Search results for: cysteine covalent binding

Authors: Ehsan Khorshidian, Afshin Farahbakhsh, Sina Aghili

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Enzymes are promising biocatalysts for many organic reactions. They have excellent features like high activity, specificity and selectivity, and can catalyze under mild and environment friendly conditions. Epoxy-activated supports are almost-ideal ones to perform very easy immobilization of proteins and enzymes at both laboratory and industrial scale. The activated epoxy supports (chitosan/alginate, Eupergit C) may be very suitable to achieve the multipoint covalent attachment of proteins and enzymes, therefore, to stabilize their three-dimensional structure. The enzyme is firstly covalently immobilized under conditions pH 7.0 and 10.0. The remaining groups of the support are blocked to stop additional interaction between the enzyme and support by mercaptoethanol or Triton X-100. The results show support allowed obtaining biocatalysts with high immobilized protein amount and hydrolytic activity. The immobilization of lipases on epoxy support may be considered as attractive tool for obtaining highly active biocatalysts to be used in both aqueous and anhydrous aqueous media.

Keywords: immobilization of enzymes, epoxy supports, enzyme multipoint covalent attachment, microbial lipases

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Authors: Sakina Fatima, Joseph-Patrick W. E. Clarke, Patricia A. Thibault, Subha Kalyaanamoorthy, Michael Levin, Aravindhan Ganesan

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Heteronuclear ribonucleoprotein (hnRNPA1 or A1) is associated with the pathology of different diseases, including neurological disorders and cancers. In particular, the aggregation and dysfunction of A1 have been identified as a critical driver for neurodegeneration (NDG) in Multiple Sclerosis (MS). Structurally, A1 includes a low-complexity domain (LCD) and two RNA-recognition motifs (RRMs), and their interdomain coordination may play a crucial role in A1 aggregation. Previous studies propose that RNA-inhibitors or nucleoside analogs that bind to RRMs can potentially prevent A1 self-association. Therefore, molecular-level understanding of the structures, dynamics, and nucleotide interactions with A1 RRMs can be useful for developing therapeutics for NDG in MS. In this work, a combination of computational modelling and biochemical experiments were employed to analyze a set of RNA-A1 RRM complexes. Initially, the atomistic models of RNA-RRM complexes were constructed by modifying known crystal structures (e.g., PDBs: 4YOE and 5MPG), and through molecular docking calculations. The complexes were optimized using molecular dynamics simulations (200-400 ns), and their binding free energies were computed. The binding affinities of the selected complexes were validated using a thermal shift assay. Further, the most important molecular interactions that contributed to the overall stability of the RNA-A1 RRM complexes were deduced. The results highlight that adenine and guanine are the most suitable nucleotides for high-affinity binding with A1. These insights will be useful in the rational design of nucleotide-analogs for targeting A1 RRMs.

Keywords: hnRNPA1, molecular docking, molecular dynamics, RNA-binding proteins

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Authors: Ashrafosadat Mousavi Laer, Elaheh Moravej

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The art of binding was simple and elementary at the beginning of Islam. This art thrived gradually and continued its development as an independent art. Identification of the binding techniques and used materials in covers and investigation of the arrays give us indexes for the better identification of different doctrines and methods of that time. The catalogers of the manuscripts usually pay attention to four items: gender, color, art elegances, injury, and exquisiteness of the cover. The criterion for classification of the covers is their art nature and gender. 15th century AD (9th century AH) was the period of the binding art development in which the most beautiful covers were produced by the so-called method of ‘burning’. At 16th century AD (10th century AH), in Safavid era, art changed completely and a fundamental evolution occurred in the technique and method of binding. The greatest change in this art was the extensive use of stamp that was made mostly of steel and copper. Theses stamps were presses against leather. These covers were called ‘beat’. In this paper, writing and bookbinding of about 32 Shahnamehs of Safavid era available in the Iranian libraries and museums are studied. An analytical-statistical study shows that four methods have been used including beat, burning, mosaic, and oily. 69 percent of the covers of these copies are cardboards with a leathery coating (goatskin) and have been produced by burning and beat methods. Its reasons are that these two methods have been common methods in Safavid era and performing them was only feasible on leather and the most desirable and commonly used leather of that time was goatskin which was the best option for cover legend durability and preserving the book and it was more durable because it had been made of goat skin. In addition, it had prepared a suitable opportunity for the binding artist’s creativity and innovation.

Keywords: Shahnameh, Safavid era, bookbinding, beat cover, burning cover

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Authors: Sonam Kumari

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Tuberculosis is the deadliest disease worldwide. Millions of people lose their lives every year due to this disease. It has turned lethal due to the erratic nature of its causative organism, Mycobacterium tuberculosis (Mtb). Mtb tends to enter into an inactive, dormant state and emerge to replicating state upon encountering favorable conditions. The mechanism by which Mtb switches from the dormant state to the replicative form is still poorly characterized. Proteome studies have given us an insight into the role of certain proteins in giving stupendous virulence to Mtb, but numerous dotsremain unconnected and unaccounted. The WhiB family of proteins is one such protein that is associated with developmental processes in actinomycetes. Mtb has seven such proteins (WhiB1 to WhiB7). WhiB proteins are transcriptional regulators; they regulate various essential genes of Mtbby binding to their promoter DNA. Biophysical parameters of the effect of DNA binding on WhiB proteins has not yet been appropriately characterized. Interaction with DNA induces conformational changes in the WhiB proteins, confirmed by steady-state fluorescence and circular dichroism spectroscopy. ITC has deduced thermodynamic parameters and the binding affinity of the interaction. Since these transcription factors are highly unstable in vitro, their stability and solubility were enhanced by the co-expression of molecular chaperones. The present study findings help determine the conditions under which the WhiB proteins interact with their interacting partner and the factors that influence their binding affinity. This is crucial in understanding their role in regulating gene expression in Mtbandin targeting WhiB proteins as a drug target to cure TB.

