An Updated Steroid Benchmark Set and Its Application in the Discovery of Novel Nanomolar Ligands of Sex Hormone-Binding Globulin
ABSTRACT A benchmark data set of steroids with known affinity for sex hormone-binding globulin (SHBG) has been widely used to validate popular molecular field-based QSAR techniques. We have expanded the data set by adding a number of nonsteroidal SHBG ligands identified both from the literature and in our previous experimental studies. This updated molecular set has been used herein to develop 4D QSAR models based on "inductive" descriptors and to gain insight into the molecular basis of protein-ligand interactions. Molecular alignment was generated by means of docking active compounds into the active site of the SHBG. Surprisingly, the alignment of the benchmark steroids contradicted the classical ligand-based alignment utilized in previous CoMFA and CoMSIA models yet afforded models with higher statistical significance and predictive power. The resulting QSAR models combined with CoMFA and CoMSiA models as well as structure-based virtual screening allowed discovering several low-micromolar to nanomolar nonsteroidal inhibitors for human SHBG.
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ABSTRACT: Plasma sex hormone-binding globulin (SHBG) regulates the access of androgens and estrogens to their target tissues and cell types. An SHBG homologue, known as the androgen-binding protein, is expressed in Sertoli cells of many mammalians, but testicular expression of human SHBG is restricted to germ cells. The primary structure of SHBG comprises tandem laminin G-like (LG) domains. The amino-terminal LG-domain includes the steroid-binding site and dimerization interface, and its tertiary structure, resolved in complex with natural and synthetic sex steroids, has revealed unanticipated mechanisms of steroid binding at the atomic level. This LG-domain interacts with fibulin-1D and fibulin-2 in a ligand-specific manner, and this is attributed to the unique way estrogens reside within the steroid-binding site, and the ordering of an otherwise flexible loop structure covering the entrance of the steroid-binding pocket. This mechanism enables estradiol to enhance the sequestration of plasma SHBG by the stroma of some tissues through binding to these extra-cellular matrix-associated proteins. The human SHBG amino-terminal LG-domain also contains several cation-binding sites, and occupancy of a zinc-binding site influences its affinity for estradiol. The complete quaternary structure of SHBG remains unresolved but structural predictions suggest that the carboxy-terminal LG-domains extend laterally from the dimerized amino-terminal LG-domains. The carboxy-terminal LG-domain contains two N-glycosylation sites, but their biological significance remains obscure. Knowledge of the SHBG tertiary structure has helped develop computational techniques based on the use of a "bench-mark data set" of steroid ligands, and created novel drug discovery and toxicology risk assessment platforms.Molecular and Cellular Endocrinology 09/2009; 316(1):13-23. DOI:10.1016/j.mce.2009.09.005 · 4.24 Impact Factor
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ABSTRACT: Anthropogenic compounds with the capacity to interact with the steroid-binding site of sex hormone binding globulin (SHBG) pose health risks to humans and other vertebrates including fish. Building on studies of human SHBG, we have applied in silico drug discovery methods to identify potential binders for SHBG in zebrafish (Danio rerio) as a model aquatic organism. Computational methods, including; homology modeling, molecular dynamics simulations, virtual screening, and 3D QSAR analysis, successfully identified 6 non-steroidal substances from the ZINC chemical database that bind to zebrafish SHBG (zfSHBG) with low-micromolar to nanomolar affinities, as determined by a competitive ligand-binding assay. We also screened 80,000 commercial substances listed by the European Chemicals Bureau and Environment Canada, and 6 non-steroidal hits from this in silico screen were tested experimentally for zfSHBG binding. All 6 of these compounds displaced the [(3)H]5alpha-dihydrotestosterone used as labeled ligand in the zfSHBG screening assay when tested at a 33 microM concentration, and 3 of them (hexestrol, 4-tert-octylcatechol, and dihydrobenzo(a)pyren-7(8H)-one) bind to zfSHBG in the micromolar range. The study demonstrates the feasibility of large-scale in silico screening of anthropogenic compounds that may disrupt or highjack functionally important protein:ligand interactions. Such studies could increase the awareness of hazards posed by existing commercial chemicals at relatively low cost.Toxicology and Applied Pharmacology 08/2008; 234(1):47-57. DOI:10.1016/j.taap.2008.07.014 · 3.63 Impact Factor
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ABSTRACT: Virtual screening is a fast, low cost method to identify potential small molecule therapeutics from large chemical databases for the vast amount of target proteins emerging from the life sciences and bioinformatics. In this work, we applied several conventional and newly developed virtual screening approaches to identify novel non-steroidal ligands for the human and zebrafish sex hormone binding globulin (SHBG). The ‘benchmark set of steroids’ is a set of steroids with known affinities for human SHBG that has been widely used for validation in the development of different virtual screening methods. We have updated this data set by including additional steroidal SHBG ligands and by modifying the predicted binding orientations of several benchmark steroids in the SHBG binding site based on the use of an improved docking protocol and information from recent crystallographic data. The new steroid binding orientations and the expanded version of the benchmark set was then used to create new in silico models which were applied in virtual screening to identify high-affinity non-steroidal human SHBG ligands from a large chemical database. Anthropogenic compounds with the capacity to interact with the steroid-binding site of SHBG pose health risks to humans and other vertebrates including fish. We constructed a homology model of SHBG from zebrafish and applied virtual screening to identify ligands for zebrafish SHBG from a set of 80 000 existing commercial substances, many of which can be exposed to the aquatic environment. Six hits from this in silico screen were tested experimentally for zebrafish SHBG binding and three of them, hexestrol, 4-tert-octylcatechol, dihydrobenzo(a)pyren-7(8H)-one demonstrated micromolar binding affinity for the zebrafish SHBG. These findings demonstrate the feasibility of using virtual screening to identify anthropogenic compounds that may disrupt or highjack functionally important protein:ligand interactions. Studies applying this new computational toxicology method could increase the awareness of hazards posed by existing commercial chemicals at relatively low cost.