Structure based drug design and in vitro metabolism study: Discovery of N-(4-methylthiophenyl)-N,2-dimethyl-cyclopenta[d]pyrimidine as a potent microtubule targeting agent

We report a series of tubulin targeting agents, some of which demonstrate potent antiproliferative activities. These analogs were designed to optimize the antiproliferative activity of 1 by varying the heteroatom substituent at the 4′-position, the basicity of the 4-position amino moiety, and conformational restriction. The potential metabolites of the active compounds were also synthesized. Some compounds demonstrated single digit nanomolar IC₅₀ values for antiproliferative effects in MDA-MB-435 melanoma cells. Particularly, the S-methyl analog 3 was more potent than 1 in MDA-MB-435 cells (IC₅₀?=?4.6?nM). Incubation of 3 with human liver microsomes showed that the primary metabolite of the S-methyl moiety of 3 was the methyl sulfinyl group, as in analog 5. This metabolite was equipotent with the lead compound 1 in MDA-MB-435 cells (IC₅₀?=?7.9?nM). Molecular modeling and electrostatic surface area were determined to explain the activities of the analogs. Most of the potent compounds overcome multiple mechanisms of drug resistance and compound 3 emerged as the lead compound for further SAR and preclinical development.     

Increasing autophagy and blocking Nrf2 suppress laminopathy‐induced age‐dependent cardiac dysfunction and shortened lifespan

Mutations in the human LMNA gene cause a collection of diseases known as laminopathies. These include myocardial diseases that exhibit age‐dependent penetrance of dysrhythmias and heart failure. The LMNA gene encodes A‐type lamins, intermediate filaments that support nuclear structure and organize the genome. Mechanisms by which mutant lamins cause age‐dependent heart defects are not well understood. To address this issue, we modeled human disease‐causing mutations in the Drosophila melanogaster Lamin C gene and expressed mutant Lamin C exclusively in the heart. This resulted in progressive cardiac dysfunction, loss of adipose tissue homeostasis, and a shortened adult lifespan. Within cardiac cells, mutant Lamin C aggregated in the cytoplasm, the CncC(Nrf2)/Keap1 redox sensing pathway was activated, mitochondria exhibited abnormal morphology, and the autophagy cargo receptor Ref2(P)/p62 was upregulated. Genetic analyses demonstrated that simultaneous over‐expression of the autophagy kinase Atg1 gene and an RNAi against CncC eliminated the cytoplasmic protein aggregates, restored cardiac function, and lengthened lifespan. These data suggest that simultaneously increasing rates of autophagy and blocking the Nrf2/Keap1 pathway are a potential therapeutic strategy for cardiac laminopathies.     

Interactions of substrates at the surface of P450s can greatly enhance substrate potency

Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.     

Biosynthesis of Al2O3 and europium-doped Al2O3 nanoparticle using Cymbopogon citratus (lemongrass) extract and its antibacterial property

Cymbopogon citratus-mediated pure aluminium oxide (Al₂O₃) and europium (Eu)-doped Al₂O₃ with different amounts of metal ion were prepared using a green synthesis method. Synthesised nanoparticles were characterised by ultraviolet (UV)-visible spectroscopy, photoluminescence (PL), Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). Synthesis of nanoparticles is confirmed by using UV-visible spectroscopy showing maximum absorption at 411 and 345 nm for Al₂O₃ and Eu-doped Al₂O₃, respectively. The antibacterial activity of prepared nanoparticles was evaluated against Pseudomonas aeruginosa, Streptococcus aureus, Escherichia coli and Klebsiella pneumoniae using a well-diffusion technique. The effect of pure Al₂O₃ and Eu-doped nanoparticles shows excellent results against P. aeruginosa, S. aureus, E. coli and K. pneumoniae.     

Promoter polymorphism MMP-1 (-1607 2G/1G) and MMP-3 (-1612 5A/6A) in development of HAND and modulation of pathogenesis of HAND

The pathogenesis of HIV-associated neurocognitive disorder (HAND) is modulated by host genetic susceptibility factors such as Matrix metalloproteinases (MMPs). Promoter polymorphism of MMP-1 and MMP-3 may modify the expression of the gene. Hence, we evaluated the association of MMP-1-16072G/1G and MMP-3-1612 5A/6A polymorphisms with development of HAND and the modulation of pathogenesis of HAND. We enrolled a total of 180 individuals, 50 HIV-infected individuals with HAND, 130 without HAND, and 150 healthy controls. Polymorphism of MMP-1 and MMP-3 were genotyped by PCR-RFLP. MMP-1-1607 2G1G, -16071G/2G-1G/1G genotypes and -1607 1G allele were associated with the development of HAND (OR = 1.64, P = 0.05; OR = 1.45, P = 0.04; OR = 1.69, P = 0.05). MMP-1-16071G1G, MMP-3-16125A5A genotypes increased the risk for the development of HAND (OR = 1.78, P = 0.25; OR = 2.39, P = 0.13). MMP-3-1612 5A5A, -1612 6A/5A-5A/5A genotypes and -1612 5A allele were associated with the reduced risk of HAND (OR = 0.40, P = 0.05; OR = 0.53, P = 0.04; OR = 0.40, P = 0.01). Haplotype 5A1G increased the risk of development of HAND (OR = 1.93, P = 0.05). As observed in advanced HIV disease stage, MMP-1-1607 1G1G genotype enhance the risk for advancement of HIV disease (OR = 1.69, P = 0.89). MMP-3-1612 6A5A genotype showed higher risk for development of HAND in alcohol users (0R = 1.65, P = 0.44). MMP-1 genotype may have an influence on development of HAND whereas MMP3-1612 5A5A genotype may reduce risk for pathogenesis of HAND.     

Detection of symbionts and virus in the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis> (Hemiptera: Aleyrodidae), vector of the <Emphasis Type="Italic">Mungbean yellow mosaic India virus</Emphasis> in Central India

Legume crops in Central India, the main soybean production area of the country, may suffer from yellow mosaic disease caused by the Mungbean yellow mosaic India virus (MYMIV). MYMIV is transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), which is a species complex composed of various genetic groups. This vector species harbors different endosymbionts among regional strains and among individuals. To elucidate fundamental aspects of this virus vector in the state of Madhya Pradesh, the infection status of the symbionts and the virus in whiteflies was studied. A polymerase chain reaction (PCR) survey of the whiteflies collected in Madhya Pradesh found four secondary endosymbionts, Arsenophonus, Hemipteriphilus, Wolbachia, and Cardinium, in addition to the primary endosymbiont Portiera. Arsenophonus and Hemipteriphilus were highly infected but the infection rates of Wolbachia and Cardinium were low. MYMIV was detected in whitefly populations collected from various host plants in Madhya Pradesh. The whitefly populations belonged to the Asia I and II genetic groups; several different Asia II populations were also distributed. Specific relations were not observed among symbiont infection status, virus infection, and the whitefly genetic groups in the populations of Madhya Pradesh, though Cardinium was highly detected in the Asia II-1 group. New primers, which can be used for PCR template validation and for discriminating two phylogenetically close endosymbionts, were designed.