Keywords: mycobacterium tuberculosis, TB, whiB proteins, ITC

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Authors: Khodzhaberdi Allaberdiev

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In order to understand the interactions on the interface polyamide fibers and epoxy matrix in fiber- reinforced composites were investigated industrial aramid fibers: armos, svm, terlon using individual epoxy matrix components, epoxies: diglycidyl ether of bisphenol A (DGEBA), three- and diglycidyl derivatives of m, p-amino-, m, p-oxy-, o, m,p-carboxybenzoic acids, the models: curing agent, aniline and the compound, that depict of the structure the primary addition reaction the amine to the epoxy resin, N-di (oxyethylphenoxy) aniline. The chemical structure of the surface of untreated and treated polyamide fibers analyzed using Fourier transform infrared spectroscopy (FTIR). The impregnation of fibers with epoxy matrix components and N-di (oxyethylphenoxy) aniline has been carried out by heating 150˚C (6h). The optimum fiber loading is at 65%.The result a thermal treatment is the covalent bonds formation , derived from a combined of homopolymerization and crosslinking mechanisms in the interfacial region between the epoxy resin and the surface of fibers. The reactivity of epoxy resins on interface in microcomposites (MC) also depends from processing aids treated on surface of fiber and the absorbance moisture. The influences these factors as evidenced by the conversion of epoxy groups values in impregnated with DGEBA of the terlons: industrial, dried (in vacuum) and purified samples: 5.20 %, 4.65% and 14.10%, respectively. The same tendency for svm and armos fibers is observed. The changes in surface composition of these MC were monitored by X-ray photoelectron spectroscopy (XPS). In the case of the purified fibers, functional groups of fibers act as well as a catalyst and curing agent of epoxy resin. It is found that the value of the epoxy groups conversion for reinforced formulations depends on aromatic polyamides nature and decreases in the order: armos >svm> terlon. This difference is due of the structural characteristics of fibers. The interfacial interactions also examined between polyglycidyl esters substituted benzoic acids and polyamide fibers in the MC. It is found that on interfacial interactions these systems influences as well as the structure and the isomerism of epoxides. The IR-spectrum impregnated fibers with aniline showed that the polyamide fibers appreciably with aniline do not react. FTIR results of treated fibers with N-di (oxyethylphenoxy) aniline fibers revealed dramatically changes IR-characteristic of the OH groups of the amino alcohol. These observations indicated hydrogen bondings and covalent interactions between amino alcohol and functional groups of fibers. This result also confirms appearance of the exo peak on Differential Scanning Calorimetry (DSC) curve of the MC. Finally, the theoretical evaluation non-covalent interactions between individual epoxy matrix components and fibers has been performed using the benzanilide and its derivative contaning the benzimidazole moiety as a models of terlon and svm,armos, respectively. Quantum-topological analysis also demonstrated the existence hydrogen bond between amide group of models and epoxy matrix components.All the results indicated that on the interface polyamide fibers and epoxy matrix exist not only covalent, but and non-covalent the interactions during the preparation of MC.

Keywords: epoxies, interface, modeling, polyamide fibers

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Authors: Hamed K. Arzani, Ahmad Amiri, Hamid K. Arzani, Salim Newaz Kazi, Ahmad Badarudin

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The new design of heat exchangers utilizing an annular distributor opens a new gateway for realizing higher energy optimization. To realize this goal, graphene nanoplatelet-based water nanofluids with promising thermophysical properties were synthesized in the presence of covalent and noncovalent functionalization. Thermal conductivity, density, viscosity and specific heat capacity were investigated and employed as a raw data for ANSYS-Fluent to be used in two-phase approach. After validation of obtained results by analytical equations, two special parameters of convective heat transfer coefficient and pressure drop were investigated. The study followed by studying other heat transfer parameters of annular pass in the presence of graphene nanopletelesbased water nanofluids at different weight concentrations, input powers and temperatures. As a result, heat transfer performance and friction loss are predicted for both synthesized nanofluids.

Keywords: heat transfer, nanofluid, turbulent flow, forced convection flow, graphene nanoplatelet

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Authors: Prasanna Ramachandran, Kareem Graham, Helena Kiefel, Sunit Jain, Todd DeSantis

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Microbes that reside inside the mammalian GI tract, commonly referred to as the gut microbiome, have been shown to have therapeutic effects in animal models of disease. We hypothesize that specific proteins produced by these microbes are responsible for this activity and may be used directly as therapeutics. To speed up the discovery of these key proteins from the big-data metagenomics, we have applied machine learning techniques. Using amino acid sequences of known epitopes and their corresponding binding partners, protein interaction descriptors (PID) were calculated, making a positive interaction set. A negative interaction dataset was calculated using sequences of proteins known not to interact with these same binding partners. Using Random Forest and positive and negative PID, a machine learning model was trained and used to predict interacting versus non-interacting proteins. Furthermore, the continuous variable, cosine similarity in the interaction descriptors was used to rank bacterial therapeutic candidates. Laboratory binding assays were conducted to test the candidates for their potential as therapeutics. Results from binding assays reveal the accuracy of the machine learning prediction and are subsequently used to further improve the model.

Keywords: protein-interactions, machine-learning, metagenomics, microbiome

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Authors: Kakali Bhadra

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Binding of small molecules to DNA and recently to RNA, continues to attract considerable attention for developing effective therapeutic agents for control of gene expression. This work focuses towards understanding interaction of harmalol, a dihydro beta-carboline alkaloid, with different nucleic acid motifs viz. double stranded CT DNA, single stranded A-form poly(A), double-stranded A-form of poly(C)·poly(G) and clover leaf tRNAphe by different spectroscopic, calorimetric and molecular modeling techniques. Results of this study converge to suggest that (i) binding constant varied in the order of CT DNA > poly(C)·poly(G) > tRNAphe > poly(A), (ii) non-cooperative binding of harmalol to poly(C)·poly(G) and poly(A) and cooperative binding with CT DNA and tRNAphe, (iii) significant structural changes of CT DNA, poly(C)·poly(G) and tRNAphe with concomitant induction of optical activity in the bound achiral alkaloid molecules, while with poly(A) no intrinsic CD perturbation was observed, (iv) the binding was predominantly exothermic, enthalpy driven, entropy favoured with CT DNA and poly(C)·poly(G) while it was entropy driven with tRNAphe and poly(A), (v) a hydrophobic contribution and comparatively large role of non-polyelectrolytic forces to Gibbs energy changes with CT DNA, poly(C)·poly(G) and tRNAphe, and (vi) intercalated state of harmalol with CT DNA and poly(C)·poly(G) structure as revealed from molecular docking and supported by the viscometric data. Furthermore, with competition dialysis assay it was shown that harmalol prefers hetero GC sequences. All these findings unequivocally pointed out that harmalol prefers binding with ds CT DNA followed by ds poly(C)·poly(G), clover leaf tRNAphe and least with ss poly(A). The results highlight the importance of structural elements in these natural beta-carboline alkaloids in stabilizing different DNA and RNA of various motifs for developing nucleic acid based better therapeutic agents.

