Advances in the Development of Shape Similarity Methods and Their Application in Drug Discovery

Molecular similarity is a key concept in drug discovery. It is based on the assumption that structurally similar molecules frequently have similar properties. Assessment of similarity between small molecules has been highly effective in the discovery and development of various drugs. Especially, two-dimensional (2D) similarity approaches have been quite popular due to their simplicity, accuracy and efficiency. Recently, the focus has been shifted toward the development of methods involving the representation and comparison of three-dimensional (3D) conformation of small molecules. Among the 3D similarity methods, evaluation of shape similarity is now gaining attention for its application not only in virtual screening but also in molecular target prediction, drug repurposing and scaffold hopping. A wide range of methods have been developed to describe molecular shape and to determine the shape similarity between small molecules. The most widely used methods include atom distance-based methods, surface-based approaches such as spherical harmonics and 3D Zernike descriptors, atom-centered Gaussian overlay based representations. Several of these methods demonstrated excellent virtual screening performance not only retrospectively but also prospectively. In addition to methods assessing the similarity between small molecules, shape similarity approaches have been developed to compare shapes of protein structures and binding pockets. Additionally, shape comparisons between atomic models and 3D density maps allowed the fitting of atomic models into cryo-electron microscopy maps. This review aims to summarize the methodological advances in shape similarity assessment highlighting advantages, disadvantages and their application in drug discovery

Identification and characterization of antibacterial compound(s) of cockroaches (Periplaneta americana)

Infectious diseases remain a significant threat to human health, contributing to more than 17 million deaths, annually. With the worsening trends of drug resistance, there is a need for newer and more powerful antimicrobial agents. We hypothesized that animals living in polluted environments are potential source of antimicrobials. Under polluted milieus, organisms such as cockroaches encounter different types of microbes, including superbugs. Such creatures survive the onslaught of superbugs and are able to ward off disease by producing antimicrobial substances. Here, we characterized antibacterial properties in extracts of various body organs of cockroaches (Periplaneta americana) and showed potent antibacterial activity in crude brain extract against methicillin-resistant Staphylococcus aureus and neuropathogenic E. coli K1. The size-exclusion spin columns revealed that the active compound(s) are less than 10 kDa in molecular mass. Using cytotoxicity assays, it was observed that pre-treatment of bacteria with lysates inhibited bacteria-mediated host cell cytotoxicity. Using spectra obtained with LC-MS on Agilent 1290 infinity liquid chromatograph, coupled with an Agilent 6460 triple quadruple mass spectrometer, tissues lysates were analyzed. Among hundreds of compounds, only a few homologous compounds were identified that contained isoquinoline group, chromene derivatives, thiazine groups, imidazoles, pyrrole containing analogs, sulfonamides, furanones, flavanones, and known to possess broad-spectrum antimicrobial properties, and possess anti-inflammatory, anti-tumour, and analgesic properties. Further identification, characterization and functional studies using individual compounds can act as a breakthrough in developing novel therapeutics against various pathogens including superbugs

AKTIVITAS ANTIFUNGI EKSTRAK METANOL DAUN SALAM (Syzygiumpolyanthum [Wight] Walp.)TERHADAP PERTUMBUHANHortaea werneckii(T1)SECARA IN VITRO

Hortaea werneckii is a fungus causes of tinea nigra. Plants that are known to have antifungal compounds are bay leaves (Syzygium polyanthum). This study was aimed to determine the best concentration of the methanol extract from bay leaves (S. polyanthum) can inhibit the growth of Hortaea werneckii (T1) in vitro. This study used a completely randomized design (CRD) with treatment levels that consisted of concentrations of 20, 25, 30, 35 and 40%, negative control (sterile distilled water) and positive control (ketoconazole 2%). The antifungal activity test was carried out using the Kirby-Bauer method. Based on the results study showed that concentration 30% is the best concentration in inhibit fungal growth Hortaea werneckii (T1)

A flavoprotein supports cell wall properties in the necrotrophic fungus Alternaria brassicicola

Background Flavin-dependent monooxygenases are involved in key biological processes as they catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. Flavoprotein monooxygenases are frequently encountered in micro-organisms, most of which require further functional and biocatalytic assessment. Here we investigated the function of the AbMak1 gene, which encodes a group A flavin monooxygenase in the plant pathogenic fungus Alternaria brassicicola, by generating a deficient mutant and examining its phenotype. Results Functional analysis indicates that the AbMak1 protein is involved in cell wall biogenesis and influences the melanization process. We documented a significant decrease in melanin content in the Δabmak1 strain compared to the wild-type and complemented strains. We investigated the cell wall morphology and physical properties in the wild-type and transformants using electron and atomic force microscopy. These approaches confirmed the aberrant morphology of the conidial wall structure in the Δabmak1 strain which had an impact on hydrophilic adhesion and conidial surface stiffness. However, there was no significant impairment in growth, conidia formation, pathogenicity or susceptibility to various environmental stresses in the Δabmak1 strain. Conclusion This study sheds new light on the function of a fungal flavin-dependent monooxygenase, which plays an important role in melanization

