Accelerating Lead Identification by High Throughput Virtual Screening: Prospective Case Studies from the Pharmaceutical Industry.

At the onset of a drug discovery program, the goal is to identify novel compounds with appropriate chemical features that can be taken forward as lead series. Here, we describe three prospective case studies, Bruton Tyrosine Kinase (BTK), RAR-Related Orphan Receptor γ t (RORγt), and Human Leukocyte Antigen DR isotype (HLA-DR) to illustrate the positive impact of high throughput virtual screening (HTVS) on the successful identification of novel chemical series. Each case represents a project with a varying degree of difficulty due to the amount of structural and ligand information available internally or in the public domain to utilize in the virtual screens. We show that HTVS can be effectively employed to identify a diverse set of potent hits for each protein system even when the gold standard, high resolution structural data or ligand binding data for benchmarking, is not available.

A novel formulation of veggies with potent liver detoxifying activity.

LXR (encoded by NR1H2 and 3) and FXR (known as bile acid receptor) encoded by NR1H4 (nuclear receptor subfamily 1, group H and member 4) are nuclear receptors in humans and are important regulators of bile acid production, cholesterol, fatty acid and glucose homeostasis hence responsible for liver detoxification. Several strategies for drug design with numerous ligands for this target have failed owing to the inability of the ligand to access the target/receptor or their early metabolisation. In this work, we have evaluated FXR and LXR structure bound with agonist and compared the binding energy affinity of active ligands present in live green-real veggies with reference drugs (ligands) present in the market. A high throughput screening combined with molecular docking, absorption, distribution, metabolism, excretion and toxicity (ADMET) predictions, log P values and percentage of human oral absorption value led to the identification of two compounds present in live green-real veggies with strong potential for liver detoxification.

Molecular decodification of gymnemic acids from Gymnema sylvestre. Discovery of a new class of liver X receptor antagonists.

The individual chemical components of commercial extract of Gymnema sylvestre, a medicinal plant used in the traditional systems of the Indian medicine for its antidiabetic and hypolipidemic properties, were isolated and evaluated for their capability to act as modulators of nuclear and membrane receptors involved in glucose and lipid homeostasis. The study disclosed for the first time that individual gymnemic acids are potent and selective antagonists for the ß isoform of LXR. Indeed the above activity was shared by the most abundant aglycone gymnemagenin (10) whereas gymnestrogenin (11) was endowed with a dual LXRα/ß antagonistic profile. Deep pharmacological investigation demonstrated that gymnestrogenin, reducing the expression of SREBP1c and ABCA1 in vitro, is able to decrease lipid accumulation in HepG2 cells. The results of this study substantiate the use of G. sylvestre extract in LXR mediated dislypidemic diseases.

A novel mutation in the NR2E3 gene associated with Goldmann-Favre syndrome and vasoproliferative tumor of the retina.

PURPOSE: Various autosomal recessive retinal dystrophies are reported to be associated with mutations in nuclear receptor subfamily 2, group E, member 3 (NR2E3, also called PNR) gene. The present study proposed to understand the clinical and genetic characteristics of the family of a patient with an ocular phenotype consistent with Goldmann-Favre syndrome (GFS) and vasoproliferative tumors of the retina (VPTRs). METHODS: Twelve family members of the proband from three generations underwent complete ophthalmic examination, including best-corrected visual acuity with Snellen optotypes, tonometry, biomicroscopic examination, indirect ophthalmoscopy after pupillary dilatation, computerized perimetry, optical coherence tomography, fundus photography, intravenous fluorescein angiography, and electroretinography (ERG). All the study subjects underwent genetic analysis of the entire coding region of the NR2E3 gene with the bidirectional DNA sequencing approach. Hundred healthy individuals were screened for the variant. RESULTS: The phenotype of the proband had features of GFS with VPTRs. The tumors showed complete resolution with cryotherapy and transpupillary thermotherapy (TTT). Sequencing of the entire coding region of the NR2E3 gene in the proband revealed a novel homozygous c.1117 A>G variant that led to the amino acid change from aspartic acid to glycine at position 406 (p.D406G). This change was present in the homozygous state in affected family members and in the heterozygous state in unaffected family members, and was undetectable in the control subjects. The identified novel p.D406G homozygous mutation was at an evolutionarily highly conserved region and may possibly affect the protein function (Sorting Intolerant From Tolerant [SIFT] score = 0.00). CONCLUSIONS: Patients with GFS may present with retinal VPTRs that respond to therapy with cryotherapy and TTT. Molecular genetic studies helped to identify a novel p.D406G mutation in the affected members, which will aid in confirming the diagnosis, for genetic counseling of family members and potentially provide some form of therapy for the affected patients.

24(S)-Saringosterol from edible marine seaweed Sargassum fusiforme is a novel selective LXRß agonist.

