Displaying publications 1 - 20 of 61 in total

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  1. Special issue: Biomolecular Modelling and Simulation
    Choong YS, Mancera R, Lee VS
    Mol Biotechnol, 2024 Apr;66(4):567.
    PMID: 38337130 DOI: 10.1007/s12033-024-01073-1
  2. Application of Computational Techniques in Antibody Fc-Fused Molecule Design for Therapeutics
    Ng CL, Lim TS, Choong YS
    Mol Biotechnol, 2024 Apr;66(4):568-581.
    PMID: 37742298 DOI: 10.1007/s12033-023-00885-x
    Since the advent of hybridoma technology in the year 1975, it took a decade to witness the first approved monoclonal antibody Orthoclone OKT39 (muromonab-CD3) in the year 1986. Since then, continuous strides have been made to engineer antibodies for specific desired effects. The engineering efforts were not confined to only the variable domains of the antibody but also included the fragment crystallizable (Fc) region that influences the immune response and serum half-life. Engineering of the Fc fragment would have a profound effect on the therapeutic dose, antibody-dependent cell-mediated cytotoxicity as well as antibody-dependent cellular phagocytosis. The integration of computational techniques into antibody engineering designs has allowed for the generation of testable hypotheses and guided the rational antibody design framework prior to further experimental evaluations. In this article, we discuss the recent works in the Fc-fused molecule design that involves computational techniques. We also summarize the usefulness of in silico techniques to aid Fc-fused molecule design and analysis for the therapeutics application.
  3. Molecular interaction analysis of anti-IL-8 scFv-10F8-6His against IL-8 monomer through molecular docking and molecular dynamic simulations
    Abdul Kadir FFN, Che Nordin MA, S M N Mydin RB, Choong YS, Che Omar MT
    J Biomol Struct Dyn, 2023 Oct 14.
    PMID: 37837430 DOI: 10.1080/07391102.2023.2269254
    Elevated interleukin 8 (IL-8) expression has been linked to unfavorable outcomes in a range of inflammatory conditions, such as rheumatoid arthritis, psoriasis, and cancer. The human monoclonal antibody (HuMab) 10F8 and the hybridoma 35B11-B bind to an epitope on human IL-8, respectively. 10F8 inhibited interaction between IL-8 and neutrophils in eczema and pustulosis palmoplantaris patients while 35B11-B decreased size lesion in rat model. The binding interaction of monoclonal antibodies and IL-8, especially how complementarity-determining region (CDR) loops could bind the N-terminal of IL-8, has not been fully deliberated at molecular-level. Here, we used a combination of molecular docking, heated and long coarse-grained molecular dynamics simulations to identify key residues of established interaction. Based on heated MD simulation, docked pose of complexes generated by ClusPro showed good binding stability throughout of 70 ns simulation. Based on long molecular dynamic simulations, key residues for the binding were identified throughout of 1000 ns simulation. TYR-53, ASP-99, and ARG-100 of heavy chain CDR together with TYR-33 of light chain CDR are among the highest contributing energy residues within the binding interaction. Meanwhile, LYS11 and TYR13 of IL-8 are important for the determination of overall binding energy. Furthermore, the result of decomposition residues analysis is in good agreement with the interaction analysis data. Current study provides a list of important interacting residues and further scrutiny on these residues is essential for future development and design of a new and stable recombinant antibody against IL-8.Communicated by Ramaswamy H. Sarma.
  4. Fulltext Subtractive panning for the isolation of monoclonal PEPITEM peptide antibody by phage display
    Alassiri M, Lai JY, Ch'ng ACW, Choong YS, Alanazi A, Lim TS
    Sci Rep, 2023 Aug 21;13(1):13627.