Keywords: calorimetry, docking, DNA/RNA-alkaloid interaction, harmalol, spectroscopy

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Authors: Nidhi Chadha, A. K. Tiwari, Marilyn D. Milton, Anil K. Mishra

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Translocator protein (18 kDa) TSPO is highly expressed during microglia activation in neuroinflammation. Although a number of PET ligands have been developed for the visualization of activated microglia, one of the advantageous approaches is to develop potential optical imaging (OI) probe. Our study involves computational screening, synthesis and evaluation of TSPO ligand through various imaging modalities namely PET/SPECT/Optical. The initial computational screening involves pharmacophore modeling from the library designing having oxo-benzooxazol-3-yl-N-phenyl-acetamide groups and synthesis for visualization of efficacy of these compounds as multimodal imaging probes. Structure modeling of monomer, Ala147Thr mutated, parallel and anti-parallel TSPO dimers was performed and docking analysis was performed for distinct binding sites. Computational analysis showed pattern of variable binding profile of known diagnostic ligands and NBMP via interactions with conserved residues along with TSPO’s natural polymorphism of Ala147→Thr, which showed alteration in the binding affinity due to considerable changes in tertiary structure. Preliminary in vitro binding studies shows binding affinity in the range of 1-5 nm and selectivity was also certified by blocking studies. In summary, this skeleton was found to be potential probe for TSPO imaging due to ease in synthesis, appropriate lipophilicity and reach to specific region of brain.

Keywords: TSPO, molecular modeling, imaging, docking

Procedia PDF Downloads 462

Authors: Sirilak Kongkaew, Pathumwadee Yodmanee, Nopporn Kaiyawet, Arthitaya Meeprasert, Thanyada Rungrotmongkol, Toshikatsu Kaburaki, Hiroshi Noguchi, Fujio Takeuch, Nawee Kungwan, Supot Hannongbua

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Behçet’s disease (BD) is a genetic autoimmune expressed by multisystemic inflammatory disorder mostly occurred at the skin, joints, gastrointestinal tract, and genitalia, including ocular, oral, genital, and central nervous systems. Most BD patients in Japan and Korea were strongly indicated by the genetic factor namely HLA-B*51 (especially, HLA-B*51:01) marker in HMC class I, while HLA-A*26:01 allele has been detected from the BD patients in Greek, Japan, and Taiwan. To understand the selective binding of the MICA-TM peptide towards the HLAs associated with BD, the molecular dynamics simulations were applied on the four HLA alleles (B*51:01, B*35:01, A*26:01, and A*11:01) in complex with such peptide. As a result, the key residues in the binding groove of HLA protein which play an important role in the MICA-TM peptide binding and stabilization were revealed. The Van der Waals force was found to be the main protein-protein interaction. Based on the binding free energy prediction by MM/PBSA method, the MICA-TM peptide interacted stronger to the HLA alleles associated to BD in the identical class by 7-12 kcal/mol. The obtained results from the present study could help to differentiate the HLA alleles and explain a source of Behçet’s disease.

Keywords: Behçet’s disease, MD simulations, HMC class I, autoimmune

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Authors: Niv Papo

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Characterizing the binding selectivity landscape of interacting proteins is crucial both for elucidating the underlying mechanisms of their interaction and for developing selective inhibitors. However, current mapping methods are laborious and cannot provide a sufficiently comprehensive description of the landscape. Here, we introduce a distinct and efficient strategy for comprehensively mapping the binding landscape of proteins using a combination of experimental multi-target selective library screening and in silico next-generation sequencing analysis. We map the binding landscape of a non-selective trypsin inhibitor, the amyloid protein precursor inhibitor (APPI), to each of four human serine proteases (kallikrein-6, mesotrypsin, and anionic and cationic trypsins). We then use this map to dissect and improve the affinity and selectivity of APPI variants toward each of the four proteases. Our strategy can be used as a platform for the development of a new generation of target-selective probes and therapeutic agents based on selective protein–protein interactions.

Keywords: drug design, directed evolution, protein engineering, protease inhibition.

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Authors: Karolis Leonavičius, Dalius Kučiauskas, Dangiras Lukošius, Arnoldas Jasiūnas, Kostas Zdanys, Rokas Stanislovas, Emilis Gegevičius, Žana Kapustina, Juozas Nainys

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Screening throughput is a common bottleneck in many research areas, including functional genomics, drug discovery, and directed evolution. High-throughput screening techniques can be classified into two main categories: (i) affinity-based screening and (ii) functional screening. The first one relies on binding assays that provide information about the affinity of a test molecule for a target binding site. Binding assays are relatively easy to establish; however, they reveal no functional activity. In contrast, functional assays show an effect triggered by the interaction of a ligand at a target binding site. Functional assays might be based on a broad range of readouts, such as cell proliferation, reporter gene expression, downstream signaling, and other effects that are a consequence of ligand binding. Screening of large cell or gene libraries based on direct activity rather than binding affinity is now a preferred strategy in many areas of research as functional assays more closely resemble the context where entities of interest are anticipated to act. Droplet sorting is the basis of high-throughput functional biological screening, yet its applicability is limited due to the technical complexity of integrating high-performance droplet analysis and manipulation systems. As a solution, the Droplet Genomics Styx platform enables custom droplet sorting workflows, which are necessary for the development of early-stage or complex biological therapeutics or industrially important biocatalysts. The poster will focus on the technical design considerations of Styx in the context of its application spectra.

