Use of Molecular Modeling to Study Spermatogenesis: An Overview Using Proteins in Sertoli Cells.

Computational structure prediction and analysis helps in understanding the structure and function of varied proteins, which otherwise becomes implausible to understand by experimental procedures. Computational techniques prove to be instrumental in understanding the molecular mechanisms that underlies physiological processes and thereby also assist in identification of potent inhibitors. Spermatogenesis, being an important cellular process that decides the fate of the progeny, holds numerous molecular interaction data, which when identified and visualized with computational structural insights, might yield a cohesive and clear-cut perception to the functionality of several proteins involved. The present chapter deals with a few selected applications of computational structure prediction towards understanding the structure of proteins and highlights how these insights are useful in providing a better understanding of different processes in spermatogenesis.

Targeting Kinase Interaction Networks: A New Paradigm in PPI Based Design of Kinase Inhibitors.

BACKGROUND: Kinases are key modulators in regulating diverse range of cellular activities and are an essential part of the protein-protein interactome. Understanding the interaction of kinases with different substrates and other proteins is vital to decode the cell signaling machinery as well as causative mechanism for disease onset and progression. OBJECTIVE: The objective of this review is to present all studies on the structure and function of few important kinases and highlight the protein-protein interaction (PPI) mechanism of kinases and the kinase specific interactome databases and how such studies could be utilized to develop anticancer drugs. METHODS: The article is a review of the detailed description of the various domains in kinases that are involved in protein-protein interactions and specific inhibitors developed targeting these PPI domains. RESULTS: The review has surfaced in depth the interacting domains in key kinases and their features and the roles of PPI in the human kinome and the various signaling cascades that are involved in certain types of cancer. CONCLUSION: The insight availed into the mechanism of existing peptide inhibitors and peptidomimetics against kinases will pave way for the design and generation of domain specific peptide inhibitors with better productivity and efficiency and the various software and servers available can be of great use for the identification and analysis of protein-protein interactions.

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.

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.

The structural analysis of MARK4 and the exploration of specific inhibitors for the MARK family: a computational approach to obstruct the role of MARK4 in prostate cancer progression.

Prostate cancer, the second most common form of cancer in adult males is generally treated using hormone therapy but the emergence of hormone refractory prostate cancer poses serious challenges to the existing therapeutic strategies. However, protein kinases are now currently identified as potent targets for treating cancer, and MARK4L, a Ser/Thr kinase in the Par-1 family, is one such kinase that is expressed primarily in the testis and is involved in the regulation of spermatid polarity during spermatogenesis. It is also associated with Wnt-induced prostate carcinogenesis, making it a promising target for the development of anti-cancer therapeutics as part of alternative therapies to counter prostate cancer. In the present work, we predicted the three dimensional structure for the kinase domain of MARK4 and analyzed its structural properties. The results illuminate the presence of the unusual DFG Asp-in/αC helix-out conformation along with the absence of an additional hydrophobic pocket adjacent to the ATP binding site in its inactive state. These structural features accentuate the need for new specific therapeutics against MARK4. Hence, a robust ligand-based pharmacophore model AARRR.9 was developed based on the three dimensional chemical features of 9-oxo-9H-acridin-10-yl derivatives which possess a high specificity towards MARK kinases. A pharmacophore based search identified six potent compounds with a better specificity and binding efficiency to MARK4 bearing stable interactions with key residues K88, A138, D199 and E106, thereby making them tough ATP competitors. The closure of the catalytic cleft observed in the ligand bound complexes and its independency to the movement of the T-loop makes them promising candidates in hampering the role of MARK4 in prostate cancer.

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.

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.

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.

Molecular dynamic simulations of the tubulin-human gamma synuclein complex: structural insight into the regulatory mechanism involved in inducing resistance against Taxol.

Members of the synuclein family (α, ß and γ synucleins) are intrinsically disordered in nature and play a crucial role in the progression of various neurodegenerative disorders and cancers. The association of γSyn with both BubR1 as well as microtubule subunits renders resistance against various anti-cancer drugs. However, the structural aspects underlying drug resistance have not been explored. In this study, the mechanism involved in the association between γSyn and microtubule subunits (αßTub) was investigated and the results reveal a strong interaction between γSyn and the tail regions of αßTub. Complexation of γSyn induces conformational rearrangements in the nucleotide binding loops (NBL), interdomain and tail regions of both α and ßTub. Moreover, in ßTub, the massive displacement observed in M and S loops significantly alters the binding site of microtubule targeting drugs like Taxol. The resulting weak association between Taxol and ßTub of the γSyn-αßTub complex was confirmed by molecular dynamic simulation studies. In addition, the effect of Taxol on NBL, M and S loops of αßTub, is reversed in the presence of γSyn. These results clearly indicate that the presence of γSyn annulled the allosteric regulation imposed by Taxol on the αßTub complex as well as preventing the binding of microtubule targeting drugs, which eventually leads to the development of resistance against these drugs in cancer cells.

