incorporating-target-protein-structure-flexibility-dynamics

Incorporating Target Protein Structure Flexibility and Dynamics in Computational Drug Discovery Using Ensemble-Based Docking Analysis

The drug discovery process is a rigorous, time-consuming, and expensive operation. The computational approach in drug discovery allows researchers to ...

Universiti Malaya

Laccase belongs to the family of blue multi-copper oxidases and are capable of oxidizing a wide range of aromatic compounds. Laccases have industrial applications in paper pulping or bleaching and hydrocarbon bioremediation as a biocatalyst. We describe the design of a laccase with broader substrate spectrum in bioremediation. The application of evolutionary trace (ET) analysis of laccase at the ligand binding site for optimal design of the enzyme is described. In this attempt, class specific sites from ET analysis were mapped onto known crystal structure of laccase. The analysis revealed 162PHE as a critical residue in structure function relationship studies.

Evolutionary trace analysis at the ligand binding site of laccase.

Laccases are industrially attractive enzymes and their applications have expanded to the field of bioremediation. The challenge of today's biotechnology in enzymatic studies is to design enzymes that not only have a higher activity but are also more stable and could fit well with the condition requirements. Laccases are known to oxidize non-natural substrates like polycyclic aromatic hydrocarbons (PAHs). We suppose by increasing the hydrophobicity of laccase, it would increase the chance of the enzyme to meet the hydrophobic substrates in a contamination site, therefore increasing the bioremediation efficacy of PAHs from environment. In this attempt, the applications of evolutionary trace (ET), molecular surface accessibility and hydrophobicity analysis on laccase sequences and laccase's crystal structure (1KYA) are described for optimal design of an enzyme with higher hydrophobicity. Our analysis revealed that Q23A, Q45I, N141A, Q237V, N262L, N301V, N331A, Q360L and Q482A could be promising exchanges to be tested in mutagenesis experiments.

A combination of evolutionary trace method, molecular surface accessibility and hydrophobicity analysis to design a high hydrophobicity laccase.

Curcumenol and curcumenone are two major constituents of the plants of medicinally important genus of Curcuma, and often govern the pharmacological effect of these plant extracts. These two compounds, isolated from C. zedoaria rhizomes were studied for their binding to human serum albumin (HSA) using the fluorescence quench titration method. Molecular docking was also performed to get a more detailed insight into their interaction with HSA at the binding site. Additions of these sesquiterpenes to HSA produced significant fluorescence quenching and blue shifts in the emission spectra of HSA. Analysis of the fluorescence data pointed toward moderate binding affinity between the ligands and HSA, with curcumenone showing a relatively higher binding constant (2.46 × 105 M-1) in comparison to curcumenol (1.97 × 104 M-1). Cluster analyses revealed that site I is the preferred binding site for both molecules with a minimum binding energy of -6.77 kcal·mol-1. However, binding of these two molecules to site II cannot be ruled out as the binding energies were found to be -5.72 and -5.74 kcal·mol-1 for curcumenol and curcumenone, respectively. The interactions of both ligands with HSA involved hydrophobic interactions as well as hydrogen bonding.

Spectrofluorometric and molecular docking studies on the binding of curcumenol and curcumenone to human serum albumin.

Autoinflammatory disorders are characterized by dysregulated innate immune response, resulting in recurrent uncontrolled systemic inflammation and fever. Gain-of-function mutations in NLRC4 have been described to cause a range of autoinflammatory disorders. We report a twelve-year-old Malay girl with recurrent fever, skin erythema, and inflammatory arthritis. Whole exome sequencing and subsequent bidirectional Sanger sequencing identified a heterozygous missense mutation in NLRC4 (NM_001199138: c.1970A > T). This variant was predicted to be damaging in silico, was absent in public and local databases and occurred in a highly conserved residue in the leucine-rich repeat (LRR) domain. Cytokine analysis showed extremely high serum IL-18 and IL-18/CXCL9 ratio, consistent with other NLRC4-MAS patients. In summary, we identified the first patient with a novel de novo heterozygous NLRC4 gene mutation contributing to autoinflammatory disease in Malaysia. Our findings reinforce the likely pathogenicity of specific LRR domain mutations in NLRC4 and expand the clinical spectrum of NLRC4 mutations.

A novel de novo NLRC4 mutation reinforces the likely pathogenicity of specific LRR domain mutation.

The potential therapeutic effect of  leaf juice has attracted wide interest from the public and scientists in relieving dengue related manifestations. Currently, there is a lack of evaluated evidence on its juice form. Therefore, this scoping review aims to critically appraise the available scientific evidence related to the efficacy of  leaf juice in dengue. A systematic search was performed using predetermined keywords on two electronic databases (PubMed and Google Scholar). Searched results were identified, screened and appraised to establish the association between  and alleviating dengue associated conditions. A total of 28 articles (ethnobotanical information: three, in vitro studies: three, ex vivo studies: one, in vivo study: 13, clinical studies: 10) were included for descriptive analysis, which covered study characteristics, juice preparation/formulations, study outcomes, and toxicity findings. Other than larvicidal activity, this review also reveals two medicinal potentials of  leaf juice on dengue infection, namely anti-thrombocytopenic and immunomodulatory effects.  leaf juice has the potential to be a new drug candidate against dengue disease safely and effectively.

