Computer-assisted Molecular Modeling Research Articles

Hapten design, monoclonal antibody preparation and immunoassay development for direct detection of the toxic metabolite of nitrovin without derivatization from fish

Nitrovin, classified as a nitrofuran drug, has been banned in numerous countries due to its carcinogenic effect on human health. However, existing analytical techniques for its detection are laborious, requiring an overnight derivatization process lasting 16 h. In this study, novel haptens were designed to prepare antibodies against aminogunidine (AGD), the metabolite of nitrovin, and then developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) method to detect AGD from fish samples without derivatization for the first time. Computer-assisted molecular modeling predicted the efficiency of novel haptens by conformational studies and electronic analysis. The results were consistent with the predicted results in producing high-affinity monoclonal antibody (mAb) against AGD. The half-inhibition concentration (IC50) value of icELISA was 0.80 ng mL−1 for AGD, with a limit of detection of 0.27 μg kg−1 and recoveries of 89.8%–97.2%. The results showed that this new hapten design and synthesis provided a great prospect to obtain high-affinity mAb for AGD. This developed icELISA substantially avoided the conventional and lengthy derivatization process and was an effective method for the direct detection of AGD in fish tissue without derivatization.

Generation of anti-idiotypic antibodies mimicking Cry2Aa toxin from an immunized mouse phage display library as potential insecticidal agents against Plutella xylostella

This study tried to generate anti-idiotypic antibodies (Ab2s) which mimic Cry2Aa toxin using a phage-display antibody library (2.8 × 107 CFU/mL). The latter was constructed from a mouse immunized with F (ab’)2 fragments digested from anti-Cry2Aa polyclonal antibodies. The F (ab’)2 fragments and Plutella xylostella (P. xylostella) brush border membrane vesicles (BBMV) were utilized as targets for selection. Eight mouse phage-display single-chain variable fragments (scFvs) were isolated and identified by enzyme-linked immunoassay (ELISA), PCR and DNA sequencing after four rounds of biopanning. Among them, M3 exhibited the highest binding affinity with F (ab’)2, while M4 bound the best with the toxin binding region of cadherin of P. xylostella (PxCad-TBR). Both of these two fragments were chosen for prokaryotic expression. The expressed M3 and M4 proteins with molecular weights of 30 kDa were purified. The M4 showed a binding affinity of 29.9 ± 2.4 nM with the PxCad-TBR and resulted in 27.8 ± 4.3 % larvae mortality against P. xylostella. Computer-assisted molecular modeling and docking analysis showed that mouse scFv M4 mimicked some Cry2Aa toxin binding sites when interacting with PxCad-TBR. Therefore, anti-idiotypic antibodies generated by BBMV-based screening could be useful for the development of new bio-insecticides as an alternative to Cry2Aa toxin for pest control.

Review on Molecular Modelling in Chemistry Education

Molecular models derived from results of quantum-chemical calculations present an important category of didactic instruments in chemistry education. These models can be used especially as tools for supporting the students’ understanding by visual learning, which can adequately address complexity of many chemical topics, incorporate appropriate didactic principles, as well as utilize the benefits brought up by the actual information technology. The proposed molecular models are non-trivial examples of didactic application of computational chemistry techniques in illustration of electron interactions in amidic group, namely the interaction of the free electron pair on the nitrogen atom with the carbonyl group and also the interaction of atoms in the amide group with other surrounding atoms in the molecule. By these molecular models it is possible to explain acid-base properties of amides applying knowledge of electron density distribution in the molecules and the resulting electrostatic potential. Presentation of the structure and properties of the amides within education is important also for the reason that amidic functions are involved in many important natural substances (e.g. proteins, peptides, nucleic acids or alkaloids), synthetic macromolecular substances (e.g. Silon) or pharmaceutical preparations (e.g. paracetamol). This paper investigated the effect of using different types of models while teaching organic chemistry on student understanding of new concepts and the spatial structure of new molecules, as well as preference of a particular model type and illustrates the possibilities of computer-assisted molecular modelling in supporting chemistry teaching and learning. Keywords: Molecular models, Acidity, Amides, Basicity, Electrostatic potential