    PMID: 37604859 DOI: 10.1038/s41598-023-40630-7
    Antibody phage display is a key tool for the development of monoclonal antibodies against various targets. However, the development of anti-peptide antibodies is a challenging process due to the small size of peptides for binding. This makes anchoring of peptides a preferred approach for panning experiments. A common approach is by using streptavidin as the anchor protein to present biotinylated peptides for panning. Here, we propose the use of recombinant expression of the target peptide and an immunogenic protein as a fusion for panning. The peptide inhibitor of trans-endothelial migration (PEPITEM) peptide sequence was fused to the Mycobacterium tuberculosis (Mtb) α-crystalline (AC) as an anchor protein. The panning process was carried out by subtractive selection of the antibody library against the AC protein first, followed by binding to the library to PEPITEM fused AC (PEPI-AC). A unique monoclonal scFv antibodies with good specificity were identified. In conclusion, the use of an alternative anchor protein to present the peptide sequence coupled with subtractive panning allows for the identification of unique monoclonal antibodies against a peptide target.
  5. Naïve antibody library derived monoclonal antibody against VP35 of Ebola virus
    Lai JY, Corona A, Ng CL, Tramontano E, Choong YS, Lim TS
    Int J Biol Macromol, 2023 Aug 01;245:125571.
    PMID: 37379953 DOI: 10.1016/j.ijbiomac.2023.125571
    Ebola virus is notorious for causing severe and even deadly haemorrhagic fever in infected humans and non-human primates. The high fatality rate of Ebola virus disease (EVD) has highlighted the need for effective diagnosis and treatment. Two monoclonal antibodies (mAbs) have been approved by USFDA for treatment of EVD. Virus surface glycoprotein is the common target for diagnostic and therapy including vaccines. Even so, VP35, a viral RNA polymerase cofactor and interferon inhibitor could be a potential target to curb EVD. The present work describes the isolation of three mAb clones from a phage-displayed human naïve scFv library against recombinant VP35. The clones showed binding against rVP35 in vitro and inhibition of VP35 in luciferase reporter gene assay. Structural modelling analysis was also carried out to identify the binding interactions involved in the antibody-antigen interaction model. This allows some insight into the "fitness" of the binding pocket between the paratope and target epitope which would be useful for the design of new mAbs through in silico means in the future. In conclusion, the information obtained from the 3 isolated mAbs could be potentially useful in the quest to improve VP35 targeting for therapeutic development in the future.
  6. Antibody Phage Display
    Nur A, Schubert M, Lai JY, Hust M, Choong YS, Isa WYHW, et al.
    Methods Mol Biol, 2023;2702:3-12.
    PMID: 37679612 DOI: 10.1007/978-1-0716-3381-6_1
    The application of antibodies has transcended across many areas of work but mainly as a research tool, for diagnostic and for therapeutic applications. Antibodies are immunoproteins from vertebrates that have the unique property of specifically binding foreign molecules and distinguish target antigens. This property allows antibodies to effectively protect the host from infections. Apart from the hybridoma technology using transgenic animals, antibody phage display is commonly considered the gold standard technique for the isolation of human monoclonal antibodies. The concept of antibody phage display surrounds the ability to display antibody fragments on the surface of M13 bacteriophage particles with the corresponding gene packaged within the particle. A repetitive in vitro affinity based selection process permits the enrichment of target specific binders. This process of recombinant human monoclonal antibody generation also enables additional engineering for various applications. This makes phage display an indispensable technique for antibody development and engineering activities.
  7. Theoretical study of the interactions between peptide tyrosine tyrosine [PYY (1-36)], a newly identified modulator in type 2 diabetes pathophysiology, with receptors NPY1R and NPY4R
    Choong YS, Lim YY, Soong JX, Savoo N, Guida C, Rhyman L, et al.
    Hormones (Athens), 2021 Sep;20(3):557-569.
    PMID: 33782920 DOI: 10.1007/s42000-021-00278-2
    PURPOSE: Diabetes mellitus is a common condition in the clinically obese. Bariatric surgery is one of the ways to put type 2 diabetes in remission. Recent findings propose the appetite-regulator peptide tyrosine tyrosine (PYY) as a therapeutic option for patients with type 2 diabetes. This novel gut hormone restores impaired insulin and glucagon secretion in pancreatic islets and is implicated in type 2 diabetes reversal after bariatric surgery. The current study elucidates the interactions between PYY and the NPY1R and NPY4R receptors using computational methods.

