Paul A. Rejto

Molecular recognition of the inhibitor AG-1343 by HIV-1 protease: conformationally flexible docking by evolutionary programming.

BACKGROUND An important prerequisite for computational structure-based drug design is prediction of the structures of ligand-protein complexes that have not yet been experimentally determined by X-ray crystallography or NMR. For this task, docking of rigid ligands is inadequate because it assumes knowledge of the conformation of the bound ligand. Docking of flexible ligands would be desirable, but requires one to search an enormous conformational space. We set out to develop a strategy for flexible docking by combining a simple model of ligand-protein interactions for molecular recognition with an evolutionary programming search technique. RESULTS We have developed an intermolecular energy function that incorporates steric and hydrogen-bonding terms. The parameters in this function were obtained by docking in three different protein systems. The effectiveness of this method was demonstrated by conformationally flexible docking of the inhibitor AG-1343, a potential new drug against AIDS, into HIV-1 protease. For this molecule, which has nine rotatable bonds, the crystal structure was reproduced within 1.5 A root-mean-square deviation 34 times in 100 simulations, each requiring eight minutes on a Silicon Graphics R4400 workstation. The energy function correctly evaluates the crystal structure as the global energy minimum. CONCLUSIONS We believe that a solution of the docking problem may be achieved by matching a simple model of molecular recognition with an efficient search procedure. The necessary ingredients of a molecular recognition model include only steric and hydrogen-bond interaction terms. Although these terms are not necessarily sufficient to predict binding affinity, they describe ligand-protein interactions faithfully enough to enable a docking program to predict the structure of the bound ligand. This docking strategy thus provides an important tool for the interdisciplinary field of rational drug design.

Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer

Gastric cancer is a heterogeneous disease with multiple environmental etiologies and alternative pathways of carcinogenesis. Beyond mutations in TP53, alterations in other genes or pathways account for only small subsets of the disease. We performed exome sequencing of 22 gastric cancer samples and identified previously unreported mutated genes and pathway alterations; in particular, we found genes involved in chromatin modification to be commonly mutated. A downstream validation study confirmed frequent inactivating mutations or protein deficiency of ARID1A, which encodes a member of the SWI-SNF chromatin remodeling family, in 83% of gastric cancers with microsatellite instability (MSI), 73% of those with Epstein-Barr virus (EBV) infection and 11% of those that were not infected with EBV and microsatellite stable (MSS). The mutation spectrum for ARID1A differs between molecular subtypes of gastric cancer, and mutation prevalence is negatively associated with mutations in TP53. Clinically, ARID1A alterations were associated with better prognosis in a stage-independent manner. These results reveal the genomic landscape, and highlight the importance of chromatin remodeling, in the molecular taxonomy of gastric cancer.

Whole-genome sequencing and comprehensive molecular profiling identify new driver mutations in gastric cancer

Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes. We performed whole-genome sequencing in 100 tumor-normal pairs, along with DNA copy number, gene expression and methylation profiling, for integrative genomic analysis. We found subtype-specific genetic and epigenetic perturbations and unique mutational signatures. We identified previously known (TP53, ARID1A and CDH1) and new (MUC6, CTNNA2, GLI3, RNF43 and others) significantly mutated driver genes. Specifically, we found RHOA mutations in 14.3% of diffuse-type tumors but not in intestinal-type tumors (P < 0.001). The mutations clustered in recurrent hotspots affecting functional domains and caused defective RHOA signaling, promoting escape from anoikis in organoid cultures. The top perturbed pathways in gastric cancer included adherens junction and focal adhesion, in which RHOA and other mutated genes we identified participate as key players. These findings illustrate a multidimensional and comprehensive genomic landscape that highlights the molecular complexity of gastric cancer and provides a road map to facilitate genome-guided personalized therapy.

Whole genome sequencing identifies recurrent mutations in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Simulating disorder–order transitions in molecular recognition of unstructured proteins: Where folding meets binding

A microscopic study of functional disorder–order folding transitions coupled to binding is performed for the p27 protein, which derives a kinetic advantage from the intrinsically disordered unbound form on binding with the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) complex. Hierarchy of structural loss during p27 coupled unfolding and unbinding is simulated by using high-temperature Monte Carlo simulations initiated from the crystal structure of the tertiary complex. Subsequent determination of the transition-state ensemble and the proposed atomic picture of the folding mechanism coupled to binding provide a microscopic rationale that reconciles the initiation recruitment of p27 at the cyclin A docking site with the kinetic benefit for a disordered α-helix in the unbound form of p27. The emerging structural polarization in the ensemble of unfolding/unbinding trajectories and in the computationally determined transition-state ensemble is not determined by the intrinsic folding preferences of p27 but rather is attributed to the topological requirements of the native intermolecular interface to order β-hairpin and β-strand of p27 that could be critical for nucleating rapid folding transition coupled to binding. In agreement with the experimental data, the disorder–order folding transition for p27 is largely determined by the functional requirement to form a specific intermolecular interface that ultimately dictates the folding mechanism and overwhelms any local folding preferences for creating a stable α-helix in the p27 structure before overcoming the major free energy barrier.