Pós-genoma de fungos patogênicos humanos : identificação de novas drogas contra os alvos moleculares TRR1 e KRE2 de Paracoccidioides lutzii

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2012.A incidência e a gravidade das micoses sistêmicas têm crescido em níveis alarmantes em todo mundo. O principal fator que tem contribuído para esta casuística é o aumento de pacientes imunocomprometidos graves ou doentes de AIDS, submetidos à quimioterapia, ou terapias imunosupressoras para transplante de órgãos ou de células hematopoiéticas. Estes pacientes, além de desenvolverem formas clínicas mais severas das micoses, geralmente fatais, estão mais susceptíveis a infecções por fungos oportunistas. Em adição, a resistência às drogas atualmente disponíveis e os efeitos colaterais causados por estas terapias, apontam para a relevante necessidade de desenvolvimento de novas drogas antifúngicas. Por genômica comparativa foi avaliada a presença de 57 genes-alvos (55 essenciais e 2 relevantes para a sobrevivência do patógeno no hospedeiro) no genoma de nove fungos patogênicos: Paracoccidioides brasiliensis (isolados Pb18 e Pb3), P. lutzii (isolado Pb01), Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans e Histoplasma capsulatum. Esta análise possibilitou a seleção de 10 genes, que apresentavam características que os descreviam como potenciais alvos para drogas. Apenas dois alvos, tioredoxina redutase (TRR1) e α-1,2- manosiltransferase (KRE2), possuem estruturas tridimensionais (3D) homólogas e depositadas no banco de dados de proteínas (PDB), com identidade de sequência primária superior a 50%. Por essa razão eles foram selecionados para realização de modelagem molecular e varredura virtual. Os modelos TRR1 e KRE2 de P. lutzii (isolate 01) e as moléculas das quimiotecas Chimiotèque Nationale e Life Chemicals foram preparados para varredura virtual. O resultado do docking possibilitou a seleção de 20 moléculas contra o alvo molecular TRR1 e 34 moléculas contra KRE2, totalizando 54 moléculas potenciais, e destas 22 foram testadas quanto a suas potenciais atividades antifúngicas contra fungos patogênicos. As melhores moléculas foram F0608-0758 (contra KRE2) e F1806-0122 (contra TRR1), nas concentrações de 2PM (1Pg/mL) e 19PM (8Pg/mL), respectivamente, para o MIC50 (capacidade de inibir 50% do crescimento de fungos do gênero Paracoccidioides spp). Em paralelo, foi realizada a expressão heteróloga em Escherichia coli da enzima TRR1 de P. lutzii (Pb01), o que permitiu iniciar os experimentos de caracterização enzimática e inibição desta flavoenzima, utilizando a tioredoxina de Saccharomyces cerevisiae (Trx3) como substrato. Os valores dos parâmetros cinéticos determinados foram Km = 4,12 PM e Vmáx = 0,008 PM/s. Ensaios iniciais de inibição da TRR1, utilizando as moléculas selecionadas por varredura virtual, sugerem uma inibição competitiva sobre o alvomolecular TRR1, o que indica que a atividade antifúngica observada nos ensaios in culture contra os fungos patogênicos humanos era devido à inibição provocada especificamente sobre o sítio catlítico do alvo-molecular. Considerando uma concentração de 0,5 PM do substrato Trx3, a porcentagem de inibição da TRR1 recombinante pela droga F0876-0030 a 65 nM foi de 35%. Para a droga F1806-0122 (155 nM inibiu cerca de 30%) foi possível calcular o valor de IC50, em cerca de 310 nM de droga. Desta forma, novas moléculas com atividade antifúngicas foram identificadas e selecionadas apontando para um novo horizonte no desenvolvimento de novas drogas para o tratamento das infecções fúngicas invasivas.The incidence and severity of mycoses have grown to alarming levels in the world. The main factor that has contributed for this is the increase of severe immunocompromised patients or patients with AIDS, undergoing chemotherapy, or therapies for organ or hematopoietic cells transplantation. These patients also develop more severe clinical mycoses forms, particularly fatal and are more susceptible to infections by opportunistic fungi. In addition, the drugs resistance and side effects caused by the available therapies demonstrated the need of new antifungal drugs development. By comparative genomics it was evaluated the presence of 57 target genes (55 essentials and 2 important for cell viability within host pathogen) in genome of nine pathogenic fungi: Paracoccidioides brasiliensis (isolates Pb18 and Pb3), P. lutzii (isolate 01), Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans and Histoplasma capsulatum. This analysis allowed the selection of 10 genes that present characteristics that describe them as potential targets for drugs. Only two targets, thioredoxin reductase (TRR1) and α-1,2-mannosyltransferase (KRE2) present homology three-dimensionall (3D) structures in protein data bank (PDB), with primary sequence identity greater than 50%. By this reason they were selected to perform molecular modeling and virtual screening. The models TRR1 and KRE2 of P.lutzii (isolate 01) and the molecules of Chimiotèque Nationale and Life Chemicals database were prepared to virtual screening. The docking results allowed the selection of 20 molecules against TRR1 molecular target and 34 molecules against TRR1, in total of 54 potential molecules, which 22 were tested for their potential antifungal activity against pathogenic fungi. The best molecules were F0608-0758 (against KRE2) e F1806-0122 (against TRR1), in concentrations of 2PM (1Pg/mL) and 19PM (8Pg/mL), respectively, for MIC50 (ability to inhibit 50% growth of Paracoccidioides spp.). In parallel, it was performed the heterologous expression in Escherichia coli of TRR1 enzyme of P. lutzii (Pb01), allowing start the experiments of enzymatic characterization and flavoprotein inhibition, using Saccharomyces cerevisiae thioredoxin (Trx3) as substrate. The kinetic parameters values determined were Km=4,12PM and Vmáx=0,008 PM/s. Preliminary inhibition assays of TRR1, using the molecules selected by virtual screening, suggest an competitive inhibition of TRR1 molecular target and indicate that the antifungal activity in culture assays against the human pathogenic fungi was due specific inhibition of molecular target active site. With Trx3 substrate at 0.5 PM, the inhibition percentage of recombinant TRR1 by F0876-0030 drug at 65 nM was 35%. For F1806-0122 drug (155 nM inhibited about 30%) it was possible calculate IC50 value around 310 nM of drug. Thus, new molecules with antifungal activity were identified and selected, pointing to a new horizon in the new drugs development for the treatment of invasive fungal infections

