Data-Driven Construction of Antitumor Agents with Controlled Polypharmacology.

Controlling which particular members of a large protein family are targeted by a drug is key to achieving a desired therapeutic response. In this study, we report a rational data-driven strategy for achieving restricted polypharmacology in the design of antitumor agents selectively targeting the TYRO3, AXL, and MERTK (TAM) family tyrosine kinases. Our computational approach, based on the concept of fragments in structural environments (FRASE), distills relevant chemical information from structural and chemogenomic databases to assemble a three-dimensional inhibitor structure directly in the protein pocket. Target engagement by the inhibitors designed led to disruption of oncogenic phenotypes as demonstrated in enzymatic assays and in a panel of cancer cell lines, including acute lymphoblastic and myeloid leukemia (ALL/AML) and nonsmall cell lung cancer (NSCLC). Structural rationale underlying the approach was corroborated by X-ray crystallography. The lead compound demonstrated potent target inhibition in a pharmacodynamic study in leukemic mice.

Chemical genetic-based phenotypic screen reveals novel regulators of gluconeogenesis in human primary hepatocytes.

Insulin resistance is a pathophysiological hallmark of type 2 diabetes and nonalcoholic fatty liver disease. Under the condition of fat accumulation in the liver, suppression of hepatic glucose production by insulin is diminished. In order to gain deeper understanding of dysregulation of glucose production in metabolic diseases, in the present study, we performed an unbiased phenotypic screening in primary human hepatocytes to discover novel mechanisms that regulate gluconeogenesis in the presence of insulin. To optimize phenotypic screening process, we used a chemical genetic screening approach by building a small-molecule library with prior knowledge of activity-based protein profiling. The "positive hits" result from the screen will be small molecules with known protein targets. This makes downstream deconvolution process (i.e., determining the relevant biological targets) less time-consuming. To unbiasedly decipher the molecular targets, we developed a novel statistical method and discovered a set of genes, including DDR3 and CACNA1E that suppressed gluconeogenesis in human hepatocytes. Further investigation, including transcriptional profiling and gene network analysis, was performed to understand the molecular functions of DRD3 and CACNA1E in human hepatocytes.

Biological Characterization of an Improved Pyrrole-Based Colchicine Site Agent Identified through Structure-Based Design.

A refined model of the colchicine site on tubulin was used to design an improved analog of the pyrrole parent compound, JG-03-14. The optimized compound, NT-7-16, was evaluated in biological assays that confirm that it has potent activities as a new colchicine site microtubule depolymerizer. NT-7-16 exhibits antiproliferative and cytotoxic activities against multiple cancer cell lines, with IC(50) values of 10-16 nM, and it is able to overcome drug resistance mediated by the expression of P-glycoprotein and the ßIII isotype of tubulin. NT-7-16 initiated the concentration-dependent loss of cellular microtubules and caused the formation of abnormal mitotic spindles, leading to mitotic accumulation. The direct interaction of NT-7-16 with purified tubulin was confirmed, and it was more potent than combretastatin A-4 in these assays. Binding studies verified that NT-7-16 binds to tubulin within the colchicine site. The antitumor effects of NT-7-16 were evaluated in an MDA-MB-435 xenograft model and it had excellent activity at concentrations that were not toxic. A second compound, NT-9-21, which contains dichloro moieties in place of the 3,5-dibromo substituents of NT-7-16, had a poorer fit within the colchicine site as predicted by modeling and the Hydropathic INTeractions score. Biological evaluations showed that NT-9-21 has 10-fold lower potency than NT-7-16, confirming the modeling predictions. These studies highlight the value of the refined colchicine-site model and identify a new pyrrole-based colchicine-site agent with potent in vitro activities and promising in vivo antitumor actions.

Systematic approach to conformational sampling for assigning absolute configuration using vibrational circular dichroism.

Systematic methods that speed-up the assignment of absolute configuration using vibrational circular dichrosim (VCD) and simplify its usage will advance this technique into a robust platform technology. Applying VCD to pharmaceutically relevant compounds has been handled in an ad hoc fashion, relying on fragment analysis and technical shortcuts to reduce the computational time required. We leverage a large computational infrastructure to provide adequate conformational exploration which enables an accurate assignment of absolute configuration. We describe a systematic approach for rapid calculation of VCD/IR spectra and comparison with corresponding measured spectra and apply this approach to assign the correct stereochemistry of nine test cases. We suggest moving away from the fragment approach when making VCD assignments. In addition to enabling faster and more reliable VCD assignments of absolute configuration, the ability to rapidly explore conformational space and sample conformations of complex molecules will have applicability in other areas of drug discovery.

Structure-activity relationships of retro-dihydrochalcones isolated from Tacca sp.