    METHODS: Protein structure prediction, molecular docking simulation, and molecular dynamics (MD) simulation were performed to elucidate the interactions of PYY with NPY1R and NPY4R.

    RESULTS: The predicted binding models of PYY-NPY receptors are in agreement with those described in the literature, although different interaction partners are presented for the C-terminal tail of PYY. Non-polar interactions are predicted to drive the formation of the protein complex. The calculated binding energies show that PYY has higher affinity for NPY4R (ΔGGBSA = -65.08 and ΔGPBSA = -87.62 kcal/mol) than for NPY1R (ΔGGBSA = -23.11 and ΔGPBSA = -50.56 kcal/mol).

    CONCLUSIONS: Based on the constructed models, the binding conformations obtained from docking and MD simulation for both the PYY-NPY1R and PYY-NPY4R complexes provide a detailed map of possible interactions. The calculated binding energies show a higher affinity of PYY for NPY4R. These findings may help to understand the mechanisms behind the improvement of diabetes following bariatric surgery.

  8. Biological evaluation the 2-aryl-2,3-dihydrobenzodiazaborinin-4(1H)-ones as potential dual α-glucosidase and α-amylase inhibitors with antioxidant properties
    Mphahlele MJ, Magwaza NM, Malindisa ST, Choong YS
    Chem Biol Drug Des, 2021 08;98(2):234-247.
    PMID: 34013660 DOI: 10.1111/cbdd.13893
    The 2-aryl-2,3-dihydrobenzodiazaborinin-4(1H)-ones (azaborininone) were synthesized as analogues of the 2-arylquinazoline-4-ones and screened through enzymatic assay in vitro for inhibitory effect against α-glucosidase and α-amylase activities. These azaborininones exhibited moderate to good inhibitory effect against these enzymes compared to acarbose used as a reference standard. The results are supported by the enzyme-ligand interactions through kinetics (in vitro) and molecular docking (in silico) studies. The test compounds also exhibited significant antioxidant activity through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) free radical scavenging assays. These azaborininone derivatives exhibited no effect on the viability of the human lung cancer (A549) cell line after 24 hr and were also not toxic towards the Vero cells.
  9. Development and structural characterisation of human scFv targeting MDM2 spliced variant MDM215kDa
    Lim CC, Chan SK, Lim YY, Ishikawa Y, Choong YS, Nagaoka Y, et al.
    Mol Immunol, 2021 07;135:191-203.
    PMID: 33930714 DOI: 10.1016/j.molimm.2021.04.016
    The murine double minute 2 (MDM2) protein is a major negative regulator of the tumour suppressor protein p53. Under normal conditions, MDM2 constantly binds to p53 transactivation domain and/or ubiquinates p53 via its role as E3 ubiquitin ligase to promote p53 degradation as well as nuclear export to maintain p53 levels in cells. Meanwhile, amplification of MDM2 and appearance of MDM2 spliced variants occur in many tumours and normal tissues making it a prognostic indicator for human cancers. The mutation or deletion of p53 protein in half of human cancers inactivates its tumour suppressor activity. However, cancers with wild type p53 have its function effectively inhibited through direct interaction with MDM2 oncoprotein. Here, we described the construction of a MDM2 spliced variant (rMDM215kDa) consisting of SWIB/MDM2 domain and its central region for antibody generation. Biopanning with a human naïve scFv library generated four scFv clones specific to rMDM215kDa. Additionally, the selected scFv clones were able to bind to the recombinant full length MDM2 (rMDM2-FL). Computational prediction showed that the selected scFv clones potentially bind to exon 7-8 of MDM2 while leaving the MDM2/SWIB domain free for p53 interaction. The developed antibodies exhibit good specificity can be further investigated for downstream biomedical and research applications.
  10. Fulltext Synthesis, Structure, Carbohydrate Enzyme Inhibition, Antioxidant Activity, In Silico Drug-Receptor Interactions and Drug-Like Profiling of the 5-Styryl-2-Aminochalcone Hybrids
    Mphahlele MJ, Agbo EN, Choong YS
    Molecules, 2021 May 04;26(9).
