelerate treatment in cancers and viral infections, due to their favorable pharmacokinetics and human compatibility. However, designing rAbs is traditionally viewed as a genetic engineering problem, with phage display and cell free systems playing a major role in sequence selection for gene synthesis. The process of antibody engineering involves complex and time-consuming laboratory techniques, which demand substantial resources and expertise. The success rate of obtaining desired antibody candidates through experimental approaches can be modest, necessitating iterative cycles of selection and optimization. With ongoing advancements in technology, in silico design of diverse antibody libraries, screening and identification of potential candidates for in vitro validation can be accelerated. To meet this need, we have developed rAbDesFlow, a unified computational workflow for recombinant antibody engineering with open-source programs and tools for ease of implementation. The workflow encompasses five computational modules to perform antigen selection, antibody library generation, antigen and antibody structure modeling, antigen-antibody interaction modeling, structure analysis, and consensus ranking of potential antibody sequences for synthesis and experimental validation. The proposed workflow has been demonstrated through design of rAbs for the ovarian cancer antigen Mucin-16 (CA-125). This approach can serve as a blueprint for designing similar engineered molecules targeting other biomarkers, allowing for a simplified adaptation to different cancer types or disease-specific antigens.

Role of HSP90 in Type 2 Diabetes Mellitus and Its Association with Liver Diseases.

Non-alcoholic fatty acid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) are the fatal liver diseases which encompass a spectrum of disease severity associated with increased risk of type 2 diabetes mellitus (T2DM), a metabolic disorder. Heat shock proteins serve as markers in early prognosis and diagnosis of early stages of liver diseases associated with metabolic disorder. This review aims to comprehensively investigate the significance of HSP90 isoforms in T2DM and liver diseases. Additionally, we explore the collective knowledge on plant-based drug compounds that regulate HSP90 isoform targets, highlighting their potential in treating T2DM-associated liver diseases. Furthermore, this review focuses on the computational systems' biology and next-generation sequencing technology approaches that are used to unravel the potential medicine for the treatment of pleiotropy of these 2 diseases and to further elucidate the mechanism.

DeepPPAPredMut: deep ensemble method for predicting the binding affinity change in protein-protein complexes upon mutation.

MOTIVATION: Protein-protein interactions underpin many cellular processes and their disruption due to mutations can lead to diseases. With the evolution of protein structure prediction methods like AlphaFold2 and the availability of extensive experimental affinity data, there is a pressing need for updated computational tools that can efficiently predict changes in binding affinity caused by mutations in protein-protein complexes. RESULTS: We developed a deep ensemble model that leverages protein sequences, predicted structure-based features, and protein functional classes to accurately predict the change in binding affinity due to mutations. The model achieved a correlation of 0.97 and a mean absolute error (MAE) of 0.35 kcal/mol on the training dataset, and maintained robust performance on the test set with a correlation of 0.72 and a MAE of 0.83 kcal/mol. Further validation using Leave-One-Out Complex (LOOC) cross-validation exhibited a correlation of 0.83 and a MAE of 0.51 kcal/mol, indicating consistent performance. AVAILABILITY AND IMPLEMENTATION: https://web.iitm.ac.in/bioinfo2/DeepPPAPredMut/index.html.

Computational approaches for identifying disease-causing mutations in proteins.

Advancements in genome sequencing have expanded the scope of investigating mutations in proteins across different diseases. Amino acid mutations in a protein alter its structure, stability and function and some of them lead to diseases. Identification of disease-causing mutations is a challenging task and it will be helpful for designing therapeutic strategies. Hence, mutation data available in the literature have been curated and stored in several databases, which have been effectively utilized for developing computational methods to identify deleterious mutations (drivers), using sequence and structure-based properties of proteins. In this chapter, we describe the contents of specific databases that have information on disease-causing and neutral mutations followed by sequence and structure-based properties. Further, characteristic features of disease-causing mutations will be discussed along with computational methods for identifying cancer hotspot residues and disease-causing mutations in proteins.

Mechanism of drug resistance in HIV-1 protease subtype C in the presence of Atazanavir.