Functional characterization of AtGLP5 and AtSUC7, and their role in plant defense and development trade-offs in Arabidopsis thaliana

This study utilized the Arabidopsis thaliana-Sclerotinia sclerotiorum pathosystem to discover valuable rapeseed stem rot disease resistance engineering traits with activation-tagging, transgenic overexpressing, and T-DNA insertion knock-out mutants. By comparing their different disease resistance phenotypes and analyzing the expression of several pathway associated genes, we have got a relatively detailed knowledge about the molecular mechanism for the anti-pathogen function of the two proteins, AtGLP5 and AtSUC7. AtGLP5 is also further demonstrated to possess SOD activity and has a pivotal role in FLS2-associated flg22 signaling particularly. AtSUC7, previously regarded as a pseudogene encoding non-functional sucrose transporter, is proved to be involved in disease resistance and flowering time control

Synthetic progress toward Parvistemonine, Spiroxins A and B, and Generation of Palmarumycin Analogues

Natural products can both challenge synthetic chemists and guide biologists. In this work, they prompted the extension of oxidative methodology to new systems, inspired the systematic modification of the palmarumycin scaffold to produce potent thioredoxin/thioredoxin reductase (Trx/TrxR) inhibitors, and demanded creative synthetic solutions to the structural challenges Mother Nature provided. The first pursuit was the total synthesis of parvistemonine, a pentacyclic azacycle isolated from Stemona plants. These plants have been used in Chinese folk medicine, and the isolated Stemona alkaloids possess therapeutic uses that range from antitussive to antiparasitic activity. The oxidative cyclization of tyrosine was incorporated into the syntheses of several natural products, and the extension of this methodology to homotyrosine for construction of the azacyclic core was investigated. Ultimately, the desired cyclization was not optimized to give synthetically viable yields, but the competing pathways and preferred reactivity was elucidated. In a separate project, a library of palmarumycin based prodrugs was synthesized. The bisnaphthospiroketal functionality, which is present in palmarumycin, is a lucrative scaffold that potently inhibits thioredoxin/thioredoxin reductase (Trx/TrxR). Some of the analogues suffered from low solubility, so various amino ester and sugar containing prodrugs were investigated. A prodrug library with improved solubility and greater plasma stability was successfully generated. One gram of the lead prodrug was needed for further biological testing, which would have been challenging with the first generation synthesis. An alternative synthesis was developed that afforded 1 g of the prodrug and decreased the total number of steps by half while significantly improving the overall yield. Finally, the total synthesis of spiroxins A and B was pursued. The spiroxins were isolated from an unidentified marine fungus and only spiroxin A was tested for biological activity. These highly oxygenated octacyclic compounds only differ in their degree of chlorination. A synthetic route was proposed that allowed access to both spiroxin A and B, and only diverged in the final chlorination step. Through a series of oxidations and reductions, this challenging core was accessed