Keywords: functional screening, droplet microfluidics, droplet sorting, dielectrophoresis

Procedia PDF Downloads 135

Authors: Don Anushka Sandaruwan Elvitigala, P. D. S. U. Wickramasinghe, Jehee Lee

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Cystatins are a large superfamily of proteins which act as reversible inhibitors of cysteine proteases. Papain proteases and cysteine cathepsins are predominant substrates of cystatins. Cystatin superfamily can be further clustered into three groups as Stefins, Cystatins, and Kininogens. Among them, stefines are also known as family 1 cystatins which harbors cystatin Bs and cystatin As. In this study, a homologue of family one cystatins more close to cystatin Bs was identified from Korean black rockfish (Sebastes schlegeli) using a prior constructed cDNA (complementary deoxyribonucleic acid) database and designated as RfCyt1. The full-length cDNA of RfCyt1 consisted of 573 bp, with a coding region of 294 bp. It comprised a 5´-untranslated region (UTR) of 55 bp, and 3´-UTR of 263 bp. The coding sequence encodes a polypeptide consisting of 97 amino acids with a predicted molecular weight of 11kDa and theoretical isoelectric point of 6.3. The RfCyt1 shared homology with other teleosts and vertebrate species and consisted conserved features of cystatin family signature including single cystatin-like domain, cysteine protease inhibitory signature of pentapeptide (QXVXG) consensus sequence and N-terminal two conserved neighboring glycine (⁸GG⁹) residues. As expected, phylogenetic reconstruction developed using the neighbor-joining method showed that RfCyt1 is clustered with the cystatin family 1 members, in which more closely with its teleostan orthologues. An SYBR Green qPCR (quantitative polymerase chain reaction) assay was performed to quantify the RfCytB transcripts in different tissues in healthy and immune stimulated fish. RfCyt1 was ubiquitously expressed in all tissue types of healthy animals with gill and spleen being the highest. Temporal expression of RfCyt1 displayed significant up-regulation upon infection with Aeromonas salmonicida. Recombinantly expressed RfCyt1 showed concentration-dependent papain inhibitory activity. Collectively these findings evidence for detectable protease inhibitory and immunity relevant roles of RfCyt1 in Sebastes schlegeli.

Keywords: Sebastes schlegeli, family 1 cystatin, immune stimulation, expressional modulation

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Authors: Matineh Sadat Hosseini Gheidari, Vahid Reza Yazdanpanah

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In this paper, we used the empirical tight-binding method (ETBM) with sp3s* approximation and considering the first nearest neighbor with spin-orbit interactions in order to model superlattice structure (SLS) of (AlₓGa₁₋ₓAs)ₘ/(GaAs)ₙ grown on GaAs (100) substrate at 300K. In the next step, we investigated the behavior of the energy gap and wavelength of this superlattice in terms of different thicknesses of core materials and Al mole fractions. As a result of this survey, we found out that as the Al composition increases, the energy gap of this superlattice has an upward trend and ranges from 1.42-1.63 eV. Also, according to the wavelength range that we gained from this superlattice in different Al mole fractions and various thicknesses, we can find a suitable semiconductor for a special light-emitting diode (LED) application.

Keywords: energy gap, empirical tight-binding method, light-emitting diode, superlattice, wavelength

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Authors: Natalia Alzate-Carvajal, Diego A. Acevedo-Guzman, Victor Meza-Laguna, Mario H. Farias, Luis A. Perez-Rey, Edgar Abarca-Morales, Victor A. Garcia-Ramirez, Vladimir A. Basiuk, Elena V. Basiuk

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Direct covalent functionalization of prefabricated free-standing graphene oxide paper (GOP) is considered as the only approach suitable for systematic tuning of thermal, mechanical and electronic characteristics of this important class of carbon nanomaterials. At the same time, the traditional liquid-phase functionalization protocols can compromise physical integrity of the paper-like material up to its total disintegration. To avoid such undesirable effects, we explored the possibility of employing an alternative, solvent-free strategy for facile and nondestructive functionalization of GOP with two representative aliphatic amines, 1-octadecylamine (ODA) and 1,12-diaminododecane (DAD), as well as with two aromatic amines, 1-aminopyrene (AP) and 1,5-diaminonaphthalene (DAN). The functionalization was performed under moderate heating at 150-180 °C in vacuum. Under such conditions, it proceeds through both amidation and epoxy ring opening reactions. Comparative characterization of pristine and amine-functionalized GOP mats was carried out by using Fourier-transform infrared, Raman, and X-ray photoelectron spectroscopy (XPS), thermogravimetric (TGA) and differential thermal analysis, scanning electron and atomic force microscopy (SEM and AFM, respectively). Besides that, we compared the stability in water, wettability, electrical conductivity and elastic (Young's) modulus of GOP mats before and after amine functionalization. The highest content of organic species was obtained in the case of GOP-ODA, followed by GOP-DAD, GOP-AP and GOP-DAN samples. The covalent functionalization increased mechanical and thermal stability of GOP, as well as its electrical conductivity. The magnitude of each effect depends on the particular chemical structure of amine employed, which allows for tuning a given GOP property. Morphological characterization by using SEM showed that, compared to pristine graphene oxide paper, amine-modified GOP mats become relatively ordered layered assemblies, in which individual GO sheets are organized in a near-parallel pattern. Financial support from the National Autonomous University of Mexico (grants DGAPA-IN101118 and IN200516) and from the National Council of Science and Technology of Mexico (CONACYT, grant 250655) is greatly appreciated. The authors also thank David A. Domínguez (CNyN of UNAM) for XPS measurements and Dr. Edgar Alvarez-Zauco (Faculty of Science of UNAM) for the opportunity to use TGA equipment.

Keywords: amines, covalent functionalization, gas-phase, graphene oxide paper

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Authors: Kah Yan How, Peh Fern Ong, Keang Peng Song

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Porphyromonas gingivalis is a black-pigmented, anaerobic Gram-negative bacterium that is important in the progression of chronic and severe periodontitis. This organism has an essential requirement for iron, which is usually obtained from hemin, using specific membrane receptors, proteases, and lipoproteins. In this study, we report the characterization of a novel 24 kDa hemin-binding protein, HmuX, in P. gingivalis W50. The hmuX gene is 651 bp long which encodes for a 217 amino acid protein. HmuX was found to be identical at the C-terminus to the previously reported HmuY protein, differing by an additional 74 amino acids at the N-terminus. Recombinant HmuX demonstrated hemin-binding ability by LDS- PAGE and TMBZ staining. Sequence analysis of HmuX revealed a putative lipoprotein attachment site, suggesting its possible role as a lipoprotein. HmuX was also localized to the outer cell surface by transmission electron microscopy. Northern analysis showed hmuX to be transcribed as a single gene and that hmuX mRNA was tightly regulated by the availability of extra-cellular hemin. P. gingivalis isogenic mutant deficient in hmuX gene exhibited significant growth retardation under hemin-limited conditions. Taken together, these results suggest that HmuX is a hemin-binding lipoprotein, important in hemin utilization for the growth of P. gingivalis.