AIDS is one of the deadliest diseases in the history of humankind caused by HIV. Despite the technological development, curtailing the viral infection inside human host still remains a challenge. Therapies such as HAART uses a combination of drugs to inhibit the viral activity. One of the important targets includes HIV protease and inhibiting its activity will minimize the production of mature structural proteins. However, the genetic diversity and the occurrence of drug resistant mutations adds complexity to effective drug design. In this study, we aimed at understanding the drug binding mechanism of one such subtype, namely subtype C and its insertion variant L38HL. We performed multiple molecular dynamics simulations along with binding free energy analysis of wild-type and L38HL bound to Atazanavir (ATV). From the analysis, we revealed that the insertion alters the hydrogen bond and hydrophobic interaction networks. The alterations in the interaction networks increase flexibility at the hinge-fulcrum interface. Further, the effects of these changes affect flap tip curling. Moreover, the changes in the hinge-fulcrum-cantilever interface alters the concerted motion of the functional regions leading to change in the direction of flap movement thus causing a subtle change in the active site volume. Additionally, formation of intramolecular hydrogen bonds in the ATV docked to L38HL restricted the movement of R1 and R2 groups thereby altering the interactions. Overall, the changes in the flexibility of flap together with the changes in the active site volume and compactness of the ligand provide insights for increased binding affinity of ATV with L38HL.

CarbDisMut: database on neutral and disease-causing mutations in human carbohydrate-binding proteins.

Protein-carbohydrate interactions are involved in several cellular and biological functions. Integrating structure and function of carbohydrate-binding proteins with disease-causing mutations help to understand the molecular basis of diseases. Although databases are available for protein-carbohydrate complexes based on structure, binding affinity and function, no specific database for mutations in human carbohydrate-binding proteins is reported in the literature. We have developed a novel database, CarbDisMut, a comprehensive integrated resource for disease-causing mutations with sequence and structural features. It has 1.17 million disease-associated mutations and 38,636 neutral mutations from 7,187 human carbohydrate-binding proteins. The database is freely available at https://web.iitm.ac.in/bioinfo2/carbdismut. The web-site is implemented using HTML, PHP and JavaScript and supports recent versions of all major browsers, such as Firefox, Chrome and Opera.

Reliable method for predicting the binding affinity of RNA-small molecule interactions using machine learning.

Ribonucleic acids (RNAs) play important roles in cellular regulation. Consequently, dysregulation of both coding and non-coding RNAs has been implicated in several disease conditions in the human body. In this regard, a growing interest has been observed to probe into the potential of RNAs to act as drug targets in disease conditions. To accelerate this search for disease-associated novel RNA targets and their small molecular inhibitors, machine learning models for binding affinity prediction were developed specific to six RNA subtypes namely, aptamers, miRNAs, repeats, ribosomal RNAs, riboswitches and viral RNAs. We found that differences in RNA sequence composition, flexibility and polar nature of RNA-binding ligands are important for predicting the binding affinity. Our method showed an average Pearson correlation (r) of 0.83 and a mean absolute error of 0.66 upon evaluation using the jack-knife test, indicating their reliability despite the low amount of data available for several RNA subtypes. Further, the models were validated with external blind test datasets, which outperform other existing quantitative structure-activity relationship (QSAR) models. We have developed a web server to host the models, RNA-Small molecule binding Affinity Predictor, which is freely available at: https://web.iitm.ac.in/bioinfo2/RSAPred/.

PRA-Pred: Structure-based prediction of protein-RNA binding affinity.