De novo CLTC variants are associated with a variable phenotype from mild to severe intellectual disability, microcephaly, hypoplasia of the corpus callosum, and epilepsy

PurposeTo delineate the genotype–phenotype correlation in individuals with likely pathogenic variants in the CLTC gene. MethodsWe describe 13 individuals with de novo CLTC variants. Causality of variants was determined by using the tolerance landscape of CLTC and computer-assisted molecular modeling where applicable. Phenotypic abnormalities observed in the individuals identified with missense and in-frame variants were compared with those with nonsense or frameshift variants in CLTC. ResultsAll de novo variants were judged to be causal. Combining our data with that of 14 previously reported affected individuals (n = 27), all had intellectual disability (ID), ranging from mild to moderate/severe, with or without additional neurologic, behavioral, craniofacial, ophthalmologic, and gastrointestinal features. Microcephaly, hypoplasia of the corpus callosum, and epilepsy were more frequently observed in individuals with missense and in-frame variants than in those with nonsense and frameshift variants. However, this difference was not significant. ConclusionsThe wide phenotypic variability associated with likely pathogenic CLTC variants seems to be associated with allelic heterogeneity. The detailed clinical characterization of a larger cohort of individuals with pathogenic CLTC variants is warranted to support the hypothesis that missense and in-frame variants exert a dominant-negative effect, whereas the nonsense and frameshift variants would result in haploinsufficiency.

Computer-aided design, synthesis, and biological studies of anticological nitrogen-containing tetraphosphonic acids against melanoma

As a part of our quest to develop new bioactive bisphosphonic acids, we synthesized a series of bis(α-aminobisphosphonic acids) in good yields (66–78%) as new entities for treating malignant melanoma. The reaction of the Schiff bases, 1,4-pheneylenediimines with the Horner–Emmons–Wadsworth reagent, tetraethyl methylene-1,1-bisphosphonate in DMF/LiOH (aq) solution led exclusively to a meso form of nitrogen-containing tetraphosphonates (NTPs) (bis(α-aminobisphosphonates)). Next, hydrolysis of the ester moieties of the tetraphosphonate products yielded the corresponding tetraphosphonic acids, which treated with MeOH/KOH (aq, 20%) to give the respective NTP-tetrapotassium salts. Prior to synthesis, the suggested structures (and others) were applied to the computer-assisted molecular modeling, PASS program to investigate their prospective biological properties. Cytotoxic properties were later evaluated against five malignant melanoma cell lines that originated from different categories of malignant melanoma primary stage (I/II), histologically advanced stage (III/IV), and metastasized malignancy. Almost all tested compounds showed antitumor activity though on different levels. Three NTP salts were found to have activity in the range of GI50 0.650–5.73 μM vs. control reference GI50: 1.745–6.50 μM. Structure activity relationship is also discussed.

Monoclonal antibody-based homogeneous immunoassay for three banned agonists and molecular modeling insight

ABSTRACTA homogeneous fluorescence polarization immunoassay (FPIA) was developed for the detection of an agonist salbutamol (SAL). Based on salbutamol-succinate-fluoresceinthiocarbamyl hexylenediamine (SAL-SUC-HDF), a fluorescein tracer with 6-carbon bridge was found to be more sensitive, the optimal FPIA showed the working range from 31.7 to 264.1 ng/mL with an IC50 value of 91.4 ng/mL and a limit of detection 16.9 ng/mL. The cross-reactivity to brombuterol and clenbuterol was unexpectedly found to reach 447.3% and 255.8%, respectively. Computer-assisted molecular modeling revealed that the steric structure and electron density surfaces’ effect appeared to be s two important factors to the broad-specific recognition to other agonists.