 Leaf Juice for Dengue: A Scoping Review.

Gossypium spp., produces economically important cotton fiber, and its yield is highly affected due to pest attacks. Insecticidal target site mutation is one of the reasons behind insecticide resistance to a wide range of pesticides. Acetylcholinesterase (AChE) protein sequences from major pests of cotton were analyzed to assess various physicochemical properties, presence of motifs, and understand evolutionary relationship. The impact of three mutant AChE1,  A216S,  F392W, and  A302S, on the strucutral stability was assessed, and F392W_AChE1 was selected based on 100 ns molecular dynamics simulation. Virtual screening of the zinc database and high-throughput virtual screening, standard precision, and extra precision docking resulted in the identification of six compounds. The six identified compounds and six known commercial pesticdes were docked with three mutant and three wild type AChE1, and one (C1) was selected based on Tice criteria. The conformational and interaction stability of the AChE1-C1 and F392W_AChE1-C1 complexes were monitored at 100 ns Gromacs simulation and were found to be thermodynamically favorable. Therefore, C1 may have the potential to bind to the resistant and susceptible strains of cotton pest, and the resistance developed by insects could be arrested. Furthermore, synthesis and field study of C1 will lead us to a better understanding of the efficacy of the identified compound.

Potential Acetylcholinesterase Inhibitor Acting on the Pesticide Resistant and Susceptible Cotton Pests.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common type of inherited cystic kidney disease. The feasibility of whole‑exome sequencing (WES) to obtain molecular diagnosis of ADPKD is still in question as previous studies showed conflicting results. Utilizing WES on a patient with ADPKD, standard bioinformatics pipeline demonstrated no pathogenic variant in the genes of interest. By visualizing read alignments using the Integrative Genomics Viewer, a region with atypical alignment of numerous soft‑clipped reads at exon 45 of polycystin 1, transient receptor potential channel interacting () gene was demonstrated. A total of four visual inspection steps were outlined to assess the origin of these soft‑clipped reads as strand bias during capture, poor mapping, sequencing error or DNA template contamination. Following assessment, the atypical alignment at  was hypothesized to be caused by an insertion/deletion mutation. Sanger sequencing confirmed the presence of a novel 20‑bp insertion in  (NM_001009944.3; c.12143_12144insTCC​CCG​CAG​TCT​TCC​CCG​CA; p.Val4048LeufsTer157), which introduced a premature stop codon and was predicted to be pathogenic. The present study demonstrated that WES could be utilized as a molecular diagnostic tool for ADPKD. Furthermore, visual inspection of read alignments was key in identifying the pathogenic variant. The proposed visual inspection steps may be incorporated into a typical WES data analysis workflow to improve the diagnostic yield.

Visual inspection reveals a novel pathogenic mutation in  missed by the variant caller in whole‑exome sequencing.

Background: Inborn errors of immunity (IEIs) are comprised of heterogeneous groups of genetic disorders affecting immune function. In this report, a 17-month-old Malay patient suspected of having Hyper IgM syndrome, a type of IEIs, was described. However, the diagnosis of Hyper IgM syndrome was excluded by the normal functional studies and the mild features of ectodermal dysplasia observed from a further clinical phenotype inspection. Methods: Whole-exome sequencing (WES) was performed to unravel the causative mutation in this patient. Results: The variant analysis demonstrated a novel missense mutation in NFKBIA (NM_020529:c.94A > T,NP_065390:p.Ser32Cys) and was predicted as damaging by in silico prediction tools. The NFKBIA gene encodes for IκBα, a member of nuclear factor kappa B (NF-κB) inhibitors, playing an important role in regulating NF-κB activity. The mutation occurred at the six degrons (Asp31-Ser36) in IκBα which were evolutionarily conserved across several species. Prediction analysis suggested that the substitution of Ser32Cys may cause a loss of the phosphorylation site at residue 32 and a gain of the sumoylation site at residue 38, resulting in the alteration of post-translational modifications of IκBα required for NF-κB activation. Conclusion: Our analysis hints that the post-translational modification in the NFKBIA Ser32Cys mutant would alter the signaling pathway of NF-κB. Our findings support the usefulness of WES in diagnosing IEIs and suggest the role of post-translational modification of IκBα.

A Novel De Novo  Missense Mutation Associated to Ectodermal Dysplasia with Dysgammaglobulinemia.