    PMID: 34064448 DOI: 10.3390/molecules26092692
    The 2-amino-5-(3/4-fluorostyryl)acetophenones were prepared and reacted with benzaldehyde derivatives to afford the corresponding 5-styryl-2-aminochalcone hybrids. The trans geometry of the styryl and α,β-unsaturated carbonyl arms, and the presence of NH…O intramolecular hydrogen bond were validated using 1H-NMR and X-ray data. The 2-amino-5-styrylacetophenones and their 5-styryl-2-aminochalcone derivatives were screened in vitro for their capability to inhibit α-glucosidase and/or α-amylase activities. Their antioxidant properties were evaluated in vitro through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) free radical scavenging assays. Kinetic studies of the most active derivatives from each series against α-glucosidase and/or α-amylase activities have been performed supported by molecular docking studies to determine plausible protein-ligand interactions on a molecular level. The key aspects of the pharmacokinetics of these compounds, i.e., absorption, distribution, metabolism, and excretion have also been simulated at theoretical level. The most active compounds from each series, namely, 2a and 3e, were evaluated for cytotoxicity against the normal monkey kidney cells (Vero cells) and the adenocarcinomic human epithelial (A549) cell line to establish their safety profile at least in vitro.
  11. Fulltext Heterocyclic Substitutions Greatly Improve Affinity and Stability of Folic Acid towards FRα. an In Silico Insight
    Al-Thiabat MG, Saqallah FG, Gazzali AM, Mohtar N, Yap BK, Choong YS, et al.
    Molecules, 2021 Feb 18;26(4).
    PMID: 33670773 DOI: 10.3390/molecules26041079
    Folate receptor alpha (FRα) is known as a biological marker for many cancers due to its overexpression in cancerous epithelial tissue. The folic acid (FA) binding affinity to the FRα active site provides a basis for designing more specific targets for FRα. Heterocyclic rings have been shown to interact with many receptors and are important to the metabolism and biological processes within the body. Nineteen FA analogs with substitution with various heterocyclic rings were designed to have higher affinity toward FRα. Molecular docking was used to study the binding affinity of designed analogs compared to FA, methotrexate (MTX), and pemetrexed (PTX). Out of 19 FA analogs, analogs with a tetrazole ring (FOL03) and benzothiophene ring (FOL08) showed the most negative binding energy and were able to interact with ASP81 and SER174 through hydrogen bonds and hydrophobic interactions with amino acids of the active site. Hence, 100 ns molecular dynamics (MD) simulations were carried out for FOL03, FOL08 compared to FA, MTX, and PTX. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of FOL03 and FOL08 showed an apparent convergence similar to that of FA, and both of them entered the binding pocket (active site) from the pteridine part, while the glutamic part was stuck at the FRα pocket entrance during the MD simulations. Molecular mechanics Poisson-Boltzmann surface accessible (MM-PBSA) and H-bond analysis revealed that FOL03 and FOL08 created more negative free binding and electrostatic energy compared to FA and PTX, and both formed stronger H-bond interactions with ASP81 than FA with excellent H-bond profiles that led them to become bound tightly in the pocket. In addition, pocket volume calculations showed that the volumes of active site for FOL03 and FOL08 inside the FRα pocket were smaller than the FA-FRα system, indicating strong interactions between the protein active site residues with these new FA analogs compared to FA during the MD simulations.
  12. Development of monoclonal antibodies against recombinant LipL21 protein of pathogenic Leptospira through phage display technology
    Mohd Ali MR, Sum JS, Aminuddin Baki NN, Choong YS, Nor Amdan NA, Amran F, et al.
    Int J Biol Macromol, 2021 Jan 31;168:289-300.