Keywords: Porphyromonas gingivalis, periodontal diseases, HmuX, protein characterization

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Authors: Sinethemba Yakobi, Lindiwe Zuma, Ofentse Pooe

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Gonococcal infections present a notable public health issue, and the major approach for treatment involves using β-lactam antibiotics that specifically target penicillin-binding protein 2 (PBP2) in Neisseria gonorrhoeae. This study examines the influence of flavonoids, namely rutin, on the structural changes of PBP2 in both penicillin-resistant (FA6140) and penicillin-susceptible (FA19) strains. The research clarifies the structural effects of particular mutations, such as inserting an aspartate residue at position 345 (Asp-345a) in the PBP2 protein. The strain FA6140, which is resistant to penicillin, shows specific changes that lead to a decrease in penicillin binding. These mutations, namely P551S and F504L, significantly impact the pace at which acylation occurs and the stability of the strain under high temperatures. Molecular docking analyses investigate the antibacterial activities of rutin and other phytocompounds, emphasizing its exceptional binding affinity and potential as an inhibitor of PBP2. Quercetin and protocatechuic acid have encouraging antibacterial effectiveness, with quercetin displaying characteristics similar to those of drugs. Molecular dynamics simulations offer a detailed comprehension of the interactions between flavonoids and PBP2, highlighting rutin's exceptional antioxidant effects and strong affinity for the substrate binding site. The study's wider ramifications pertain to the pressing requirement for antiviral treatments in the context of the ongoing COVID-19 epidemic. Flavonoids have a strong affinity for binding to PBP2, indicating their potential as inhibitors to impair cell wall formation in N. gonorrhoeae. Ultimately, this study provides extensive knowledge on the interactions between proteins and ligands, the dynamics of the structure, and the ability of flavonoids to combat penicillin-resistant N. gonorrhoeae bacteria. The verified simulation outcomes establish a basis for creating potent inhibitors and medicinal therapies to combat infectious illnesses.

Keywords: phytochemicals, penicillin-binding protein 2, gonococcal infection, ligand-protein interaction, binding energy, neisseria gonorrhoeae FA19, neisseria gonorrhoeae FA6140, flavonoids

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Authors: Muhamad Azuddin Hassan, Siti Shafura Karim, Ambri Mohamed, Iskandar Yahya

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Human serum albumin plays a significant part in the physiological functions of the human body system (HSA).HSA level monitoring is critical for early detection of HSA-related illnesses. The goal of this study is to show that a field effect transistor (FET)-based immunosensor can assess HSA using high aspect ratio carbon nanotubes network (CNT) as a transducer. The CNT network were deposited using air brush technique, and the FET device was made using a shadow mask process. Field emission scanning electron microscopy and a current-voltage measurement system were used to examine the morphology and electrical properties of the CNT network, respectively. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy were used to confirm the surface alteration of the CNT. The detection process is based on covalent binding interactions between an antibody and an HSA target, which resulted in a change in the manufactured biosensor's drain current (Id).In a linear range between 1 ng/ml and 10zg/ml, the biosensor has a high sensitivity of 0.826 mA (g/ml)-1 and a LOD value of 1.9zg/ml.HSA was also identified in a genuine serum despite interference from other biomolecules, demonstrating the CNT-FET immunosensor's ability to quantify HSA in a complex biological environment.

Keywords: carbon nanotubes network, biosensor, human serum albumin

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Authors: German Hernandez-Alonso, Antonio Lazcano, Arturo Becerra

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Until today, viruses remain controversial and poorly understood; about their origin, this problem represents an enigma and one of the great challenges for the contemporary biology. Three main theories have tried to explain the origin of viruses: regressive evolution, escaped host gene, and pre-cellular origin. Under the perspective of the escaped host gene theory, it can be assumed a cellular origin of viral components, like protein RNA-binding domains. These universal distributed RNA-binding domains are related to the RNA metabolism processes, including transcription, processing, and modification of transcripts, translation, RNA degradation and its regulation. In the case of viruses, these domains are present in important viral proteins like helicases, nucleases, polymerases, capsid proteins or regulation factors. Therefore, they are implicated in the replicative cycle and parasitic processes of viruses. That is why it is possible to think that those domains present low levels of divergence due to selective pressures. For these reasons, the main goal for this project is to create a catalogue of the RNA-binding domains found in all the available viral proteomes, using bioinformatics tools in order to analyze its evolutionary process, and thus shed light on the general virus evolution. ProDom database was used to obtain larger than six thousand RNA-binding domain families that belong to the three cellular domains of life and some viral groups. From the sequences of these families, protein profiles were created using HMMER 3.1 tools in order to find distant homologous within greater than four thousand viral proteomes available in GenBank. Once accomplished the analysis, almost three thousand hits were obtained in the viral proteomes. The homologous sequences were found in proteomes of the principal Baltimore viral groups, showing interesting distribution patterns that can contribute to understand the evolution of viruses and their host-virus interactions. Presence of cellular RNA-binding domains within virus proteomes seem to be explained by closed interactions between viruses and their hosts. Recruitment of these domains is advantageous for the viral fitness, allowing viruses to be adapted to the host cellular environment.

Keywords: bioinformatics tools, distant homology, RNA-binding domains, viral evolution

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Authors: Nicole C. Valdez, Vincent L. Borromeo, Conrad C. Chong, Ahmad F. Mazahery