Dietary phytosterols have been successfully used for lowering cholesterol levels, which correlates with the fact that some phytosterols are able to act as liver X receptor (LXR) agonists. Sargassum fusiforme is an edible marine seaweed well-known for its antiatherosclerotic function in traditional Chinese medicine. In this study, seven phytosterols including fucosterol (1), saringosterol (2), 24-hydroperoxy-24-vinyl-cholesterol (3), 29-hydroperoxy-stigmasta-5,24(28)-dien-3ß-ol (4), 24-methylene-cholesterol (5), 24-keto-cholesterol (6), and 5α,8α-epidioxyergosta-6,22-dien-3ß-ol (7) were purified and evaluated for their actions on LXR-mediated transcription using a reporter assay. Among these phytosterols, 2 was the most potent compound in stimulating the transcriptional activities of LXRα by (3.81±0.15)-fold and LXRß by (14.40±1.10)-fold, respectively. Two epimers of 2, 24(S)-saringosterol (2a) and 24(R)-saringosterol (2b), were subsequently separated by semipreparative high-performance liquid chromatography. Interestingly, 2a was more potent than 2b in LXRß-mediated transactivation ((3.50±0.17)-fold vs (1.63±0.12)-fold) compared with control. Consistently, 2a induced higher expression levels of LXR target genes including key players in reverse cholesterol transport in six cell lines. These data along with molecular modeling suggested that 2a acts as a selective LXRß agonist and is a potent natural cholesterol-lowering agent. This study also demonstrated that phytosterols in S. fusiforme contributed to the well-known antiatherosclerotic function.

Structure of the retinoid X receptor α-liver X receptor ß (RXRα-LXRß) heterodimer on DNA.

Nuclear receptors (NRs) are conditional transcription factors with common multidomain organization that bind diverse DNA elements. How DNA sequences influence NR conformation is poorly understood. Here we report the crystal structure of the human retinoid X receptor α-liver X receptor ß (RXRα-LXRß) heterodimer on its cognate element, an AGGTCA direct repeat spaced by 4 nt. The complex has an extended X-shaped arrangement, with DNA- and ligand-binding domains crossed, in contrast to the parallel domain arrangement of other NRs that bind an AGGTCA direct repeat spaced by 1 nt. The LXRß core binds DNA via canonical contacts and auxiliary DNA contacts that enhance affinity for the response element. Comparisons of RXRα-LXRßs in the crystal asymmetric unit and with previous NR structures reveal flexibility in NR organization and suggest a role for RXRα in adaptation of heterodimeric complexes to DNA.

Discovery of new liver X receptor agonists by pharmacophore modeling and shape-based virtual screening.

Agonists of liver X receptors (LXR) α and ß are important regulators of cholesterol metabolism, but agonism of the LXRα subtype appears to cause hepatic lipogenesis, suggesting LXRß-selective activators are attractive new lipid lowering drugs. In this work, pharmacophore modeling and shape-based virtual screening were combined to predict new LXRß-selective ligands. Out of the 10 predicted compounds, three displayed significant LXR activity. Two activated both LXR subtypes. The third compound activated LXRß 1.8-fold over LXRα.

Molecular evidence for the involvement of RORα and RORγ in immune response in teleost.

In mammals, retinoid-related orphan receptors (ROR) consist of three members as RORα, RORß and RORγ. It is well known that RORα plays a critical role in cerebellum development while RORγt directs T helper 17 (Th17) cell differentiation. So far, the knowledge on fish ROR family is limited as only zebrafish ROR family members have been characterized, showing that they play roles in embryonic and cerebellar development. In this study, we have cloned two paralogues for RORα (RORα1 and RORα2) and RORγ (RORγ1 and RORγ2) from grass carp (Ctenopharyngodon idellus). Phylogenetic analysis showed that grass carp RORα and RORγ were grouped in the clade of zebrafish RORα and RORγ, respectively. Real-time RT-PCR assay revealed that these four ROR transcripts exhibited similar expression patterns, in particular the high levels in pituitary, brain and some immune-related tissues, suggesting that all of them may play a role in endocrine and immune system of teleost. To explore the immune roles of grass carp RORα and RORγ, their expression was detected in periphery blood lymphocytes (PBLs) challenged by immune stimuli. Results showed that both RORα and RORγ mRNA levels were up-regulated by PHA but not LPS in PBLs, suggesting that their expression may be subject to different immune processes. In the same cell model, poly I:C stimulation induced RORγ1/2 but not RORα1/2 expression, pointing to the different roles of grass carp RORα and RORγ in immune response. Consistently, bacterial challenge significantly up-regulated the expression of these four ROR genes in spleen, headkidney and thymus. These results not only contribute to elucidate the roles of ROR in fish immunity but also facilitate to further clarify the existence of Th17-like cells in fish.