    PMID: 33310091 DOI: 10.1016/j.ijbiomac.2020.12.062
    Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs and symptoms of leptospirosis are usually varied, allowing it to be mistaken for other causes of acute febrile syndromes. Thus, early diagnosis and identification of a specific agent in clinical samples is crucial for effective treatment. This study was aimed to develop specific monoclonal antibodies against LipL21 antigen for future use in leptospirosis rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was optimized for expression and evaluated for immunogenicity. Then, a naïve phage antibody library was utilized to identify single chain fragment variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were analysed through monoclonal phage ELISA. However, after taking into consideration the background OD405 values, only 4 clones were sent for sequencing to determine human germline sequences. The sequence analysis showed that all 4 clones are identical. The in silico analysis of scFv-lip-1 complex indicated that the charged residues of scFv CDRs are responsible for the recognition with rLipL21 epitopes. The generated monoclonal antibody against rLipL21 will be evaluated as a detection reagent for the diagnosis of human leptospirosis in a future study.
  13. Fulltext Broad specificity of immune helminth scFv library to identify monoclonal antibodies targeting Strongyloides
    Rahumatullah A, Balachandra D, Noordin R, Baharudeen Z, Lim YY, Choong YS, et al.
    Sci Rep, 2021 01 28;11(1):2502.
    PMID: 33510342 DOI: 10.1038/s41598-021-82125-3
    Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.
  14. Fulltext Demystifying the catalytic pathway of Mycobacterium tuberculosis isocitrate lyase
    Ibeji CU, Salleh NAM, Sum JS, Ch'ng ACW, Lim TS, Choong YS
    Sci Rep, 2020 11 03;10(1):18925.
    PMID: 33144641 DOI: 10.1038/s41598-020-75799-8
    Pulmonary tuberculosis, caused by Mycobacterium tuberculosis, is one of the most persistent diseases leading to death in humans. As one of the key targets during the latent/dormant stage of M. tuberculosis, isocitrate lyase (ICL) has been a subject of interest for new tuberculosis therapeutics. In this work, the cleavage of the isocitrate by M. tuberculosis ICL was studied using quantum mechanics/molecular mechanics method at M06-2X/6-31+G(d,p): AMBER level of theory. The electronic embedding approach was applied to provide a better depiction of electrostatic interactions between MM and QM regions. Two possible pathways (pathway I that involves Asp108 and pathway II that involves Glu182) that could lead to the metabolism of isocitrate was studied in this study. The results suggested that the core residues involved in isocitrate catalytic cleavage mechanism are Asp108, Cys191 and Arg228. A water molecule bonded to Mg2+ acts as the catalytic base for the deprotonation of isocitrate C(2)-OH group, while Cys191 acts as the catalytic acid. Our observation suggests that the shuttle proton from isocitrate hydroxyl group C(2) atom is favourably transferred to Asp108 instead of Glu182 with a lower activation energy of 6.2 kcal/mol. Natural bond analysis also demonstrated that pathway I involving the transfer of proton to Asp108 has a higher intermolecular interaction and charge transfer that were associated with higher stabilization energy. The QM/MM transition state stepwise catalytic mechanism of ICL agrees with the in vitro enzymatic assay whereby Asp108Ala and Cys191Ser ICL mutants lost their isocitrate cleavage activities.
  15. Fulltext An intermolecular-split G-quadruplex DNAzyme sensor for dengue virus detection
    Ida J, Kuzuya A, Choong YS, Lim TS
    RSC Adv, 2020 Sep 07;10(55):33040-33051.
    PMID: 35515051 DOI: 10.1039/d0ra05439a
    Nucleic acids have special ability to organize themselves into various non-canonical structures, including a four-stranded DNA structure termed G-quadruplex (G4) that has been utilized for diagnostic and therapeutic applications. Herein, we report the ability of G4 to distinguish dengue virus (DENV) based on its serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) using a split G4-hemin DNAzyme configuration. In this system, two separate G-rich oligonucleotides are brought together upon target DNA strand hybridization to form a three-way junction architecture, allowing the formation of a G4 structure. The G4 formation in complexation with hemin can thus provide a signal readout by generating a DNAzyme that is able to catalyze H2O2-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). This results in a change of color providing a sensing platform for the colorimetric detection of DENV. In our approach, betaine and dimethyl sulfoxide were utilized for better G4 generation by enhancing the target-probe hybridization. In addition to this serotype-specific assay, a multi-probe cocktail assay, which is an all-in-one assay was also examined for DENV detection. The system highlights the potential of split G-quadruplex configurations for the development of DNA-based detection and serotyping systems in the future.