This study explores the plausible molecular interaction between a potent hepatitis C virus inhibitor, PSI-6206 (PSI), and human serum albumin (HSA), a primary transporter in blood plasma. Results obtained from both computational viz. molecular docking and molecular dynamics (MD) simulation and wet lab techniques such as UV absorption, fluorescence, circular dichroism (CD), and atomic force microscopy (AFM) complemented each other. While docking results identified PSI binding to subdomain IIA (Site I) of HSA by forming six hydrogen bonds, MD simulations signified the complex stability throughout the 50,000 ps. A consistent cutback in the Stern-Volmer quenching constant (K) along with rising temperatures supported the static mode of fluorescence quenching in response to PSI addition and implied the development of the PSI-HSA complex. This discovery was backed by the alteration of the HSA UV absorption spectrum, a larger value (>10 M.s) of the bimolecular quenching rate constant (kq) and the AFM-guided swelling of the HSA molecule, in the presence of PSI. Moreover, the fluorescence titration results revealed a modest binding affinity (4.27-6.25×10 M) in the PSI-HSA system, involving hydrogen bonds, van der Waals and hydrophobic interactions, as inferred from ΔS = + 22.77 J mol K and ΔH = - 11.02 KJ molvalues. CD and 3D fluorescence spectra reminded significant adjustment in the 2° and 3° structures and modification in the Tyr/Trp microenvironment of the protein in the PSI-bound state. The results obtained from drug competing experiments also advocated the binding location of PSI in HSA as Site I.

Use of computational and wet lab techniques to examine the molecular association between a potent hepatitis C virus inhibitor, PSI-6206 and human serum albumin.

Bruton's tyrosine kinase (BTK) is a cytoplasmic protein involved in the B cell development. X-linked agammaglobulinemia (XLA) is caused by mutation in the  gene, which results in very low or absent B cells. Affected males have markedly reduced immunoglobulin levels, which render them susceptible to recurrent and severe bacterial infections. Methods: Patients suspected with X-linked agammaglobulinemia were enrolled during the period of 2010-2018. Clinical summary, and immunological profiles of these patients were recorded. Peripheral blood samples were collected for monocyte BTK protein expression detection and  genetic analysis. The medical records between January 2020 and June 2023 were reviewed to investigate COVID-19 in XLA.

Clinical features and mutational analysis of X-linked agammaglobulinemia patients in Malaysia.

The binding mode of antineoplastic antimetabolite, floxuridine (FUDR), with human serum albumin (HSA), the leading carrier in blood circulation, was ascertained using multi-spectroscopic, microscopic, and computational techniques. A static fluorescence quenching was established due to decreased K values with rising temperatures, suggesting FUDR-HSA complexation. UV-vis absorption spectral results also supported this conclusion. The binding constant, K values, were found within 9.7-7.9 × 10 M at 290, 300, and 310 K, demonstrating a moderate binding affinity for the FUDR-HSA system. Thermodynamic data (ΔS = +46.35 J.mol.K and ΔH = -8.77 kJ.mol) predicted the nature of stabilizing forces (hydrogen-bonds, hydrophobic, and van der Waals interactions) for the FUDR-HSA complex. Circular dichroism spectra displayed a minor disruption in the protein's 2° and 3° structures. At the same time, atomic force microscopy images proved variations in the FUDR-HSA surface morphology, confirming its complex formation. The protein's microenvironment around Trp/Tyr residues was also modified, as judged by 3-D fluorescence spectra. FUDR-bound HSA showed better resistance against thermal stress. As disclosed from ligand displacement studies, the FUDR binding site was placed in subdomain IIA (Site I). Further, the molecular docking analysis corroborated the competing displacement studies. Molecular dynamics evaluations revealed that the complex achieved equilibrium during simulations, confirming the FUDR-HSA complex's stability.

Deciphering the binding mode and structural perturbations in floxuridine-human serum albumin complexation with spectroscopic, microscopic, and computational techniques.

Article	Year
Evolutionary trace analysis at the ligand binding site of laccase. Bioinformation\| PubMed ID: 18795108	2008
A combination of evolutionary trace method, molecular surface accessibility and hydrophobicity analysis to design a high hydrophobicity laccase. In silico biology\| PubMed ID: 22430288	2010
Spectrofluorometric and molecular docking studies on the binding of curcumenol and curcumenone to human serum albumin. International journal of molecular sciences\| PubMed ID: 25756376	2015
A novel de novo NLRC4 mutation reinforces the likely pathogenicity of specific LRR domain mutation. Clinical immunology (Orlando, Fla.)\| PubMed ID: 31870725	2020
Leaf Juice for Dengue: A Scoping Review. Nutrients\| PubMed ID: 35458146	2022
Potential Acetylcholinesterase Inhibitor Acting on the Pesticide Resistant and Susceptible Cotton Pests. ACS omega\| PubMed ID: 35755373	2022
Visual inspection reveals a novel pathogenic mutation in missed by the variant caller in whole‑exome sequencing. Molecular medicine reports\| PubMed ID: 36281931	2022
A Novel De Novo Missense Mutation Associated to Ectodermal Dysplasia with Dysgammaglobulinemia. Genes\| PubMed ID: 36292785	2022
Use of computational and wet lab techniques to examine the molecular association between a potent hepatitis C virus inhibitor, PSI-6206 and human serum albumin. Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy\| PubMed ID: 36868020	2023
Clinical features and mutational analysis of X-linked agammaglobulinemia patients in Malaysia. Frontiers in immunology\| PubMed ID: 37809070	2023
Deciphering the binding mode and structural perturbations in floxuridine-human serum albumin complexation with spectroscopic, microscopic, and computational techniques. Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy\| PubMed ID: 38061108	2024

Saharuddin Bin Mohamad

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