Developing efficient protocols for synthesis, antiosteoarthritic, antiinflammatory assessments and docking studies of nitrogen-containing bisphosphonate derivatives

We report herein, two synthetic approaches to three types of nitrogen-containing bisphosphonates (N-BPs) in moderate to high yields (58–88%). Ester cleavage of selected bisphosphonates was undertaken to obtain their N-BP-acid analogs. Based on the reported bisphosphonate properties and the prospective biological prediction using computer-assisted molecular modeling (CAMM), new compounds were evaluated in a mouse model of antigen-induced arthritis (AIA) and the delayed-type hypersensitivity granuloma reaction (DTH-GRA) for chronic inflammation. Pharmacological results showed that N-BP-acids are more favorable for antiarthritis activity than N-BP-esters. On the other hand, the majority of N-BPs revealed good antiinflammatory potency compared to their acid analogs. The results also showed that the presence of a free thiol group in a molecule enhances the anti-inflammatory activity. Furthermore, bioscreening results were in good agreement with the prediction investigation. A hypothesis of molecular modeling study, including fitting of the synthesized compounds into 3D-pharmacophore using Discovery Studio 2.5 software and their docking into the human farnesyl pyrophosphate synthase (hFPPS, PDB code: 2F8C protein) showed good results consistent with the observed pharmacological properties.

Design, synthesis and pharmacological screening of β-amino-, thiadiazole/thiadiazine-phosphonate based triazole motifs as antimicrobial/cytotoxic agents.

Three different series of phosphonate derivatives, β-amino- and fused thiadiazolo/thiadiazine-phosphonates have been synthesized using the addition and/or addition-cyclization protocol of Horner-Wadsworth-Emmons (HWE) reagents to 1,2,4-triazole-3-thiols. The design of potentially antimicrobial and anticancer phosphor esters relied on the results of computer-assisted molecular modeling. All synthesized phosphonates were evaluated for their in vitro antimicrobial activities while anticancer properties were determined for eight out of twenty new phosphonates. The tested phosphonates, except for compounds that have a nitrile moiety, exhibited moderate to significant antimicrobial activity. Nevertheless, the most active compounds were fused thiadiazole-phosphonates, which inhibited the growth of both Gram-negative and Gram-positive bacteria better than β-aminophosphonates and fused thiadiazolophosphonates. In parallel, the antitumor activity screenings of selected phosphonates from each series and substrate 1 were also done. Their antitumor properties against ten carcinoma cell lines, including breast (MCF7, MDA-MB- 231/ ATCC, MDA-MB-435, BT-549), ovarian (IGROVI, OVCAR-3, SK-OV-3), prostate (PX-3, PU-145), and liver (HEPG2), were investigated. The results showed that all synthesized compounds reflected remarkable antitumor activity against breast (especially MDA-MB-231/ATCC and BT-549), and prostate carcinoma cell lines (PC-3 and DU-145), whereas a moderate to good effect on ovarian and liver cancer cells was observed.

Three decades of structure- and property-based molecular design.

Roche has pioneered structure- and property-based molecular design to drug discovery. While this is an ongoing development, the past three decades feature key events that have revolutionized the way drug discovery is conducted in Big Pharma industry. It has been a great privilege to have been involved in this transformation process, to have been able to collaborate with, direct, guide, or simply encourage outstanding experts in various disciplines to build and further develop what has become a major pillar of modern small-molecule drug discovery. This article is an account of major events that took place since the early decision of Roche to implement computer-assisted molecular modeling 32 years ago and is devoted to the key players involved. It highlights the internal build-up of structural biology, with protein X-ray structure determination at its core, and the early setup of bioinformatics. It describes the strategic shift to large compound libraries and high-throughput screening with the development of novel compound storage and ultra-high-throughput screening facilities, as well as the strategic return to focused screening of small motif-based compound libraries. These developments were accompanied by the rise of miniaturized parallel compound property analytics which resulted in a major paradigm shift in medicinal chemistry from linear to multi-dimensional lead optimization. The rapid growth of huge collections of property data stimulated the development of various novel data mining concepts with 'matched molecular pair' analysis and novel variants thereof playing crucial roles. As compound properties got more prominent in molecular design, exploration of specific structural motifs for property modulation became a research activity complementary to target-oriented medicinal chemistry. The exploration of oxetane is given as an example. For the sake of brevity, this account cannot detail all further developments that have taken place in each individual area of structure- and property-based drug discovery and it can only hint at important developments in other disciplines that have equally contributed to major paradigm shifts in Roche's small-molecule drug discovery efforts.