A novel selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor prevents human adipogenesis

Glucocorticoid excess increases fat mass, preferentially within omental depots; yet circulating cortisol concentrations are normal in most patients with metabolic syndrome (MS). At a pre-receptor level, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates cortisol from cortisone locally within adipose tissue, and inhibition of 11β-HSD1 in liver and adipose tissue has been proposed as a novel therapy to treat MS by reducing hepatic glucose output and adiposity. Using a transformed human subcutaneous preadipocyte cell line (Chub-S7) and human primary preadipocytes, we have defined the role of glucocorticoids and 11β-HSD1 in regulating adipose tissue differentiation. Human cells were differentiated with 1·0 μM cortisol (F), or cortisone (E) with or without 100 nM of a highly selective 11β-HSD1 inhibitor PF-877423. 11β-HSD1 mRNA expression increased across adipocyte differentiation (P<0·001, n=4), which was paralleled by an increase in 11β-HSD1 oxo-reductase activity (from nil on day 0 to 5·9±1.9 pmol/mg per h on day 16, P<0·01, n=7). Cortisone enhanced adipocyte differentiation; fatty acid-binding protein 4 expression increased 312-fold (P<0·001) and glycerol-3-phosphate dehydrogenase 47-fold (P<0·001) versus controls. This was abolished by co-incubation with PF-877423. In addition, cellular lipid content decreased significantly. These findings were confirmed in the primary cultures of human subcutaneous preadipocytes. The increase in 11β-HSD1 mRNA expression and activity is essential for the induction of human adipogenesis. Blocking adipogenesis with a novel and specific 11β-HSD1 inhibitor may represent a novel approach to treat obesity in patients with MS.

Genomic landscape of copy number aberrations enables the identification of oncogenic drivers in hepatocellular carcinoma

Cancer is a genetic disease with frequent somatic DNA alterations. Studying recurrent copy number aberrations (CNAs) in human cancers would enable the elucidation of disease mechanisms and the prioritization of candidate oncogenic drivers with causal roles in oncogenesis. We have comprehensively and systematically characterized CNAs and the accompanying gene expression changes in tumors and matched nontumor liver tissues from 286 hepatocellular carcinoma (HCC) patients. Our analysis identified 29 recurrently amplified and 22 recurrently deleted regions with a high level of copy number changes. These regions harbor established oncogenes and tumor suppressors, including CCND1 (cyclin D1), MET (hepatocyte growth factor receptor), CDKN2A (cyclin‐dependent kinase inhibitor 2A) and CDKN2B (cyclin‐dependent kinase inhibitor 2B), as well as many other genes not previously reported to be involved in liver carcinogenesis. Pathway analysis of cis‐acting genes in the amplification and deletion peaks implicates alterations of core cancer pathways, including cell‐cycle, p53 signaling, phosphoinositide 3‐kinase signaling, mitogen‐activated protein kinase signaling, Wnt signaling, and transforming growth factor beta signaling, in a large proportion of HCC patients. We further credentialed two candidate driver genes (BCL9 and MTDH) from the recurrent focal amplification peaks and showed that they play a significant role in HCC growth and survival. Conclusion: We have demonstrated that characterizing the CNA landscape in HCC will facilitate the understanding of disease mechanisms and the identification of oncogenic drivers that may serve as potential therapeutic targets for the treatment of this devastating disease. (Hepatology 2013;58:706–717)

Demonstration of Proof of Mechanism and Pharmacokinetics and Pharmacodynamic Relationship with 4-Cyano-biphenyl- 4-sulfonic Acid (6-Amino-pyridin-2-yl)-amide (PF-915275), an Inhibitor of 11-Hydroxysteroid Dehydrogenase Type 1, in Cynomolgus Monkeys