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Breast cancer is the most prevalent cancer worldwide, where the majority of cases are estrogen-receptor positive and involve 2 receptor proteins. The binding of estrogen to estrogen receptor alpha (ERα) promotes breast cancer growth, while it's binding to estrogen-receptor beta (ERβ) inhibits tumor growth. While natural products have been a promising source of chemotherapeutic agents, the challenge remains in finding a bioactive compound that specifically targets cancer cells, minimizing side effects on normal cells. Flavonoids are natural products that act as phytoestrogens and induce the same response as estrogen. They are able to compete with estrogen for binding to ERα; however, it has a higher binding affinity for ERβ. Their abundance in nature and low toxicity make them a potential candidate for breast cancer treatment. This study aimed to determine which particular flavonoids can specifically recognize ERβ and potentially be used for breast cancer treatment through molecular docking. A total of 206 flavonoids comprised of 97 isoflavones and 109 flavanones were collected from ZINC15, while the 3D structures of ERβ and ERα were obtained from Protein Data Bank. These flavonoid subclasses were chosen as they bind more strongly to ERs due to their chemical structure. The structures of the flavonoid ligands were converted using Open Babel, while the estrogen receptor protein structures were prepared using Autodock MGL Tools. The optimal binding site was found using BIOVIA Discovery Studio Visualizer before docking all flavonoids on both ERβ and ERα through Autodock Vina. Genistein is a flavonoid that exhibits anticancer effects by binding to ERβ, so its binding affinity was used as a baseline. Eriodictyol and 4”,6”-Di-O-Galloylprunin both exceeded genistein’s binding affinity for ERβ and was lower than its binding affinity for ERα. Of the two, eriodictyol was pursued due to its antitumor properties on a lung cancer cell line and on glioma cells. It is able to arrest the cell cycle at the G2/M phase by inhibiting the mTOR/PI3k/Akt cascade and is able to induce apoptosis via the PI3K/Akt/NF-kB pathway. Protein pathway and gene analysis were also conducted using ChEMBL and PANTHER and it was shown that eriodictyol might induce anticancer effects through the ROS1, CA7, KMO, and KDM1A genes which are involved in cell proliferation in breast cancer, non-small cell lung cancer, and other diseases. The high binding affinity of eriodictyol to ERβ, as well as its potential affected genes and antitumor effects, therefore, make it a candidate for the development of new breast cancer treatment. Verification through in vitro experiments such as checking the upregulation and downregulation of genes through qPCR and checking cell cycle arrest using a flow cytometry assay is recommended.

Keywords: breast cancer, estrogen receptor, flavonoid, molecular docking

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Authors: M. Kowalik, J. Masternak, K. Kazimierczuk, O. V. Khavryuchenko, B. Kupcewicz, B. Barszcz

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Recently, the growing interest of chemists in metal-organic coordination polymers (MOCPs) is primarily derived from their intriguing structures and potential applications in catalysis, gas storage, molecular sensing, ion exchanges, nonlinear optics, luminescence, etc. Currently, we are devoting considerable effort to finding the proper method of synthesizing new coordination polymers containing S- or N-heteroaromatic carboxylates as linkers and characterizing the obtained Pb(II) compounds according to their structural diversity, luminescence, and thermal properties. The choice of Pb(II) as the central ion of MOCPs was motivated by several reasons mentioned in the literature: i) a large ionic radius allowing for a wide range of coordination numbers, ii) the stereoactivity of the 6s2 lone electron pair leading to a hemidirected or holodirected geometry, iii) a flexible coordination environment, and iv) the possibility to form secondary bonds and unusual non-covalent interactions, such as classic hydrogen bonds and π···π stacking interactions, as well as nonconventional hydrogen bonds and rarely reported tetrel bonds, Pb(lone pair)···π interactions, C–H···Pb agostic-type interactions or hydrogen bonds, and chelate ring stacking interactions. Moreover, the construction of coordination polymers requires the selection of proper ligands acting as linkers, because we are looking for materials exhibiting different network topologies and fluorescence properties, which point to potential applications. The reaction of Pb(NO₃)₂ with 1H-pyrrole-2-carboxylic acid (2prCOOH) leads to the formation of a new four-nuclear Pb(II) polymer, [Pb4(2prCOO)₈(H₂O)]ₙ, which has been characterized by CHN, FT-IR, TG, PL and single-crystal X-ray diffraction methods. In view of the primary Pb–O bonds, Pb1 and Pb2 show hemidirected pentagonal pyramidal geometries, while Pb2 and Pb4 display hemidirected octahedral geometries. The topology of the strongest Pb–O bonds was determined as the (4·8²) fes topology. Taking the secondary Pb–O bonds into account, the coordination number of Pb centres increased, Pb1 exhibited a hemidirected monocapped pentagonal pyramidal geometry, Pb2 and Pb4 exhibited a holodirected tricapped trigonal prismatic geometry, and Pb3 exhibited a holodirected bicapped trigonal prismatic geometry. Moreover, the Pb(II) lone pair stereoactivity was confirmed by DFT calculations. The 2D structure was expanded into 3D by the existence of non-covalent O/C–H···π and Pb···π interactions, which was confirmed by the Hirshfeld surface analysis. The above mentioned interactions improve the rigidity of the structure and facilitate the charge and energy transfer between metal centres, making the polymer a promising luminescent compound.

Keywords: coordination polymers, fluorescence properties, lead(II), lone electron pair stereoactivity, non-covalent interactions

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Authors: Tengfei He, Mengjie Zhang, Baosheng Jin

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In this study, the low-cost halloysite (Hal) was utilized for the first time to enhance the solid-phase enrichment and stability of heavy metals (HMs) during solid waste pyrolysis through experimental and theoretical methods, and compared with kaolinite (Kao). Experimental results demonstrated that Hal was superior to Kao in improving the solid-phase enrichment of HMs. Adding Hal reduced the proportion of HMs in the unstable fraction (F1+F2), consequently lowering the environmental risk of biochar and the extractable state of HMs. Through Grand canonical Monte Carlo and Density Functional Theory (DFT) simulations, the adsorption amounts and adsorption mechanisms of Cd/Pb compound on Hal/Kao surfaces were analyzed. The adsorption amounts of HMs by Hal were significantly higher than Kao and decreased with increasing temperature, and the difference in adsorption performance caused by structural bending was negligible. The DFT results indicated that Cd/Pb monomers were stabilized by establishing covalent bonds with OH or reactive O atoms on the Al-(0 0 1) surface, whereas the covalent bonds with ionic bonding properties formed between Cl atoms and unsaturated Al atoms played a crucial role in stabilizing HM chlorides. This study highlights the potential of Hal in stabilizing HMs during pyrolysis without requiring any modifications.