Role of surface residue 184 in the catalytic activity of NADH oxidase from Streptococcus pyogenes

Nicotinamide adenine dinucleotide (NADH) oxidase from Streptococcus pyogenes (SpNox) is a flavoprotein harboring one molecule of noncovalently bound flavin adenine dinucleotide. It catalyzes the oxidation of NADH by reducing molecular O2 to H2O directly through a four-electron reduction. In this study, we selected the lysine residues on the surface of SpNox and mutated them into arginine residues to study the effect on the enzyme activity. A single-point mutation (K184R) at the surface of SpNox enhanced NADH oxidase activity by approximately 50% and improved thermostability with 46.6% longer half life at 30°C. Further insights into the function of residue K184 were obtained by substituting it with other nonpolar, polar, positively charged, and negatively charged residues. To elucidate the role of this residue, computer-assisted molecular modeling and substrate docking were performed. The results demonstrate that even a single mutation at the surface of the enzyme induces changes in the interaction at the active site and affects the activity and stability. Additionally, the data also suggest that the K184R mutant can be used as an effective biocatalyst for NAD(+) regeneration in L-rare sugar production.

Folded Conformation, Cyclic Pentamer, Nanostructure, and PAD4 Binding Mode of YW3-56

The physical and chemical mechanisms of small molecules with pharmacological activity forming nano-structures are developing into a new field of nano-medicine. By using ROESY 2D NMR spectroscopy, trandem mass spectroscopy, transmission electron microscopy and computer-assisted molecular modeling, this paper demonstrated the contribution of the folded conformation, the intra- and intermolecular π-π stacking, the intra- and intermolecular hydrogen bonds, and the receptor binding free energy of 6-dimethylaminonaph-2-yl-{N-S-[1-benzylcarba-moyl-4-(2-chloroacetamidobutyl)]-carboxamide (YW3-56) to the rapid formation of nano-rings and the slow formation of nano-capsules. Thus we have developed a strategy that makes it possible to elucidate the physical and chemical mechanisms of bioactive small molecules forming nano-structures.

Structure-activity relationships of peptides incorporating a bioactive reverse-turn heterocycle at the melanocortin receptors: identification of a 5800-fold mouse melanocortin-3 receptor (mMC3R) selective antagonist/partial agonist versus the mouse melanocortin-4 receptor (mMC4R).

The melanocortin-3 (MC3) and melanocortin-4 (MC4) receptors regulate energy homeostasis, food intake, and associated physiological conditions. The melanocortin-4 receptor (MC4R) has been studied extensively. Less is known about specific physiological roles of the melanocortin-3 receptor (MC3R). A major obstacle to this lack of knowledge is attributed to a limited number of identified MC3R selective ligands. We previously reported a spatial scanning approach of a 10-membered thioether-heterocycle ring incorporated into a chimeric peptide template that identified a lead nM MC4R ligand. Upon the basis of those results, 17 compounds were designed and synthesized that focused upon modification in the pharmacophore domain. Notable results include the identification of a 0.13 nM potent 5800-fold mMC3R selective antagonist/slight partial agonist versus a 760 nM mMC4R full agonist (ligand 11). Biophysical experiments (two-dimensional (1)H NMR and computer-assisted molecular modeling) of this ligand resulted in the identification of an inverse γ-turn secondary structure in the ligand pharmacophore domain.