  16. Synthesis of furocoumarin-stilbene hybrids as potential multifunctional drugs against multiple biochemical targets associated with Alzheimer's disease
    Agbo EN, Gildenhuys S, Choong YS, Mphahlele MJ, More GK
    Bioorg Chem, 2020 08;101:103997.
    PMID: 32554280 DOI: 10.1016/j.bioorg.2020.103997
    A series of furocoumarin-stilbene hybrids has been synthesized and evaluated in vitro for inhibitory effect against acetylcholinesterase (AChE), butyrylcholinestarase (BChE), β-secretase, cyclooxygenase-2 (COX-2), and lipoxygenase-5 (LOX-5) activities including free radical-scavenging properties. Among these hybrids, 8-(3,5-dimethoxyphenyl)-4-(3,5-dimethoxystyryl)furochromen-2-one 4h exhibited significant anticholinesterase activity and inhibitory effect against β-secretase, COX-2 and LOX-5 activities. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and an in vitro cell-based antioxidant activity assay involving lipopolysaccharide induced reactive oxygen species production revealed that 4h has capability of scavenging free radicals. Molecular docking into AChE, BChE, β-secretase, COX-2 and LOX-5 active sites has also been performed.
  17. Fulltext Synthesis, In Vitro Evaluation and Molecular Docking of the 5-Acetyl-2-aryl-6-hydroxybenzo[b]furans against Multiple Targets Linked to Type 2 Diabetes
    Mphahlele MJ, Choong YS, Maluleka MM, Gildenhuys S
    Biomolecules, 2020 03 07;10(3).
    PMID: 32156083 DOI: 10.3390/biom10030418
    The 5-acetyl-2-aryl-6-hydroxybenzo[b]furans 2a-h have been evaluated through in vitro enzymatic assay against targets which are linked to type 2 diabetes (T2D), namely, α-glucosidase, protein tyrosine phosphatase 1B (PTP1B) and β-secretase. These compounds have also been evaluated for antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging method. The most active compounds against α-glucosidase and/or PTP1B, namely, 4-fluorophenyl 2c, 4-methoxyphenyl 2g and 3,5-dimethoxyphenyl substituted 2h derivatives were also evaluated for potential anti-inflammatory properties against cyclooxygenase-2 activity. The Lineweaver-Burk and Dixon plots were used to determine the type of inhibition on compounds 2c and 2h against α-glucosidase and PTP1B receptors. The interactions were investigated in modelled complexes against α-glucosidase and PTP1B via molecular docking.
  18. Fulltext A Direct Role for the CD1b Endogenous Spacer in the Recognition of a Mycobacterium tuberculosis Antigen by T-Cell Receptors
    Camacho F, Moreno E, Garcia-Alles LF, Chinea Santiago G, Gilleron M, Vasquez A, et al.
    Front Immunol, 2020;11:566710.
    PMID: 33162982 DOI: 10.3389/fimmu.2020.566710
    Lipids, glycolipids and lipopeptides derived from Mycobacterium tuberculosis (Mtb) are presented to T cells by monomorphic molecules known as CD1. This is the case of the Mtb-specific sulfoglycolipid Ac2SGL, which is presented by CD1b molecules and is recognized by T cells found in tuberculosis (TB) patients and in individuals with latent infections. Our group, using filamentous phage display technology, obtained two specific ligands against the CD1b-Ac2SGL complex: (i) a single chain T cell receptor (scTCR) from a human T cell clone recognizing the CD1b-AcSGL complex; and (ii) a light chain domain antibody (dAbκ11). Both ligands showed lower reactivity to a synthetic analog of Ac2SGL (SGL12), having a shorter acyl chain as compared to the natural antigen. Here we put forward the hypothesis that the CD1b endogenous spacer lipid (EnSpacer) plays an important role in the recognition of the CD1b-Ac2SGL complex by specific T cells. To support this hypothesis we combined: (a) molecular binding assays for both the scTCR and the dAbκ11 antibody domain against a small panel of synthetic Ac2SGL analogs having different acyl chains, (b) molecular modeling of the CD1b-Ac2SGL/EnSpacer complex, and (c) modeling of the interactions of this complex with the scTCR. Our results contribute to understand the mechanisms of lipid presentation by CD1b molecules and their interactions with T-cell receptors and other specific ligands, which may help to develop specific tools targeting Mtb infected cells for therapeutic and diagnostic applications.