Understanding crucial factors that affect the binding affinity of protein-RNA complexes is vital for comprehending their recognition mechanisms. This study involved compiling experimentally measured binding affinity (ΔG) values of 217 protein-RNA complexes and extracting numerous structure-based features, considering RNA, protein, and interactions between protein and RNA. Our findings indicate the significance of RNA base-step parameters, interaction energies, number of atomic contacts in the complex, hydrogen bonds, and contact potentials in understanding the binding affinity. Further, we observed that these factors are influenced by the type of RNA strand and the function of the protein in a protein-RNA complex. Multiple regression equations were developed for different classes of complexes to perform the prediction of the binding affinity between the protein and RNA. We evaluated the models using the jack-knife test and achieved an overall correlation 0.77 between the experimental and predicted binding affinities with a mean absolute error of 1.02 kcal/mol. Furthermore, we introduced a web server, PRA-Pred, intended for the prediction of protein-RNA binding affinity, and it is freely accessible through https://web.iitm.ac.in/bioinfo2/prapred/. We propose that our approach could function as a potential resource for investigating protein-RNA recognitions and developing therapeutic strategies.

MPA-Pred: A machine learning approach for predicting the binding affinity of membrane protein-protein complexes.

Membrane protein-protein interactions are essential for several functions including cell signaling, ion transport, and enzymatic activity. These interactions are mainly dictated by their binding affinities. Although several methods are available for predicting the binding affinity of protein-protein complexes, there exists no specific method for membrane protein-protein complexes. In this work, we collected the experimental binding affinity data for a set of 114 membrane protein-protein complexes and derived several structure and sequence-based features. Our analysis on the relationship between binding affinity and the features revealed that the important factors mainly depend on the type of membrane protein and the functional class of the protein. Specifically, aromatic and charged residues at the interface, and aromatic-aromatic and electrostatic interactions are found to be important to understand the binding affinity. Further, we developed a method, MPA-Pred, for predicting the binding affinity of membrane protein-protein complexes using a machine learning approach. It showed an average correlation and mean absolute error of 0.83 and 0.91 kcal/mol, respectively, using the jack-knife test on a set of 114 complexes. We have also developed a web server and it is available at https://web.iitm.ac.in/bioinfo2/MPA-Pred/. This method can be used for predicting the affinity of membrane protein-protein complexes at a large scale and aid to improve drug design strategies.

Predicting the immune escape of SARS-CoV-2 neutralizing antibodies upon mutation.

COVID-19 has resulted in millions of deaths and severe impact on economies worldwide. Moreover, the emergence of SARS-CoV-2 variants presented significant challenges in controlling the pandemic, particularly their potential to avoid the immune system and evade vaccine immunity. This has led to a growing need for research to predict how mutations in SARS-CoV-2 reduces the ability of antibodies to neutralize the virus. In this study, we assembled a set of 1813 mutations from the interface of SARS-CoV-2 spike protein's receptor binding domain (RBD) and neutralizing antibody complexes and developed a machine learning model to classify high or low escape mutations using interaction energy, inter-residue contacts and predicted binding free energy change. Our approach achieved an Area under the Receiver Operating Characteristics (ROC) Curve (AUC) of 0.91 using the Random Forest classifier on the test dataset with 217 mutations. The model was further utilized to predict the escape mutations on a dataset of 29,165 mutations located at the interface of 83 RBD-neutralizing antibody complexes. A small subset of this dataset was also validated based on available experimental data. We found that top 10 % high escape mutations were dominated by charged to nonpolar mutations whereas low escape mutations were dominated by polar to nonpolar mutations. We believe that the present method will allow prioritization of high/low escape mutations in the context of neutralizing antibodies targeting SARS-CoV-2 RBD region and assist antibody design for current and emerging variants.

Investigating Neuron Degeneration in Huntington's Disease Using RNA-Seq Based Transcriptome Study.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused due to a CAG repeat expansion in the huntingtin (HTT) gene. The primary symptoms of HD include motor dysfunction such as chorea, dystonia, and involuntary movements. The primary motor cortex (BA4) is the key brain region responsible for executing motor/movement activities. Investigating patient and control samples from the BA4 region will provide a deeper understanding of the genes responsible for neuron degeneration and help to identify potential markers. Previous studies have focused on overall differential gene expression and associated biological functions. In this study, we illustrate the relationship between variants and differentially expressed genes/transcripts. We identified variants and their associated genes along with the quantification of genes and transcripts. We also predicted the effect of variants on various regulatory activities and found that many variants are regulating gene expression. Variants affecting miRNA and its targets are also highlighted in our study. Co-expression network studies revealed the role of novel genes. Function interaction network analysis unveiled the importance of genes involved in vesicle-mediated transport. From this unified approach, we propose that genes expressed in immune cells are crucial for reducing neuron death in HD.