Plasmon enhanced fluoro-immunoassay using egg yolk antibodies for ultra-sensitive detection of herbicide diuron

Plasmon enhanced fluorescence immunoassay (PEFI) format has been reported in developing a sensitive heterogeneous fluoroimmunoassay for monitoring the phenylurea herbicide diuron. Computer-assisted molecular modeling was carried out to study the conformational and electrostatic effects of synthesized hapten for producing highly specific egg yolk antibody against a phenyl urea herbicide diuron. The generated antibodies were labeled with fluorescein isothiocyanate at different molar ratios and used as tracer in the developed fluorescence based immunoassay. The sensitivity of the assay format was enhanced by using silver nanoparticles tagged with bovine serum albumin as a new blocking reagent in the developed PEFI format. Enhancer treatment on the developed immunoassay showed a significant improvement of fluorescence signal intensity with approximately 10 fold increase in assay sensitivity. The immunoassay has a detection limit of 0.01 ng mL(-1) with good signal precision (~2%) in the optimum working concentration range between 1 pg mL(-1) to 10 μg mL(-1) of diuron. These findings facilitate high throughput fluorescence-based processes that could be useful in biology, drug discovery and compound screening applications.

Synthesis, Quantitative Structure–Activity Relationship, and Anti-Inflammatory Profiles of Substituted 5- and 6-N-Heterocycle Bisphosphonate Esters

A modified multicomponent reaction in a one-pot synthesis of a new series of substituted N-heterocyclic aminomethylene bisphosphonates, in high rates, was described. Accordingly, synthesis of α-aminobisphosphonates was achieved, via the Kabachnik–Fields reaction, from the substituted amine with triethyl orthoformate, and followed by treating the product mixture with a toluene solution containing diethyl phosphite–sodium salt. The design of potentially anti-inflammatory bisphosphonates (BPs) relied on the results of computer-assisted molecular modeling. If Later, new BPs were evaluated in animal model of chronic inflammation using the delayed-type hypersensitivity granuloma reaction.

Multi-Analyte Immunoassay for Pesticides: A Review

The current trend of pesticide immunoassay is developing multi-analyte immunoassays, that is, more than one target can be detected per test. In this mini-review, the strategies to achieve multi-analyte immunoassays, which include multi-antibody strategy, broad-specificity antibody strategy, and others, are briefly introduced. In addition, the recent developments of multi-analyte immunoassays for pesticides were summarized. At last, we give a future outlook of this area, which includes rational design generic hapten with the assist of computer-assisted molecular modeling (CAMM), further improving the properties of broad-specificity antibody by recombinant antibody (rAb) technology and developing a noncompetitive immunoassay format.

The Human Lactoferrin-Derived Peptide hLF1-11 Exerts Immunomodulatory Effects by Specific Inhibition of Myeloperoxidase Activity

Because of their ability to eliminate pathogens and to modulate various host immune responses, antimicrobial peptides are considered as candidate agents to fight infections by (antibiotic-resistant) pathogens. We recently reported that hLF1-11 (GRRRRSVQWCA), an antimicrobial peptide derived from the N terminus of human lactoferrin, displays diverse modulatory activities on monocytes, thereby enhancing their actions in innate immune responses. The aim of this study was to identify the cellular target of hLF1-11 that mediates these effects. Results revealed that hLF1-11 binds and subsequently penetrates human monocytes, after which it inhibits the enzymatic activities of myeloperoxidase (MPO). Moreover, a chemical inhibitor of MPO (aminobenzoic acid hydrazide) mimicked the effects of hLF1-11 on the inflammatory response by monocytes and on monocyte-macrophage differentiation. Computer-assisted molecular modeling predicted that hLF1-11 can bind to the edge of and within the crevice of the active site of MPO. Experiments with a set of hLF1-11 peptides with amino acid substitutions identified the stretch of arginines and the cysteine at position 10 as pivotal in these immunomodulatory properties of hLF1-11. We conclude that hLF1-11 may exert its modulatory effects on human monocytes by specific inhibition of MPO activity.