Keywords: heavy metals, halloysite, density functional theory, grand canonical Monte Carlo

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Authors: Han-Bin Kim, Sooim Shin, Moonsung Choi

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There is a growing interest in introducing a desired functional group on enzymes in the field of protein engineering. In here, various redox centers were newly created using histidine tag, which is widely used for protein purification, plus cobalt in one of cupredoxins, amicyanin. The coordination of Cobalt-His tag and reactivity of the Co²⁺ loaded His-tag also were characterized. 3xHis-tag, 6xHis-tag, and 9xHis-tag were introduced on amicyanin by site-directed mutagenesis, and then Co²⁺ was loaded on each His-tagged amicyanin. The spectral changes at 330 nm corresponding to cobalt binding on His-tag site indicated the binding ratio of 3xHis-tag, 6xHis-tag, and 9xHis-tag to cobalt as 1:1, 1:2, 1:3 respectively. Based on kinetic studies of binding cobalt to 3xHis-tag, 6xHis-tag, and 9xHis-tagged amicyanin, the nature of the sites was elucidated. In addition, internal electron transfer properties between Cu¹⁺ site and engineered site of amicyanin were determined. These results provide insight into improvement of metal coordination and alternation of the redox properties of metal as a new catalytic site on proteins.

Keywords: amicyanin, cobalt, histidine, protein engineering

Procedia PDF Downloads 162

Authors: Kai-Cheng Hsu, Tzu-Ying Sung, Jinn-Moon Yang

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Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Although several drugs have been developed to treat colorectal cancer, such as Regorafenib and 5-FU, their efficacy is often limited by the development of drug resistance. Therefore, development of new drugs with new scaffolds is necessary to treat CRC. Here, we used site-moiety maps to identify inhibitors against PIM1, LIMK1, SRC, and mTOR, which are often overexpressed in CRC. A site-moiety map represents physicochemical properties and moiety preferences of a binding site through anchors. An anchor contains three elements: (1) conserved interacting residues of a binding pocket; (2) moiety preference of the binding pocket; and (3) the type (e.g., hydrogen-bonding or van der Waals interactions) of interaction between the moieties and the binding pocket. Then, we performed a structure-based virtual screening of ~260,000 compounds and selected compound candidates with high site-moiety map scores for bioassays. Among these candidates, compound 1 and compound 2 inhibited the growth of CRC cells with IC50 values of <10 μM. The experimental result of enzyme-based assays indicated that compound 1 is a dual inhibitor against PIM1 (IC50 6 μM) and LIMK1(IC50 11 μM). Compound 2 was predicted as a SRC inhibitor and will be further validated. The compounds inhibited different protein targets compared to the current drugs. We believe that the compounds provide a starting point to design new drugs for CRC treatment.

Keywords: colorectal cancer, drug discovery, site-moiety map, virtual screening, PIM1, LIMK1

Procedia PDF Downloads 246

Authors: Xing Lu, Yi-ming Wu, Yan-hong Zhu, Zhen-feng Chen, Hong Liang, Yan Peng

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[Eu(BMQ)2(NO3)3(CH3OH)(H2O)] (1),and [Er(BMQ)2(NO3)3(CH3OH)(H2O)] (2),were synthesized. Compounds 1 and 2 exhibit a certain extent cytotoxicity against Hep G2, Hela 229, MGC80-3 and BEL-7404 cell lines invitro, with IC50 values in the14.51±1.41μM to 52.49±4.01μM range. Compound 1 exhibited significantly enhanced cytotoxicity against MGC80-3 cell line, comparing with free 3-(1H-benzimidazol-2-yl)-6-methyl-2(1H)- quinolinone. The binding abilities of 1 to DNA were stronger than that of 2. Intercalation is the most probable binding mode for both the complexes.

Keywords: quinolinone, Eu(II) complex, Er(III) complex, cytotoxicity.

Procedia PDF Downloads 598

Authors: Harish Rajak, Swati Singh

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A series of novel pyrimidine based semicarbazone were designed and synthesized on the basis of semicarbazone based pharmacophoric model to satisfy the structural prerequisite crucial for antiepileptic activity. The semicarbazones based pharmacophoric model consists of following four essential binding sites: (i) An aryl hydrophobic binding site with halo substituent; (ii) A hydrogen bonding domain; (iii) An electron donor group and (iv) Another hydrophobic-hydrophilic site controlling the pharmacokinetic features of the anticonvulsant. The aryl semicarbazones has been recognized as a structurally novel class of compounds with remarkable anticonvulsant activity. In the present study, all the test semicarbazones were subjected to molecular docking using Glide v5.8. Some of the compounds were found to interact with ARG192, GLU270 and THR353 residues of 1OHV protein, present in GABA-AT receptor. The chemical structures of the synthesized molecules were characterized by elemental and spectral (IR, 1H NMR, 13C NMR and MS) analysis. The anticonvulsant activities of the compounds were investigated using maximal electroshock seizure (MES) and subcutaneous pentylenetrtrazole (scPTZ) models. The neurotoxicity was evaluated in mice by the rotorod test. The attempts were also made to establish structure-activity relationships among synthesized compounds. The results of the present study confirmed that the pharmacophore model with four binding sites is essential for antiepileptic activity.

Keywords: pyrimidine, semicarbazones, anticonvulsant activity, neurotoxicity

Procedia PDF Downloads 253

Authors: Agnieszka Krzemińska, Katarzyna Świderek, Vicente Molinier, Piotr Paneth

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In order to understand in details the interactions between ligands and the enzyme isotope effects were studied between clinically used drugs that bind in the active site of Human Immunodeficiency Virus Reverse Transcriptase, HIV-1 RT, as well as triazole-based inhibitor that binds in the allosteric pocket of this enzyme. The magnitudes and origins of the resulting binding isotope effects were analyzed. Subsequently, binding isotope effect of the same triazole-based inhibitor bound in the active site were analyzed and compared. Together, these results show differences in binding origins in two sites of the enzyme and allow to analyze binding mode and place of newly synthesized inhibitors. Typical protocol is described below on the example of triazole ligand in the allosteric pocket. Triazole was docked into allosteric cavity of HIV-1 RT with Glide using extra-precision mode as implemented in Schroedinger software. The structure of HIV-1 RT was obtained from Protein Data Bank as structure of PDB ID 2RKI. The pKa for titratable amino acids was calculated using PROPKA software, and in order to neutralize the system 15 Cl- were added using tLEaP package implemented in AMBERTools ver.1.5. Also N-terminals and C-terminals were build using tLEaP. The system was placed in 144x160x144Å3 orthorhombic box of water molecules using NAMD program. Missing parameters for triazole were obtained at the AM1 level using Antechamber software implemented in AMBERTools. The energy minimizations were carried out by means of a conjugate gradient algorithm using NAMD. Then system was heated from 0 to 300 K with temperature increment 0.001 K. Subsequently 2 ns Langevin−Verlet (NVT) MM MD simulation with AMBER force field implemented in NAMD was carried out. Periodic Boundary Conditions and cut-offs for the nonbonding interactions, range radius from 14.5 to 16 Å, are used. After 2 ns relaxation 200 ps of QM/MM MD at 300 K were simulated. The triazole was treated quantum mechanically at the AM1 level, protein was described using AMBER and water molecules were described using TIP3P, as implemented in fDynamo library. Molecules 20 Å apart from the triazole were kept frozen, with cut-offs established on range radius from 14.5 to 16 Å. In order to describe interactions between triazole and RT free energy of binding using Free Energy Perturbation method was done. The change in frequencies from ligand in solution to ligand bounded in enzyme was used to calculate binding isotope effects.

