Quantum biochemical analysis of the binding interactions between a potential inhibitory drug and the Ebola viral glycoprotein.

Ebola virus disease (EVD) causes outbreaks and epidemics in West Africa that persist until today. The envelope glycoprotein of Ebola virus (GP) consists of two subunits, GP1 and GP2, and plays a key role in anchoring or fusing the virus to the host cell in its active form on the virion surface. Toremifene (TOR) is a ligand that mainly acts as an estrogen receptor antagonist; however, a recent study showed a strong and efficient interaction with GP. In this context, we aimed to evaluate the energetic affinity features involved in the interaction between GP and toremifene by computer simulation techniques using the Molecular Fractionation Method with Conjugate Caps (MFCC) scheme and quantum-mechanical (QM) calculations, as well as missense mutations to assess protein stability. We identified ASP522, GLU100, TYR517, THR519, LEU186, LEU515 as the most attractive residues in the EBOV glycoprotein structure that form the binding pocket. We divided toremifene into three regions and evaluated that region i was more important than region iii and region ii for the formation of the TOR-GP1/GP2 complex, which might control the molecular remodeling process of TOR. The mutations that caused more destabilization were ARG134, LEU515, TYR517 and ARG559, while those that caused stabilization were GLU523 and ASP522. TYR517 is a critical residue for the binding of TOR, and is highly conserved among EBOV species. Our results may help to elucidate the mechanism of drug action on the GP protein of the Ebola virus and subsequently develop new pharmacological approaches against EVD.Communicated by Ramaswamy H. Sarma.

Identification of a new antifungal compound against isocitrate lyase of Paracoccidioides brasiliensis.

Aim: To perform virtual screening of compounds based on natural products targeting isocitrate lyase of Paracoccidioides brasiliensis. Materials & methods: Homology modeling and molecular dynamics simulations were applied in order to obtain conformational models for virtual screening. The selected hits were tested in vitro against enzymatic activity of ICL of the dimorphic fungus P. brasiliensis and growth of the Paracoccidioides spp. The cytotoxicity and selectivity index of the compounds were defined. Results & conclusion: Carboxamide, lactone and ß-carboline moieties were identified as interesting chemical groups for the design of new antifungal compounds. The compounds inhibited ICL of the dimorphic fungus P. brasiliensis activity. The compound 4559339 presented minimum inhibitory concentration of 7.3 µg/ml in P. brasiliensis with fungicidal effect at this concentration. Thus, a new potential antifungal against P. brasiliensis is proposed.

Mycobacterium abscessus subsp. massiliense mycma_0076 and mycma_0077 Genes Code for Ferritins That Are Modulated by Iron Concentration.

Mycobacterium abscessus complex has been characterized in the last decade as part of a cluster of mycobacteria that evolved from an opportunistic to true human pathogen; however, the factors responsible for pathogenicity are still undefined. It appears that the success of mycobacterial infection is intrinsically related with the capacity of the bacteria to regulate intracellular iron levels, mostly using iron storage proteins. This study evaluated two potential M. abscessus subsp. massiliense genes involved in iron storage. Unlike other opportunist or pathogenic mycobacteria studied, M. abscessus complex has two genes similar to ferritins from M. tuberculosis (Rv3841), and in M. abscessus subsp. massiliense, those genes are annotated as mycma_0076 and mycma_0077. Molecular dynamic analysis of the predicted expressed proteins showed that they have a ferroxidase center. The expressions of mycma_0076 and mycma_0077 genes were modulated by the iron levels in both in vitro cultures as well as infected macrophages. Structural studies using size-exclusion chromatography, circular dichroism spectroscopy and dynamic light scattering showed that r0076 protein has a structure similar to those observed in the ferritin family. The r0076 forms oligomers in solution most likely composed of 24 subunits. Functional studies with recombinant proteins, obtained from heterologous expression of mycma_0076 and mycma_0077 genes in Escherichia coli, showed that both proteins were capable of oxidizing Fe2+ into Fe3+, demonstrating that these proteins have a functional ferroxidase center. In conclusion, two ferritins proteins were shown, for the first time, to be involved in iron storage in M. abscessus subsp. massiliense and their expressions were modulated by the iron levels.

Molecular dynamics simulations of Zika virus NS3 helicase: Insights into RNA binding site activity.

America is still suffering with the outbreak of Zika virus (ZIKV) infection. Congenital ZIKV syndrome has already caused a public health emergency of international concern. However, there are still no vaccines to prevent or drugs to treat the infection caused by ZIKV. The ZIKV NS3 helicase (NS3h) protein is a promising target for drug discovery due to its essential role in viral genome replication. NS3h unwinds the viral RNA to enable the replication of the viral genome by the NS5 protein. NS3h contains two important binding sites: the NTPase binding site and the RNA binding site. Here, we used molecular dynamics (MD) simulations to study the molecular behavior of ZIKV NS3h in the presence and absence of ssRNA and the potential implications for NS3h activity and inhibition. Although there is conformational variability and poor electron densities of the RNA binding loop in various apo flaviviruses NS3h crystallographic structures, the MD trajectories of NS3h-ssRNA demonstrated that the RNA binding loop becomes more stable when NS3h is occupied by RNA. Our results suggest that the presence of RNA generates important interactions with the RNA binding loop, and these interactions stabilize the loop sufficiently that it remains in a closed conformation. This closed conformation likely keeps the ssRNA bound to the protein for a sufficient duration to enable the unwinding/replication activities of NS3h to occur. In addition, conformational changes of this RNA binding loop can change the nature and location of the optimal ligand binding site, according to ligand binding site prediction results. These are important findings to help guide the design and discovery of new inhibitors of NS3h as promising compounds to treat the ZIKV infection.

ß-Carboline alkaloids from Galianthe ramosa inhibit malate synthase from Paracoccidioides spp.

As part of our continuing chemical and biological analyses of Rubiaceae species from Cerrado, we isolated novel alkaloids 1 and 2, along with known compounds epicatechin, ursolic acid, and oleanolic acid, from Galianthe ramosa. Alkaloid 2 inhibited malate synthase from the pathogenic fungus Paracoccidioides spp. This enzyme is considered an important molecular target because it is not found in humans. Molecular docking simulations were used to describe the interactions between the alkaloids and malate synthase.