Small molecule inhibition of RAS/MAPK signaling ameliorates developmental pathologies of Kabuki Syndrome.

Kabuki Syndrome (KS) is a rare disorder characterized by distinctive facial features, short stature, skeletal abnormalities, and neurodevelopmental deficits. Previously, we showed that loss of function of RAP1A, a RAF1 regulator, can activate the RAS/MAPK pathway and cause KS, an observation recapitulated in other genetic models of the disorder. These data suggested that suppression of this signaling cascade might be of therapeutic benefit for some features of KS. To pursue this possibility, we performed a focused small molecule screen of a series of RAS/MAPK pathway inhibitors, where we tested their ability to rescue disease-relevant phenotypes in a zebrafish model of the most common KS locus, kmt2d. Consistent with a pathway-driven screening paradigm, two of 27 compounds showed reproducible rescue of early developmental pathologies. Further analyses showed that one compound, desmethyl-Dabrafenib (dmDf), induced no overt pathologies in zebrafish embryos but could rescue MEK hyperactivation in vivo and, concomitantly, structural KS-relevant phenotypes in all KS zebrafish models (kmt2d, kmd6a and rap1). Mass spectrometry quantitation suggested that a 100 nM dose resulted in sub-nanomolar exposure of this inhibitor and was sufficient to rescue both mandibular and neurodevelopmental defects. Crucially, germline kmt2d mutants recapitulated the gastrulation movement defects, micrognathia and neurogenesis phenotypes of transient models; treatment with dmDf ameliorated all of them significantly. Taken together, our data reinforce a causal link between MEK hyperactivation and KS and suggest that chemical suppression of BRAF might be of potential clinical utility for some features of this disorder.

Quantifying, Visualizing, and Monitoring Lead Optimization.

Although lead optimization (LO) is by definition a process, process-centric analysis and visualization of this important phase of pharmaceutical R&D has been lacking. Here we describe a simple statistical framework to quantify and visualize the progression of LO projects so that the vital signs of LO convergence can be monitored. We refer to the resulting visualizations generated by our methodology as the "LO telemetry" of a project. These visualizations can be automated to provide objective, holistic, and instantaneous analysis and communication of LO progression. This enhances the ability of project teams to more effectively drive LO process, while enabling management to better coordinate and prioritize LO projects. We present the telemetry of five LO projects comprising different biological targets and different project outcomes, including clinical compound selection, termination due to preclinical safety/tox, and termination due to lack of tractability. We demonstrate that LO progression is accurately captured by the telemetry. We also present metrics to quantify LO efficiency and tractability.

Comparison of the specificity of interaction of cellular and viral zinc-binding domains with 2-mercaptobenzamide thioesters.

The interactions of two 2-mercaptobenzamide thioester compounds with six diverse zinc-binding domains (ZBDs) have been analyzed by UV/visible spectroscopy, NMR spectroscopy, and nucleic acid binding assays. These thioester compounds serve as useful tools for probing the intrinsic chemical stability of ZBDs that exist within a variety of cellular and viral proteins. In our studies, the classical (Cys(2)His(2)) zinc finger ZBDs, the interleaved RING like ZBDs of protein kinase C delta (Cys(2)HisCys and HisCys(3)), and the carboxyl-terminal (Cys(2)HisCys) ZBD of Mouse Mammary Tumor Virus nucleocapsid protein (MMTV NCp10) were resistant to reaction with the thioester compounds. In contrast, the thioester compounds were able to efficiently eject zinc from the amino-terminal (Cys(2)HisCys) ZBD of MMTV NCp10, a Cys(2)HisCys ZBD from Friend of GATA-1 (FOG-1), and from both Cys(4) ZBDs of GATA-1. In all cases, zinc ejection led to a loss of protein structure. Interestingly, GATA-1 was resistant to reaction with the thioester compounds when bound to its target DNA sequence. The electronic and steric screening was calculated for select ZBDs to further explore their reactivity. Based on these results, it appears that both first and second zinc-coordination shell interactions within ZBDs, as well as nucleic acid binding, play important roles in determining the chemical stability and reactivity of ZBDs. These studies not only provide information regarding the relative reactivity of cysteine residues within structural ZBDs but also are crucial for the design of future therapeutic agents that selectively target ZBDs, such as those that occur in the HIV-1 nucleocapsid protein.

Statistical tools for virtual screening.

In large-scale virtual screening (VS) campaigns, data are often computed for millions of compounds to identify leads, but there remains the task of prioritizing VS "hits" for experimental assays and the dilemma of assessing true/false positives. We present two statistical methods for mining large databases: (1) a general scoring metric based on the VS signal-to-noise level within a compound neighborhood; (2) a neighborhood-based sampling strategy for reducing database size, in lieu of property-based filters.

Suppression of breast cancer by chemical modulation of vulnerable zinc fingers in estrogen receptor.

Current antiestrogen therapy for breast cancer is limited by the mixed estrogenic and antiestrogenic activity of selective estrogen receptor modulators. Here we show that the function of zinc fingers in the estrogen receptor DNA-binding domain (DBD) is susceptible to chemical inhibition by electrophilic disulfide benzamide and benzisothiazolone derivatives, which selectively block binding of the estrogen receptor to its responsive element and subsequent transcription. These compounds also significantly inhibit estrogen-stimulated cell proliferation, markedly reduce tumor mass in nude mice bearing human MCF-7 breast cancer xenografts, and interfere with cell-cycle and apoptosis regulatory gene expression. Functional assays and computational analysis support a molecular mechanism whereby electrophilic agents preferentially disrupt the vulnerable C-terminal zinc finger, thus suppressing estrogen receptor-mediated breast carcinoma progression. Our results provide the proof of principle for a new strategy to inhibit breast cancer at the level of DNA binding, rather than the classical antagonism of estrogen binding.