Pranitha Jenardhanan

De novo deleterious genetic variations target a biological network centered on Aβ peptide in early-onset Alzheimer disease.

We hypothesized that de novo variants (DNV) might participate in the genetic determinism of sporadic early-onset Alzheimer disease (EOAD, onset before 65 years). We investigated 14 sporadic EOAD trios first by array-comparative genomic hybridization. Two patients carried a de novo copy number variation (CNV). We then performed whole-exome sequencing in the 12 remaining trios and identified 12 non-synonymous DNVs in six patients. The two de novo CNVs (an amyloid precursor protein (APP) duplication and a BACE2 intronic deletion) and 3/12 non-synonymous DNVs (in PSEN1, VPS35 and MARK4) targeted genes from a biological network centered on the Amyloid beta (Aβ) peptide. We showed that this a priori-defined genetic network was significantly enriched in amino acid-altering DNV, compared with the rest of the exome. The causality of the APP de novo duplication (which is the first reported one) was obvious. In addition, we provided evidence of the functional impact of the following three non-synonymous DNVs targeting this network: the novel PSEN1 variant resulted in exon 9 skipping in patient’s RNA, leading to a pathogenic missense at exons 8–10 junction; the VPS35 missense variant led to partial loss of retromer function, which may impact neuronal APP trafficking and Aβ secretion; and the MARK4 multiple nucleotide variant resulted into increased Tau phosphorylation, which may trigger enhanced Aβ-induced toxicity. Despite the difficulty to recruit Alzheimer disease (AD) trios owing to age structures of the pedigrees and the genetic heterogeneity of the disease, this strategy allowed us to highlight the role of de novo pathogenic events, the putative involvement of new genes in AD genetics and the key role of Aβ network alteration in AD.

A computational study of CYP3A4 mediated drug interaction profiles for anti-HIV drugs

AbstractMolecular docking is a reliable method with which to identify the binding conformations of substrates, inducers and inhibitors of cytochrome P450 (CYP) enzymes. We used the docking method to explore possible binding modes of an entry inhibitor (maraviroc) and non-nucleoside reverse transcriptase inhibitors (delavirdine, efavirenz and etravirine) to cytochrome P450 3A4 (CYP3A4). In addition, docking results were compared with the binding conformations of HIV protease drugs to infer the binding site residues and potential drug–drug interaction profiles for combination therapy in the treatment of AIDS. We observed that efavirenz and etravirine induce metabolism of co-administered drugs by binding to a unique position in the active site of CYP3A4. Dosage adjustment is required for delavirdine and maraviroc when combined with HIV protease drugs. The present results are in good agreement with experimental data from drug interaction profiles. The information provided in this paper will be helpful in furthering our understanding the functions of CYP3A4, and could aid in the design of new drugs that would be metabolized easily without having any drug–drug interaction profile. FigureThe docked poses of etravirine (ETR) inside the active site of cytochrome P450 3A4 (CYP3A4). The binding site residues are colored by atom type: gray carbon, red oxygen, blue nitrogen, cyan hydrogen, yellow sulfur. The sticks of the ligands and heme are colored in green.

Effect of environmental contaminants on spermatogenesis.

Indiscriminate use of synthetic chemical compounds and the unregulated presence of heavy metals threatens the integral reproducibility of mankind and other living organisms. The toxicity of these compounds far outweighs the usefulness of these compounds. Male reproductive health is linked to the process of spermatogenesis and there is a general consensus that males are more sensitive to these environmental contaminants and so significantly affected when compared to their female counterparts. The review discusses the various toxic contaminants polluting the environment and the effect of these compounds on spermatogenesis and its relevance on male infertility in humans. It provides a detailed report on the chemical nature of few selected reprotoxicants like estrogen analogues, phthalates, dioxins, heavy metals and their action mechanism on various cellular targets that play a role in spermatogenesis with special highlights at the genetic and molecular levels. Understanding the toxicity of these compounds serves a dual purpose; to develop counter measures to protect ourselves from cellular damage and to use these compounds as a model to better understand the intricate process of spermatogenesis. The review would also help researchers formulate stringent regulations and usage restrictions in the synthesis of new compounds.