TMKit: a Python interface for computational analysis of transmembrane proteins.

Transmembrane proteins are receptors, enzymes, transporters and ion channels that are instrumental in regulating a variety of cellular activities, such as signal transduction and cell communication. Despite tremendous progress in computational capacities to support protein research, there is still a significant gap in the availability of specialized computational analysis toolkits for transmembrane protein research. Here, we introduce TMKit, an open-source Python programming interface that is modular, scalable and specifically designed for processing transmembrane protein data. TMKit is a one-stop computational analysis tool for transmembrane proteins, enabling users to perform database wrangling, engineer features at the mutational, domain and topological levels, and visualize protein-protein interaction interfaces. In addition, TMKit includes seqNetRR, a high-performance computing library that allows customized construction of a large number of residue connections. This library is particularly well suited for assigning correlation matrix-based features at a fast speed. TMKit should serve as a useful tool for researchers in assisting the study of transmembrane protein sequences and structures. TMKit is publicly available through https://github.com/2003100127/tmkit and https://tmkit-guide.herokuapp.com/doc/overview.

Deep learning-based method for predicting and classifying the binding affinity of protein-protein complexes.

Protein-protein interactions (PPIs) play a critical role in various biological processes. Accurately estimating the binding affinity of PPIs is essential for understanding the underlying molecular recognition mechanisms. In this study, we employed a deep learning approach to predict the binding affinity (ΔG) of protein-protein complexes. To this end, we compiled a dataset of 903 protein-protein complexes, each with its corresponding experimental binding affinity, which belong to six functional classes. We extracted 8 to 20 non-redundant features from the sequence information as well as the predicted three-dimensional structures using feature selection methods for each protein functional class. Our method showed an overall mean absolute error of 1.05 kcal/mol and a correlation of 0.79 between experimental and predicted ΔG values. Additionally, we evaluated our model for discriminating high and low affinity protein-protein complexes and it achieved an accuracy of 87% with an F1 score of 0.86 using 10-fold cross-validation on the selected features. Our approach presents an efficient tool for studying PPIs and provides crucial insights into the underlying mechanisms of the molecular recognition process. The web server can be freely accessed at https://web.iitm.ac.in/bioinfo2/DeepPPAPred/index.html.

Pairwise and Multi-chain Protein Docking Enhanced Using LZerD Web Server.

Interactions of proteins with other macromolecules have important structural and functional roles in the basic processes of living cells. To understand and elucidate the mechanisms of interactions, it is important to know the 3D structures of the complexes. Proteomes contain numerous protein-protein complexes, for which experimentally determined structures often do not exist. Computational techniques can be a practical alternative to obtain useful complex structure models. Here, we present a web server that provides access to the LZerD and Multi-LZerD protein docking tools, which can perform both pairwise and multi-chain docking. The web server is user-friendly, with options to visualize the distribution and structures of binding poses of top-scoring models. The LZerD web server is available at https://lzerd.kiharalab.org . This chapter dictates the algorithm and step-by-step procedure to model the monomeric structures with AttentiveDist, and also provides the detail of pairwise LZerD docking, and multi-LZerD. This also provided case studies for each of the three modules.

Comment on 'Thermodynamic database supports deciphering protein-nucleic acid interactions'.

Mei and colleagues reported a thermodynamic database, PNATDB for protein-nucleic acid interactions, which contains 12 635 experimentally determined thermodynamic parameters. They claimed that extracting data from existing databases is difficult. ProNAB, which has more than 20 000 experimental data points for binding affinities of protein-nucleic acid complexes and other information, was not discussed.

MutBLESS: A tool to identify disease-prone sites in cancer using deep learning.

