Identification of bicyclic compounds that act as dual inhibitors of Bcl-2 and Mcl-1.

Elevated expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1 contributes to poor prognosis and resistance to current treatment modalities in multiple cancers. Here, we report the design, synthesis and characterization of benzimidazole chalcone and flavonoid scaffold-derived bicyclic compounds targeting both Bcl-2 and Mcl-1 by optimizing the structural differences in the binding sites of both these proteins. Initial docking screen of Bcl-2 and Mcl-1 with pro-apoptotic protein Bim revealed possible hits with optimal binding energies. All the optimized bicyclic compounds were screened for their in vitro cytotoxic activity against two oral cancer cell lines (AW8507 and AW13516) which express high levels of Bcl-2 and Mcl-1. Compound 4d from the benzimidazole chalcone series and compound 6d from the flavonoid series exhibited significant cytotoxic activity (IC50 7.12 µM and 17.18 µM, respectively) against AW13516 cell line. Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) analysis further demonstrated that compound 4d and compound 6d could effectively inhibit the Bcl-2 and Mcl-1 proteins by displacing their BH3 binding partners. Both compounds exhibited potent activation of canonical pathway of apoptosis evident from appearance of cleaved Caspase-3 and PARP. Further, treatment of oral cancer cells with the inhibitors induced dissociation of the BH3 only protein Bim from Mcl-1 and Bak from Bcl-2 but failed to release Bax from Bcl-xL thereby confirming the nature of compounds as BH3-mimetics selectively targeting Bcl-2 and Mcl-1. Our study thus identifies bicyclic compounds as promising candidates for anti-apoptotic Bcl-2/Mcl-1 dual inhibitors with a potential for further development.

Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins.

We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized, unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-mediated conformational change in different proteins associated with CAG triplet repeat disorders.

Integrating Ligand and Target-Driven Based Virtual Screening Approaches With in vitro Human Cell Line Models and Time-Resolved Fluorescence Resonance Energy Transfer Assay to Identify Novel Hit Compounds Against BCL-2.

Antiapoptotic members of B-cell leukemia/lymphoma-2 (BCL-2) family proteins are one of the overexpressed proteins in cancer cells that are oncogenic targets. As such, targeting of BCL-2 family proteins raises hopes for new therapeutic discoveries. Thus, we used multistep screening and filtering approaches that combine structure and ligand-based drug design to identify new, effective BCL-2 inhibitors from a small molecule database (Specs SC), which includes more than 210,000 compounds. This database is first filtered based on binary "cancer-QSAR" model constructed with 886 training and 167 test set compounds and common 26 toxicity quantitative structure-activity relationships (QSAR) models. Predicted non-toxic compounds are considered for target-driven studies. Here, we applied two different approaches to filter and select hit compounds for further in vitro biological assays and human cell line experiments. In the first approach, a molecular docking and filtering approach is used to rank compounds based on their docking scores and only a few top-ranked molecules are selected for further long (100-ns) molecular dynamics (MD) simulations and in vitro tests. While docking algorithms are promising in predicting binding poses, they can be less prone to precisely predict ranking of compounds leading to decrease in the success rate of in silico studies. Hence, in the second approach, top-docking poses of each compound filtered through QSAR studies are subjected to initially short (1 ns) MD simulations and their binding energies are calculated via molecular mechanics generalized Born surface area (MM/GBSA) method. Then, the compounds are ranked based on their average MM/GBSA energy values to select hit molecules for further long MD simulations and in vitro studies. Additionally, we have applied text-mining approaches to identify molecules that contain "indol" phrase as many of the approved drugs contain indole and indol derivatives. Around 2700 compounds are filtered based on "cancer-QSAR" model and are then docked into BCL-2. Short MD simulations are performed for the top-docking poses for each compound in complex with BCL-2. The complexes are again ranked based on their MM/GBSA values to select hit molecules for further long MD simulations and in vitro studies. In total, seven molecules are subjected to biological activity tests in various human cancer cell lines as well as Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay. Inhibitory concentrations are evaluated, and biological activities and apoptotic potentials are assessed by cell culture studies. Four molecules are found to be limiting the proliferation capacity of cancer cells while increasing the apoptotic cell fractions.

Development of second generation EP2 antagonists with high selectivity.

EP2 receptor has emerged as an important biological target for therapeutic intervention. In particular, it has been shown to exacerbate disease progression of a variety of CNS and peripheral diseases. Deletion of the EP2 receptor in mouse models recapitulates several features of the COX-2 inhibition, thus presenting a new avenue for anti-inflammatory therapy which could bypass some of the adverse side effects observed by the COX-2 inhibition therapy. We have recently reported a cinnamic amide class of EP2 antagonists with high potency, but these compounds exhibited a moderate selectivity against prostanoid receptor DP1. Moreover they possess acrylamide moiety in the structure, which may result in liver toxicity over longer period of use in a chronic disease model. Thus, we now developed a second generation compounds that devoid of the acrylamide functionality and possess high potency and improved (>1000-fold) selectivity to EP2 over other prostanoid receptors.

A transgenic minipig model of Huntington's Disease.

BACKGROUND: Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. OBJECTIVE: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter. METHODS: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. RESULTS: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. CONCLUSIONS: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.