Microtubule affinity-regulating kinase 4 (MARK4) is a component of the ectoplasmic specialization in the rat testis.

During the seminiferous epithelial cycle of spermatogenesis, the ectoplasmic specialization (ES, a testis-specific adherens junction, AJ, type) maintains the polarity of elongating/elongated spermatids and confers adhesion to Sertoli cells in the seminiferous epithelium, and known as the apical ES. On the other hand, the ES is also found at the Sertoli-Sertoli cell interface at the blood-testis barrier (BTB) known as basal ES, which together with the tight junction (TJ), maintains Sertoli cell polarity and adhesion, creating a functional barrier that limits paracellular transport of substances across the BTB. However, the apical and basal ES are segregated and restricted to the adluminal compartment and the BTB, respectively. During the transit of preleptotene spermatocytes across the BTB and the release of sperm at spermiation at stage VIII of the seminiferous epithelial cycle, both the apical and basal ES undergo extensive restructuring to facilitate cell movement at these sites. The regulation of these events, in particular their coordination, remains unclear. Studies in other epithelia have shown that the tubulin cytoskeleton is intimately related to cell movement, and MARK [microtubule-associated protein (MAP)/microtubule affinity-regulating kinase] family kinases are crucial regulators of tubulin cytoskeleton stability. Herein MARK4, the predominant member of the MARK protein family in the testis, was shown to be expressed by both Sertoli and germ cells. MARK4 was also detected at the apical and basal ES, displaying highly restrictive spatiotemporal expression at these sites, as well as co-localizing with markers of the apical and basal ES. The expression of MARK4 was found to be stage-specific during the epithelial cycle, structurally associating with α-tubulin and the desmosomal adaptor plakophilin-2, but not with actin-based BTB proteins occludin, ß-catenin and Eps8 (epidermal growth factor receptor pathway substrate 8, an actin bundling and barbed end capping protein). More importantly, it was shown that the expression of MARK4 tightly associated with the integrity of the apical ES because a diminished expression of MARK4 associated with apical ES disruption that led to the detachment of elongating/elongated spermatids from the epithelium. These findings thus illustrate that the integrity of apical ES, an actin-based and testis-specific AJ, is dependent not only on the actin filament network, but also on the tubulin-based cytoskeleton.

Role of P-glycoprotein at the blood-testis barrier on adjudin distribution in the testis: a revisit of recent data.

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in mammals including rodents and humans. It is used to sequester meiosis I and II, postmeiotic spermatid development via spermiogenesis and the release of sperm at spermiation from the systemic circulation, such that these events take place in an immune-privileged site in the adluminal (apical) compartment behind the BTB, segregated from the host immune system. Additionally, drug transporters, namely efflux (e.g., P-glycoprotein) and influx (e.g., Oatp3) pumps, many of which are integral membrane proteins in Sertoli cells at the BTB also work cooperatively to restrict the entry of drugs, toxicants, chemicals, steroids and other xenobiotics into the adluminal compartment. As such, the BTB that serves as an important physiological and selective barrier to protect germ cell development also poses a "hurdle" in male contraceptive development. For instance, adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, a potential nonhormonal male contraceptive that exerts its effects on germ cell adhesion, most notably at the Sertoli cell-spermatid interface, to induce "premature" germ cell loss from the seminiferous epithelium mimicking spermiation, has a relatively poor bioavailability largely because of the BTB. Since male contraceptives (e.g., adjudin) will be used by healthy men for an extended period of his life span after puberty, a better understanding on the BTB is necessary in order to effectively deliver drugs across this blood-tissue barrier in particular if these compounds exert their effects on developing germ cells in the adluminal compartment. This can also reduce long-term toxicity and health risk if the effective dosing can be lowered in order to widen the margin between its safety and efficacy. Herein, we summarize latest findings in this area of research, we also provide a critical evaluation on research areas that deserve attention in future studies.

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

Molecular 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.