Adjudin disrupts spermatogenesis by targeting drug transporters Lesson from the breast cancer resistance protein (BCRP)

For non-hormonal male contraceptives that exert their effects in the testis locally instead of via the hypothalamic-pituitary-testicular axis, such as adjudin that disrupts germ cell adhesion, a major hurdle in their development is to improve their bioavailability so that they can be efficiently delivered to the seminiferous epithelium by transporting across the blood-testis barrier (BTB). If this can be done, it would widen the gap between their efficacy and general toxicity. However, Sertoli cells that constitute the BTB, peritubular myoid cells in the tunica propria, germ cells at different stages of their development, as well as endothelial cells that constitute the microvessels in the interstitium are all equipped with multiple drug transporters, most notably efflux drug transporters, such as P-glycoprotein, multidrug resistance-related protein 1 (MRP1) and breast cancer resistance protein (BCRP) that can actively prevent drugs (e.g., adjudin) from entering the seminiferous epithelium to exert their effects. Recent studies have shown that BCRP is highly expressed by endothelial cells of the microvessels in the interstitium in the testis and also peritubular myoid cells in tunica propria even though it is absent from Sertoli cells at the site of the BTB. Furthermore, BCRP is also expressed spatiotemporally by Sertoli cells and step 19 spermatids in the rat testis and stage-specifically, limiting to stage VII‒VIII of the epithelial cycle, and restricted to the apical ectoplasmic specialization [apical ES, a testis-specific F-actin-rich adherens junction (AJ)]. Interestingly, adjudin was recently shown to be capable of downregulating BCRP expression at the apical ES. In this Opinion article, we critically discuss the latest findings on BCRP; in particular, we provide some findings utilizing molecular modeling to define the interacting domains of BCRP with adjudin. Based on this information, it is hoped that the next generation of adjudin analogs to be synthesized can improve their efficacy in downregulating BCRP and perhaps other drug efflux transporters in the testis to improve their efficacy to traverse the BTB by modifying their interacting domains.

Kinases as targets for chemical modulators: Structural aspects and their role in spermatogenesis.

Protein phosphorylation and de-phosphorylation events are crucial in deciding the fate of cells. They regulate cellular growth, differentiation and cell death, and kinases are the key players of these events. The members of ser/thr kinases and tyrosine kinases form the majority of protein kinase family, exerting their regulatory mechanism in almost all cells. In testis, they impact signal transduction events, regulate all stages of sperm development from mitosis through fertilization. Understanding the function of these kinases at the structural level and studying their interactions with inhibitors can help in understanding the machinery of spermatogenesis. In view of this, we have reviewed some of the prominent kinases that are known to play a role in spermatogenesis. A better understanding of the impacts of kinase inhibition on spermatogenesis should aid in the interpretation of lesions and hopefully further the development of more efficient and potent drug candidates.

Computational analysis of CYP3A4-mediated metabolism to investigate drug interactions between anti-TB and anti-HIV drugs in HIV/TB co-infection

The treatment of human immunodeficiency virus (HIV)-related tuberculosis (TB) includes combination therapy, in which antiretroviral drugs and anti-TB drugs are co-administered. The complexities associated with the treatment of dual infection by Mycobacterium tuberculosis and HIV include occurrence of drug–drug interactions, in addition to pill burden, overlapping drug toxicity, and immune reconstitution inflammatory syndrome. Drug–drug interactions can occur between these drugs toward cytochrome P450 3A4 (CYP3A4), a drug-metabolizing enzyme. A thorough understanding of these interactions can prevent occurrence of treatment failures and drug toxicity. Molecular docking studies were carried out for FDA-approved drugs, to predict binding mechanism of anti-TB drugs with CYP3A4 and to compare them with our previous studies on antiretroviral drugs, in order to infer possible occurrence of drug–drug interactions. The core regimen of anti-TB treatment viz., rifampin (RIF), isoniazid (INH), and pyrazinamide (PZA) showed similar binding mode (i.e., competitive binding) via utilizing the same binding residue, Arg212 in CYP3A4. This regimen also shared similar binding mode with antiretroviral protease inhibitors except tipranavir, nelfinavir, lopinavir, and atazanavir. Contraindicated drug interactions were not observed between non-nucleoside reverse transcriptase inhibitors, delavirdine, efavirenz, and etravirine; and anti-TB drugs, RIF, INH, and PZA. The contraindications occurring within anti-TB drugs can be negated with inhibitory effect of INH to induction effect of RIF toward CYP3A4. We evaluated the importance of Arg212 along with Ser119, Ala370, Arg372, and heme moiety for mediating oxidative metabolism of drugs. These drug interaction details were incorporated into our web server by creating a database called “CYP3A4 DDIDB” and it is open accessible from http://cyp.bicpu.edu.in.