Several biologically active compounds have been identified from Tacca species, including glycosides, diarylheptanoids, saponins, withanolides, and the taccalonolide class of microtubule stabilizers. More recently, two cytotoxic retro-dihydrochalcones named evelynin A (7) and taccabulin A (6) were isolated and their biological activities characterized, including the finding that taccabulin has microtubule destabilizing effects. Here we describe the identification and characterization of five new retro-chalcones, named taccabulins B-E (1-4) and evelynin B (5) from Tacca sp. extracts. Their structures were determined using 1D and 2D NMR as well as mass spectroscopic data and modeled into the colchicine binding site of tubulin. The antiproliferative and microtubule effects of each compound were determined experimentally and found to be well correlated with modeling studies. The isolation and biological characterization of several retro-dihydrochalcones facilitated preliminary structure-activity relationships for this compound class concerning its antiproliferative and microtubule depolymerizing activities.

How to deal with low-resolution target structures: using SAR, ensemble docking, hydropathic analysis, and 3D-QSAR to definitively map the αß-tubulin colchicine site.

αß-Tubulin colchicine site inhibitors (CSIs) from four scaffolds that we previously tested for antiproliferative activity were modeled to better understand their effect on microtubules. Docking models, constructed by exploiting the SAR of a pyrrole subset and HINT scoring, guided ensemble docking of all 59 compounds. This conformation set and two variants having progressively less structure knowledge were subjected to CoMFA, CoMFA+HINT, and CoMSIA 3D-QSAR analyses. The CoMFA+HINT model (docked alignment) showed the best statistics: leave-one-out q(2) of 0.616, r(2) of 0.949, and r(2)pred (internal test set) of 0.755. An external (tested in other laboratories) collection of 24 CSIs from eight scaffolds were evaluated with the 3D-QSAR models, which correctly ranked their activity trends in 7/8 scaffolds for CoMFA+HINT (8/8 for CoMFA). The combination of SAR, ensemble docking, hydropathic analysis, and 3D-QSAR provides an atomic-scale colchicine site model more consistent with a target structure resolution much higher than the ~3.6 Å available for αß-tubulin.

Developing novel C-4 analogues of pyrrole-based antitubulin agents: weak but critical hydrogen bonding in the colchicine site.

The synthesis, biological evaluation and molecular modeling of a series of pyrrole compounds related to 3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid that evaluates and optimizes C-4 substituents are reported. The key factor for microtubule depolymerization activity appears to be the presence of an appropriately positioned acceptor for Cys241ß in the otherwise hydrophobic subpocket A.

Correct protonation states and relevant waters = better computational simulations?

The unique physicochemical properties of water make it the most important molecule for life. Water molecules have many roles, direct and indirect, related to both biological structure and function. This paper: 1) reviews tools for the prediction of water conservation in and around protein active sites, by empirical (knowledge-based) algorithms and by methods based on thermodynamics principles; 2) reviews principles and approaches to predict pK(a) for both protein residue ensembles and for ligands; and 3) discusses the HINT biomolecular interaction model and forcefield - based on experimental measurements of LogP(o/w), the 1-octanol/water partition coefficient, which implicitly incorporates all solution phenomena like these, and others like tautomerism and entropy. Lastly, it must be considered that the "real" biological environment is a continuum of nano-states and it may not be possible to represent it as a single discrete all-atom model.

Pyrrole-Based Antitubulin Agents: Two Distinct Binding Modalities are Predicted for C-2 Analogs in the Colchicine Site.

3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester is a promising antitubulin lead agent that targets the colchicine site of tubulin. C-2 analogs were synthesized and tested for microtubule depolymerizing and antiproliferative activity. Molecular modeling studies using both GOLD docking and HINT (Hydropathic INTeraction) scoring revealed two distinct binding modes that explain the structural-activity relationships and are in accord with the structural basis of colchicine binding to tubulin. The binding mode of higher activity compounds is buried deeper in the site and overlaps well with rings A and C of colchicine, while the lower activity binding mode shows fewer critical contacts with tubulin. The model distinguishes highly active compounds from those with weaker activities and provides novel insights into the colchicine site and compound design.

Pharmacophore modeling of human adenosine receptor A(2A) antagonists.

Three-dimensional pharmacophore models of human adenosine receptor A(2A) antagonists were developed based on 23 diverse compounds selected from a large number of A(2A) antagonists. The best pharmacophore model, Hypo1, contained five features: one hydrogen bond donor , three hydrophobic points and one ring aromatic. Its correlation coefficient, root mean square deviation, and cost difference values were 0.955, 0.921 and 84.4, respectively, suggested that the Hypo1 model was reasonable and reliable. This model was validated by three methods: a test set of 106 diverse compounds, a simulated virtual screening, and superimposition with the crystal structure of A(2A) receptor. The results showed that Hypo1 was not only in agreement with the A(2A) crystal structure and literature reports, but also well identified active A(2A) antagonists from the virtual database. This methodology provides helpful information and a robust tool for the discovery of potent A(2A) antagonists.