Crystallographic study and molecular modeling on the oxidation product of N-[(8R)-2-methoxy-5,6,7,8,9,10-hexahydro-6,9-methanocyclohepta[b]indol-8-yl]acetamide

Crystallization of N-[(8R)-2-methoxy-5,6,7,8,9,10-hexahydro-6,9-methanocyclohepta[b]indol-8-yl]acetamide was accompanied by oxidation at the C5a–C10a bond with formation of N-[(5S)-10-methoxy-2,8-dioxo-1,2,3,4,5,6,7,8-octahydro-3,6-methano-1-benzazecin-5-yl]acetamide whose structure was determined by X-ray analysis. Docking of this compound into melatonin-binding pocket of MT1A receptor was simulated by computer-assisted molecular modeling.

Activation of the Isotypes of Protein Kinase C-α, β1, β2, γ, Δ, ε and ζ by Mezerein, a Second-stage Tumour-promoting Agent: Correlation with Computer-assisted Molecular Modelling

The second-stage tumour promoter mezerein was compared with the full promoter, tetra-decanoyl phorbol acetate (TPA) and the non-promoter, resiniferatoxin for its ability to activate the isotypes of protein kinase C of the c, n and a groups. Mezerein was a selectively weaker activator of the n-PKC group of isotypes, Δ and e, than TPA whilst resiniferatoxin failed to activate PKC up to maximum concentration of 1000 nM. None of the diterpenes used were found to activate the PKC isotype ζ. These results were correlated with computer generated minimum free energy values (CAMM) and Log D, a measure of their hydrophobicity. There was little difference in minimum free energy values of TPA and mezerein; however mezerein had only about 50% of the hydrophobicity of TPA.

Incorporation of a Bioactive Reverse-Turn Heterocycle into a Peptide Template Using Solid-Phase Synthesis To Probe Melanocortin Receptor Selectivity and Ligand Conformations by 2D 1H NMR

By use of a solid-phase synthetic approach, a bioactive reverse turn heterocycle was incorporated into a cyclic peptide template to probe melanocortin receptor potency and ligand structural conformations. The five melanocortin receptor isoforms (MC1R-MC5R) are G-protein-coupled receptors (GPCRs) that are regulated by endogenous agonists and antagonists. This pathway is involved in pigmentation, weight, and energy homeostasis. Herein, we report novel analogues of the chimeric AGRP-melanocortin peptide template integrated with a small molecule moiety to probe the structural and functional consequences of the core His-Phe-Arg-Trp peptide domain using a reverse-turn heterocycle. A series of six compounds are reported that result in inactive to full agonists with nanomolar potency. Biophysical structural analysis [2D (1)H NMR and computer-assisted molecular modeling (CAMM)] were performed on selected analogues, resulting in the identification that these peptide-small molecule hybrids possessed increased flexibility and fewer discrete conformational families compared to the reference peptide and result in a novel template for further structure-function studies.

A humanized anti-osteopontin antibody protects from Concanavalin A induced-liver injury in mice

Osteopontin has been implicated in various inflammatory diseases including rheumatoid arthritis, multiple sclerosis, Crohn's disease, and fulminant hepatitis. Increased expression of osteopontin has been detected in pathological foci of these diseases. RA and fulminant hepatitis have been successfully treated by administration of neutralizing anti-osteopontin antibody in mice. However, rodent antibodies are highly immunogenic in humans and therefore limited in their clinical application. Here, a murine monoclonal antibody 23C3 against human osteopontin, was humanized by complementarity-determining region grafting method based on computer-assisted molecular modeling. The humanized version of 23C3, denoted as Hu23C3, was shown to possess affinity comparable to that of its parental antibody. Hu23C3 could also inhibit monocyte migration in response to osteopontin in vitro. Furthermore, in vivo data showed that Hu23C3 significantly protects mice from Concanavalin A (Con A) induced-liver injury in association with the reduction of transaminase activities and improvement of liver injury. Mechanistic studies demonstrated that Hu23C3 inhibited T and NKT cell infiltration, and activation of nuclear factor κB (NF-κB) in the liver, resulting in reduction of TNF-α and IFN-γ production. Thus, our data strongly support that the humanized anti-osteopontin antibody, Hu23C3, may have a potential for the treatment of T cell mediated-hepatitis in human.