Computational Methods Involved in Evaluating the Toxicity of the Reproductive Toxicants in Sertoli Cell

The Sertoli cell, the somatic component of seminiferous tubule, provides nutritional support and immunological protection and supports overall growth and division of germ cells. Cytoskeletons, junction proteins, and kinases in Sertoli cells are prime targets for reproductive toxicants and other environmental contaminants. Among the varied targets, the kinases that are crucial for regulating varied activities in spermatogenesis such as assembly/disassembly of blood-testis barrier and apical ES and those that are involved in conferring polarity are highly targeted. In an attempt to study the effect of toxicants on these kinases, the present chapter deals with computational methodology concerning their three-dimensional structure prediction, identification of inhibitors, and understanding of conformational changes induced by these inhibitors.

Interaction of Oligomeric Breast Cancer Resistant Protein (BCRP) with Adjudin: A Male Contraceptive with Anti-Cancer Activity

Breast cancer resistant protein (BCRP, ABCG2) is an ATP-binding cassette (ABC) transporter, which together with two other ABC efflux drug pumps, namely P-glycoprotein (P-gp, ABCB1) and multidrug resistance-related protein 1 (MRP1, ABCC1) is the most important multidrug resistance protein foun d in eukaryotic cells including cells in the testis. However, unlike P-gp and MRP1, which are components of the Sertoli cell blood-testis barrier (BTB), BCRP is not expressed at the BTB in rodents and human testes. Instead, BCRP is expressed by peritubular myoid cells and endothelial cells of the lymphatic vessel in the tunica propria, residing outside the BTB. As such, the testis is equipped with two levels of defense against xenobiotics or drugs, preventing these harmful substances from entering the adluminal compartment to perturb meiosis and post-meiotic spermatid development: one at the level of the BTB conferred by P-gp and MRP1 and one at the tunica propria conferred by BCRP. The presence of drug transporters at the tunica propria as well as at the Sertoli cell BTB thus poses significant obstacles in developing non-hormonal contraceptives if these drugs (e.g., adjudin) exert their effects in germ cells behind the BTB, such as in the adluminal (apical) compartment of the seminiferous epithelium. Herein, we summarize recent findings pertinent to adjudin, a non-hormonal male contraceptive, and molecular interactions of adjudin with BCRP so that this information can be helpful to devise delivery strategies to evade BCRP in the tunica propria to improve its bioavailability in the testis.

Environmental Impact on Gametogenesis and Embryogenesis: An Overview

Gametogenesis and embryogenesis are critical for normal propagation of life. Any adverse condition arising due to environmental impacts, such as lifestyle (smoking, alcoholism, and obesity etc.…) or exposure to environmental contaminants during these stages greatly alters the normal reproductive health. Recently, decline in the quality of reproductive health including sperm count in male has been attributed to industrialization and release of endocrine disruptors in environment. It is now widely accepted that there are a plethora of ways in which the environmental contaminants can potentially act on the endocrine system affecting gametogenesis and embryogenesis. Most of the critical periods of gametogenesis and embryogenesis depend upon the availability of various hormones. It is, therefore, quite predictable that the environmental contaminants possessing endocrine-disrupting properties either directly or indirectly target the reproductive potential by affecting various stages of development.

Nonreceptor Protein Kinases c-Src, c-Yes, and FAK Are Biomarkers for Male Contraceptive Research

A major obstacle in male contraceptive research and development is the lack of reliable and sensitive biomarkers to monitor the efficacy and potency of candidate compounds under investigation. Since the use of routine andrology techniques/analyses, such as sperm count, sperm motility, sperm morphology, sperm DNA integrity, sperm metabolism, and other semen characteristics (e.g., semen volume, pH, bacterial content) are tedious, representing the combined changes that take place in the testis and the male reproductive tract including the epididymis, rete testis, efferent ducts, prostate, and seminal vesicles. As such, the number of compounds that can be rapidly screened and tested is severely limited. Also, the outcomes are often difficult to interpret since it is not known if a compound under investigation exerts its effects mostly in the testis, the epididymis, another accessory sex organ or a combination of these organs. Herein, we summarize recent findings in the field regarding the use of nonreceptor protein kinases c-Src, c-Yes, and FAK as possible biomarkers for male contraceptive development based on our experience with adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly known as AF-2364). This information should pave the way of using these, and possibly other, markers for male contraceptive research.