Mario G. Cardozo

MM-GB/SA Rescoring of Docking Poses in Structure-Based Lead Optimization

The critical issues in docking include the prediction of the correct binding pose and the accurate estimation of the corresponding binding affinity. Different docking methodologies have all been successful in reproducing the crystallographic binding modes but struggle when predicting the corresponding binding affinities. The aim of this work is to evaluate the performance of the MM-GB/SA rescoring of docking poses in structure-based lead optimization. To accomplish that, a diverse set of pharmaceutically relevant targets, including CDK2, FactorXa, Thrombin, and HIV-RT were selected. The correlation between the MM-GB/SA results and experimental data in all cases is remarkable. It even qualifies this approach as a more attractive alternative for rank-ordering than the Free Energy Perturbation and Thermodynamic Integration methodologies because, while as accurate, it can handle more structurally dissimilar ligands and provides results at a fraction of the computational cost. On the technical side, the benefit of performing a conformational analysis and having an ensemble of conformers to represent each ligand in the unbound state during the MM-GB/SA rescoring procedure was investigated. In addition, the estimation of conformational entropy penalties for the ligands upon binding, computed from the Boltzmann distribution in water, was evaluated and compared to a commonly used approach employed by many docking scoring functions.

Identification and functional characterization of allosteric agonists for the G protein-coupled receptor FFA2.

FFA2 (GPR43) has been identified as a receptor for short-chain fatty acids (SCFAs) that include acetate and propionate. FFA2 is highly expressed in islets, a subset of immune cells, and adipocytes. Although the potential roles of FFA2 activation in these tissues have previously been described, the physiological functions are still unclear. The potency for SCFAs on FFA2 is low, in the high micromolar to millimolar concentrations. To identify better pharmacological tools to study receptor function, we used high-throughput screening (HTS) to discover a series of small molecule phenylacetamides as novel and more potent FFA2 agonists. This series is specific for FFA2 over FFA1 (GPR40) and FFA3 (GPR41), and it is able to activate both the Gαq and Gαi pathways in vitro on Chinese hamster ovary cells stably expressing FFA2. Treatment of adipocytes with these compounds also resulted in Gαi-dependent inhibition of lipolysis similar to that of endogenous ligands (SCFAs). It is noteworthy that these compounds not only acted as FFA2 agonists but also exhibited positive cooperativity with acetate or propionate. The observed allosteric modulation was consistent in all the functional assays that we have explored, including cAMP, calcium mobilization, guanosine 5′-[γ-thio]triphosphate binding, and lipolysis. Molecular modeling analysis of FFA2 based on human β2-adrenergic receptor structure revealed potential nonoverlapping binding sites for the endogenous and synthetic ligands, further providing insight into the binding pocket for the allosteric interactions. This is the first report describing the identification of novel allosteric modulators with agonist activity for FFA2, and these compounds may serve as tools for further unraveling the physiological functions of the receptor and its involvement in various diseases.

Significance Analysis and Multiple Pharmacophore Models for Differentiating P-Glycoprotein Substrates

P-glycoprotein (Pgp) mediated drug efflux affects the absorption, distribution, and clearance of a broad structural variety of drugs. Early assessment of the potential of compounds to interact with Pgp can aid in the selection and optimization of drug candidates. To differentiate nonsubstrates from substrates of Pgp, a robust predictive pharmacophore model was targeted in a supervised analysis of three-dimensional (3D) pharmacophores from 163 published compounds. A comprehensive set of pharmacophores has been generated from conformers of whole molecules of both substrates and nonsubstrates of P-glycoprotein. Four-point 3D pharmacophores were employed to increase the amount of shape information and resolution, including the ability to distinguish chirality. A novel algorithm of the pharmacophore-specific t-statistic was applied to the actual structure-activity data and 400 sets of artificial data (sampled by decorrelating the structure and Pgp efflux activity). The optimal size of the significant pharmacophore set was determined through this analysis. A simple classification tree using nine distinct pharmacophores was constructed to distinguish nonsubstrates from substrates of Pgp. An overall accuracy of 87.7% was achieved for the training set and 87.6% for the external independent test set. Furthermore, each of nine pharmacophores can be independently utilized as an accurate marker for potential Pgp substrates.

Structure guided design of a series of sphingosine kinase (SphK) inhibitors.

Sphingosine-1-phosphate (S1P) signaling plays a vital role in mitogenesis, cell migration and angiogenesis. Sphingosine kinases (SphKs) catalyze a key step in sphingomyelin metabolism that leads to the production of S1P. There are two isoforms of SphK and observations made with SphK deficient mice show the two isoforms can compensate for each others loss. Thus, inhibition of both isoforms is likely required to block SphK dependent angiogenesis. A structure based approach was used to design and synthesize a series of SphK inhibitors resulting in the identification of the first potent inhibitors of both isoforms of human SphK. Additionally, to our knowledge, this series of inhibitors contains the only sufficiently potent inhibitors of murine SphK1 with suitable physico-chemical properties to pharmacologically interrogate the role of SphK1 in rodent models and to reproduce the phenotype of SphK1 (-/-) mice.

Synthesis and optimization of substituted furo[2,3-d]-pyrimidin-4-amines and 7H-pyrrolo[2,3-d]pyrimidin-4-amines as ACK1 inhibitors.

Two classes of ACK1 inhibitors, 4,5,6-trisubstituted furo[2,3-d]pyrimidin4-amines and 4,5,6-trisubstituted 7H-pyrrolo[2,3-d]pyrimidin-4-amines, were discovered and evaluated as ACK1 inhibitors. Further structural refinement led to the identification of potent and selective dithiolane inhibitor 37.

Discovery and in vivo evaluation of (S)-N-(1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine (AMG319) and related PI3Kδ inhibitors for inflammation and autoimmune disease.

The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.

Discovery and in Vivo Evaluation of Dual PI3Kβ/δ Inhibitors

Structure-based rational design led to the synthesis of a novel series of potent PI3K inhibitors. The optimized pyrrolopyridine analogue 63 was a potent and selective PI3Kβ/δ dual inhibitor that displayed suitable physicochemical properties and pharmacokinetic profile for animal studies. Analogue 63 was found to be efficacious in animal models of inflammation including a keyhole limpet hemocyanin (KLH) study and a collagen-induced arthritis (CIA) disease model of rheumatoid arthritis. These studies highlight the potential therapeutic value of inhibiting both the PI3Kβ and δ isoforms in the treatment of a number of inflammatory diseases.

Identification and optimization of N3,N6-diaryl-1H-pyrazolo[3,4-d]pyrimidine-3,6-diamines as a novel class of ACK1 inhibitors.

A new series of pyrazolo[3,4-d]pyrimidine-3,6-diamines was designed and synthesized as potent and selective inhibitors of the nonreceptor tyrosine kinase, ACK1. These compounds arose from efforts to rigidify an earlier series of N-aryl pyrimidine-5-carboxamides. The synthesis and structure-activity relationships of this new series of inhibitors are reported. The most promising compounds were also profiled for their pharmacokinetic properties.

The synthesis and SAR of novel diarylsulfone 11β-HSD1 inhibitors.

In this communication, human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitory activities of a novel series of diarylsulfones are described. Optimization of this series resulted in several highly potent 11β-HSD1 inhibitors with excellent pharmacokinetic (PK) properties. Compound (S)-25 showed excellent efficacy in a non-human primate ex vivo pharmacodynamic model.

Discovery, Optimization, and in Vivo Evaluation of Benzimidazole Derivatives AM-8508 and AM-9635 as Potent and Selective PI3Kδ Inhibitors

Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inhibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.