Understanding the molecular basis and impact of mutations at different stages of cancer are long-standing challenges in cancer biology. Identification of driver mutations from experiments is expensive and time intensive. In the present study, we collected the data for experimentally known driver mutations in 22 different cancer types and classified them into six categories: breast cancer (BRCA), acute myeloid leukaemia (LAML), endometrial carcinoma (EC), stomach cancer (STAD), skin cancer (SKCM), and other cancer types which contains 5747 disease prone and 5514 neutral sites in 516 proteins. The analysis of amino acid distribution along mutant sites revealed that the motifs AAA and LR are preferred in disease-prone sites whereas QPP and QF are dominant in neutral sites. Further, we developed a method using deep neural networks to predict disease-prone sites with amino acid sequence-based features such as physicochemical properties, secondary structure, tri-peptide motifs and conservation scores. We obtained an average AUC of 0.97 in five cancer types BRCA, LAML, EC, STAD and SKCM in a test dataset and 0.72 in all other cancer types together. Our method showed excellent performance for identifying cancer-specific mutations with an average sensitivity, specificity, and accuracy of 96.56 %, 97.39 %, and 97.64 %, respectively. We developed a web server for identifying cancer-prone sites, and it is available at https://web.iitm.ac.in/bioinfo2/MutBLESS/index.html. We suggest that our method can serve as an effective method to identify disease-prone sites and assist to develop therapeutic strategies.

Understanding Drug Resistance of Wild-Type and L38HL Insertion Mutant of HIV-1 C Protease to Saquinavir.

Acquired immunodeficiency syndrome (AIDS) is one of the most challenging infectious diseases to treat on a global scale. Understanding the mechanisms underlying the development of drug resistance is necessary for novel therapeutics. HIV subtype C is known to harbor mutations at critical positions of HIV aspartic protease compared to HIV subtype B, which affects the binding affinity. Recently, a novel double-insertion mutation at codon 38 (L38HL) was characterized in HIV subtype C protease, whose effects on the interaction with protease inhibitors are hitherto unknown. In this study, the potential of L38HL double-insertion in HIV subtype C protease to induce a drug resistance phenotype towards the protease inhibitor, Saquinavir (SQV), was probed using various computational techniques, such as molecular dynamics simulations, binding free energy calculations, local conformational changes and principal component analysis. The results indicate that the L38HL mutation exhibits an increase in flexibility at the hinge and flap regions with a decrease in the binding affinity of SQV in comparison with wild-type HIV protease C. Further, we observed a wide opening at the binding site in the L38HL variant due to an alteration in flap dynamics, leading to a decrease in interactions with the binding site of the mutant protease. It is supported by an altered direction of motion of flap residues in the L38HL variant compared with the wild-type. These results provide deep insights into understanding the potential drug resistance phenotype in infected individuals.

AutoPLP: A Padlock Probe Design Pipeline for Zoonotic Pathogens.

Emergence of novel zoonotic infections among the human population has increased the burden on global healthcare systems to curb their spread. To meet the evolutionary agility of pathogens, it is essential to revamp the existing diagnostic methods for early detection and characterization of the pathogens at the molecular level. Padlock probes (PLPs), which can leverage the power of isothermal nucleic acid amplification techniques (NAAT) such as rolling circle amplification (RCA), are known for their high sensitivity and specificity in detecting a diverse pathogen panel of interest. However, due to the complexity involved in deciding the target regions for PLP design and the need for optimization of multiple experimental parameters, the applicability of RCA has been limited in point-of-care testing for pathogen detection. To address this gap, we have developed a novel and integrated PLP design pipeline named AutoPLP, which can automate the probe design process for a diverse pathogen panel of interest. The pipeline is composed of three modules which can perform sequence data curation, multiple sequence alignment, conservation analysis, filtration based on experimental parameters (Tm, GC content, and secondary structure formation), and in silico probe validation via potential cross-hybridization check with host genome. The modules can also take into account the backbone and restriction site information, appropriate combinations of which are incorporated along with the probe arms to design a complete probe sequence. The potential applications of AutoPLP are showcased through the design of PLPs for the detection of rabies virus and drug-resistant strains of Mycobacterium tuberculosis.