  19. Structural approaches for the DNA binding motifs prediction in Bacillus thuringiensis sigma-E transcription factor (σETF)
    Lim YY, Lim TS, Choong YS
    J Mol Model, 2019 Sep 05;25(10):301.
    PMID: 31486892 DOI: 10.1007/s00894-019-4192-3
    The sigma-E transcription factor (σETF) can be found in most of the bacteria cells including Bacillus thuringiensis. However, the cellular regulatory mechanisms of these transcription factors in the mass production of δ-endotoxins during sporulation stage are yet to be revealed. In addition, the recognition of DNA towards σETF DNA binding motifs that led to the transcription activities is also being poorly studied. Therefore, this work studied the possible DNA binding motifs of σETF by utilising in silico approaches. The structure of σETF was first built via three different computational methods. A cognate DNA sequence was then docked to the predicted σETF DNA-binding motifs. The binding free energy calculated using molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) for triplicate 50 ns simulation of σETF-DNA complex revealed favourable binding energy of DNA to σETF (average ∆Gbind = -34.57 kcal/mol) mainly driven by non-polar interactions. This study revealed that σETF LYS131, ARG133, PHE138, TRP146, ARG222, LYS225 and ARG226 are most likely the key residues upon the binding and recognition of DNA prior to transcription actives. Since determination of genome-regulating protein which recognises specific DNA sequence is important to discriminate between the proteins preferences for different genes, this study might provide some understanding on the possible σETF-DNA recognition prior to transcription initiated for the δ-endotoxins production.
  20. Applications of Ensemble Docking in Potential Inhibitor Screening for Mycobacterium tuberculosis Isocitrate Lyase Using a Local Plant Database
    Lee YV, Choi SB, Wahab HA, Lim TS, Choong YS
    J Chem Inf Model, 2019 05 28;59(5):2487-2495.
    PMID: 30840452 DOI: 10.1021/acs.jcim.8b00963
    Isocitrate lyase (ICL) is a persistent factor for the survival of dormant stage Mycobacterium tuberculosis (MTB), thus a potential drug target for tuberculosis treatment. In this work, ensemble docking approach was used to screen for potential inhibitors of ICL. The ensemble conformations of ICL active site were obtained from molecular dynamics simulation on three dimer form systems, namely the apo ICL, ICL in complex with metabolites (glyoxylate and succinate), and ICL in complex with substrate (isocitrate). Together with the ensemble conformations and the X-ray crystal structures, 22 structures were used for the screening against Malaysian Natural Compound Database (NADI). The top 10 compounds for each ensemble conformation were selected. The number of compounds was then further narrowed down to 22 compounds that were within the Lipinski's Rule of Five for drug-likeliness and were also docked into more than one ensemble conformation. Theses 22 compounds were furthered evaluate using whole cell assay. Some compounds were not commercially available; therefore, plant crude extracts were used for the whole cell assay. Compared to itaconate (the known inhibitor of ICL), crude extracts from Manilkara zapota, Morinda citrifolia, Vitex negundo, and Momordica charantia showed some inhibition activity. The MIC/MBC value were 12.5/25, 12.5/25, 0.78/1.6, and 0.39/1.6 mg/mL, respectively. This work could serve as a preliminary study in order to narrow the scope for high throughput screening in the future.
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