Glucocorticoids, through activation of the glucocorticoid receptor (GR), regulate hepatic gluconeogenesis. Elevated hepatic expression and activity of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) play a key role in ligand-induced activation of the GR through the production of cortisol. Evidence from genetically modified mice suggests that inhibition of 11βHSD1 might be a therapeutic approach to treat the metabolic syndrome. We have identified a potent 11βHSD1 inhibitor, 4′-cyano-biphenyl-4-sulfonic acid (6-amino-pyridin-2-yl)-amide (PF-915275), that is selective for the primate and human enzymes. The objective of this study was to demonstrate target inhibition with PF-915275 and to quantify the relationship between target inhibition and drug exposure in monkeys. We characterized the ability of PF-915275 to inhibit the conversion of prednisone, a synthetic cortisone analog that can be distinguished from the endogenous substrate cortisone, enabling a direct measure of substrate to product conversion without the complication of feedback. Adult cynomolgus monkeys were administered either vehicle or various doses of PF-915275 followed by a 10-mg/kg dose of prednisone. Prednisone conversion to prednisolone and the concentrations of PF-915275 were measured by liquid chromatography/tandem mass spectrometry. PF-915275 dose-dependently inhibited 11βHSD1-mediated conversion of prednisone to prednisolone, with a maximum of 87% inhibition at a 3-mg/kg dose. An exposure-response relationship was demonstrated, with an estimated EC50 of 391 nM (total) and 17 nM (free). Insulin levels were also reduced in a dose-related manner. These results should enable the development of a biomarker for evaluating target modulation in humans that will aid in identifying 11βHSD1 inhibitors to treat diabetes and other related metabolic diseases.

An integrated genomic approach to identify predictive biomarkers of response to the aurora kinase inhibitor PF-03814735.

PF-03814735 is a novel, reversible inhibitor of Aurora kinases A and B that finished a phase I clinical trial for the treatment of advanced solid tumors. To find predictive biomarkers of drug sensitivity, we screened a diverse panel of 87 cancer cell lines for growth inhibition upon PF-03814735 treatment. Small cell lung cancer (SCLC) and, to a lesser extent, colon cancer lines were very sensitive to PF-03814735. The status of the Myc gene family and retinoblastoma pathway members significantly correlated with the efficacy of PF-03814735. Whereas RB1 inactivation, intact CDKN2A/p16, and normal CCND1/Cyclin D1 status are hallmarks of SCLC, activation or amplification of any of the three Myc genes (MYC, MYCL1, and MYCN) clearly differentiated cell line sensitivity within the SCLC panel. By contrast, we found that expression of Aurora A and B were weak predictors of response. We observed a decrease in histone H3 phosphorylation and polyploidization of sensitive lines, consistent with the phenotype of Aurora B inhibition. In vivo experiments with two SCLC xenograft models confirmed the sensitivity of Myc gene-driven models to PF-03814735 and a possible schedule dependence of MYC/c-Myc–driven tumors. Altogether our results suggest that SCLC and other malignancies driven by the Myc family genes may be suitable indications for treatment by Aurora B kinase inhibitors. Mol Cancer Ther; 11(3); 710–9. ©2012 AACR.

A Comprehensive Characterization of Genome-Wide Copy Number Aberrations in Colorectal Cancer Reveals Novel Oncogenes and Patterns of Alterations

To develop a comprehensive overview of copy number aberrations (CNAs) in stage-II/III colorectal cancer (CRC), we characterized 302 tumors from the PETACC-3 clinical trial. Microsatellite-stable (MSS) samples (n = 269) had 66 minimal common CNA regions, with frequent gains on 20 q (72.5%), 7 (41.8%), 8 q (33.1%) and 13 q (51.0%) and losses on 18 (58.6%), 4 q (26%) and 21 q (21.6%). MSS tumors have significantly more CNAs than microsatellite-instable (MSI) tumors: within the MSI tumors a novel deletion of the tumor suppressor WWOX at 16 q23.1 was identified (p<0.01). Focal aberrations identified by the GISTIC method confirmed amplifications of oncogenes including EGFR, ERBB2, CCND1, MET, and MYC, and deletions of tumor suppressors including TP53, APC, and SMAD4, and gene expression was highly concordant with copy number aberration for these genes. Novel amplicons included putative oncogenes such as WNK1 and HNF4A, which also showed high concordance between copy number and expression. Survival analysis associated a specific patient segment featured by chromosome 20 q gains to an improved overall survival, which might be due to higher expression of genes such as EEF1B2 and PTK6. The CNA clustering also grouped tumors characterized by a poor prognosis BRAF-mutant-like signature derived from mRNA data from this cohort. We further revealed non-random correlation between CNAs among unlinked loci, including positive correlation between 20 q gain and 8 q gain, and 20 q gain and chromosome 18 loss, consistent with co-selection of these CNAs. These results reinforce the non-random nature of somatic CNAs in stage-II/III CRC and highlight loci and genes that may play an important role in driving the development and outcome of this disease.