Keywords: binding isotope effects, molecular dynamics, HIV, reverse transcriptase

Procedia PDF Downloads 431

Authors: Bright Chukwunwike Uzuegbunam, Wojciech Paslawski, Hans Agren, Christer Halldin, Wolfgang Weber, Markus Luster, Thomas Arzberger, Behrooz Hooshyar Yousefi

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There is a need to develop a PET tracer that will enable to diagnosis and track the progression of Alpha-synucleinopathies (Parkinson’s disease [PD], dementia with Lewy bodies [DLB], multiple system atrophy [MSA]) in living subjects over time. Alpha-synuclein aggregates (a-syn), which are present in all the stages of disease progression, for instance, in PD, are a suitable target for in vivo PET imaging. For this reason, we have developed some promising a-syn tracers based on a disarylbisthiazole (DABTA) scaffold. The precursors are synthesized via a modified Hantzsch thiazole synthesis. The precursors were then radiolabeled via one- or two-step radiofluorination methods. The ligands were initially screened using a combination of molecular dynamics and quantum/molecular mechanics approaches in order to calculate the binding affinity to a-syn (in silico binding experiments). Experimental in vitro binding assays were also performed. The ligands were further screened in other experiments such as log D, in vitro plasma protein binding & plasma stability, biodistribution & brain metabolite analyses in healthy mice. Radiochemical yields were up to 30% - 72% in some cases. Molecular docking revealed possible binding sites in a-syn and also the free energy of binding to those sites (-28.9 - -66.9 kcal/mol), which correlated to the high binding affinity of the DABTAs to a-syn (Ki as low as 0.5 nM) and selectivity (> 100-fold) over Aβ and tau, which usually co-exist with a-synin some pathologies. The log D values range from 2.88 - 2.34, which correlated with free-protein fraction of 0.28% - 0.5%. Biodistribution experiments revealed that the tracers are taken up (5.6 %ID/g - 7.3 %ID/g) in the brain at 5 min (post-injection) p.i., and cleared out (values as low as 0.39 %ID/g were obtained at 120 min p.i. Analyses of the mice brain 20 min p.i. Revealed almost no radiometabolites in the brain in most cases. It can be concluded that in silico study presents a new venue for the rational development of radioligands with suitable features. The results obtained so far are promising and encourage us to further validate the DABTAs in autoradiography, immunohistochemistry, and in vivo imaging in non-human primates and humans.

Keywords: alpha-synuclein aggregates, alpha-synucleinopathies, PET imaging, tracer development

Procedia PDF Downloads 235

Authors: Usha Kiran, M. Z. Abdin

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Osmotin is an abundant cationic multifunctional protein discovered in cells of tobacco (Nicotiana tabacum L. var Wisconsin 38) adapted to an environment of low osmotic potential. It provides plants protection from pathogens, hence placed in the PRP family of proteins. The osmotin induced proline accumulation has been reported in plants including transgenic tomato and strawberry conferring tolerance against both biotic and abiotic stresses. The exact mechanism of induction of proline by osmotin is however, not known till date. These observations have led us to hypothesize that osmotin induced proline accumulation could be due to its involvement as transcription factor and/or cell signal pathway modulator in proline biosynthesis. The present investigation was therefore, undertaken to analyze the osmotin protein as transcription factor /cell signalling modulator using bioinformatics tools. The results of available online DNA binding motif search programs revealed that osmotin does not contain DNA-binding motifs. The alignment results of osmotin protein with the protein sequence from DATF showed the homology in the range of 0-20%, suggesting that it might not contain a DNA binding motif. Further to find unique DNA-binding domain, the superimposition of osmotin 3D structure on modeled Arabidopsis transcription factors using Chimera also suggested absence of the same. We, however, found evidence implicating osmotin in cell signaling. With these results, we concluded that osmotin is not a transcription factor but regulating proline biosynthesis and accumulation through cell signaling during abiotic stresses.

Keywords: osmotin, cell signaling modulator, bioinformatic tools, protein

Procedia PDF Downloads 272

Authors: Deepika Saini, Sandeep Jain, Ajay Kumar

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The quinoline scaffold is one of the most widely studied in the discovery of derivatives with various heterocyclic moieties due to its potential antimalarial activities. In the present study, a chalcone series of quinoline derivatives clubbed with pyrazole were synthesized to evaluate their antimalarial property by in vitro schizont maturation inhibition assay against both chloroquine sensitive, 3D7 and chloroquine resistant, RKL9 strain of Plasmodium falciparum. Further, top five compounds were studied for in vivo preclinical study for antimalarial potential against P. berghei in Swiss albino mice. To understand the mechanism of synthesized analogues, they were screened computationally by molecular docking techniques. Compounds were docked into the active site of a protein receptor, Plasmodium falciparum Cysteine Protease Falcipain-2. The compounds were successfully synthesized, and structural confirmation was performed by FTIR, 1H-NMR, mass spectrometry and elemental analysis. In vitro study suggested that the compounds 5b, 5g, 5l, 5s and 5u possessed best antimalarial activity and further tested for in vivo screening. Compound 5u (CH₃ on both rings) with EC₅₀ 0.313 & 0.801 µg/ml against CQ-S & CQ-R strains of P. falciparum respectively and 78.01% suppression of parasitemia. The molecular docking studies of the compounds helped in understanding the mechanism of action against falcipain-2. The present study reveals the binding signatures of the synthesized ligands within the active site of the protein, and it explains the results from in vitro study in their EC₅₀ values and percentage parasitemia.

Keywords: antimalarial activity, chalcone, docking, quinoline

Procedia PDF Downloads 409