Vincent Madison

Three-Dimensional Models of Wild-Type and Mutated Forms of Cytochrome P450 14α-Sterol Demethylases from Aspergillus fumigatus and Candida albicans Provide Insights into Posaconazole Binding

ABSTRACT The cytochrome P450 sterol 14α-demethylase enzyme (CYP51) is the target of azole antifungals. Azoles block ergosterol synthesis, and thereby fungal growth, by binding in the active-site cavity of the enzyme and ligating the iron atom of the heme cofactor through a nitrogen atom of the azole. Mutations in and around the CYP51 active site have resulted in azole resistance. In this work, homology models of the CYP51 enzymes from Aspergillus fumigatus and Candida albicans were constructed based on the X-ray crystal structure of CYP51 from Mycobacterium tuberculosis. Using these models, binding modes for voriconazole (VOR), fluconazole (FLZ), itraconazole (ITZ), and posaconazole (POS) were predicted from docking calculations. Previous work had demonstrated that mutations in the vicinity of the heme cofactor had a greater impact on the binding of FLZ and VOR than on the binding of POS and ITZ. Our modeling data suggest that the long side chains of POS and ITZ occupy a specific channel within CYP51 and that this additional interaction, which is not available to VOR and FLZ, serves to stabilize the binding of these azoles to the mutated CYP51 proteins. The model also predicts that mutations that were previously shown to specifically impact POS susceptibility in A. fumigatus and C. albicans act by interfering with the binding of the long side chain.

AKT crystal structure and AKT-specific inhibitors.

AKT kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. This review summarizes studies that support the rationale for targeting AKT kinases in new drug discovery efforts. Structural features of AKT kinase in its inactive and active states, as determined by crystal structure analysis, are described. Recent efforts in the development and biological evaluation of small molecule inhibitors of AKT, and the challenges remaining are summarized. Inhibitors targeting the ATP binding site, PH domain and protein substrate binding site, as well as isoform selective allosteric inhibitors are reviewed. Structure-based design using PKA mutants as surrogates and computer modeling in the discovery of selective inhibitors is discussed. The issues and challenges facing the development of different classes of inhibitors as therapeutics are also discussed.

Human members of the eukaryotic protein kinase family

BackgroundEukaryotic protein kinases (EPKs) constitute one of the largest recognized protein families represented in the human genome. EPKs, which are similar to each other in sequence, structure and biochemical properties, are important players in virtually every signaling pathway involved in normal development and disease. Near completion of projects to sequence the human genome and transcriptome provide an opportunity to identify and perform sequence analysis on a nearly complete set of human EPKs.ResultsPublicly available genetic sequence data were searched for human sequences that potentially represent EPK family members. After removal of duplicates, splice variants and pseudogenes, this search yielded 510 sequences with recognizable similarity to the EPK family. Protein sequences of putative EPK catalytic domains identified in the search were aligned, and a phonogram was constructed based on the alignment. Representative sequence records in GenBank were identified, and derived information about gene mapping and nomenclature was summarized.ConclusionsThis work represents a nearly comprehensive census and early bioinformatics overview of the EPKs encoded in the human genome. Evaluation of the sequence relationships between these proteins contributes contextual information that enhances understanding of individual family members. This curation of human EPK sequences provides tools and a framework for the further characterization of this important class of enzymes.

Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases

Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation.

Crystal structures of the pro-inflammatory cytokine interleukin-23 and its complex with a high-affinity neutralizing antibody

Interleukin (IL)-23 is a pro-inflammatory cytokine playing a key role in the pathogenesis of several autoimmune and inflammatory diseases. We have determined the crystal structures of the heterodimeric p19-p40 IL-23 and its complex with the Fab (antigen-binding fragment) of a neutralizing antibody at 2.9 and 1.9 A, respectively. The IL-23 structure closely resembles that of IL-12. They share the common p40 subunit, and IL-23 p19 overlaps well with IL-12 p35. Along the hydrophilic heterodimeric interface, fewer charged residues are involved for IL-23 compared with IL-12. The binding site of the Fab is located exclusively on the p19 subunit, and comparison with published cytokine-receptor structures suggests that it overlaps with the IL-23 receptor binding site.

Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach-Part 2.

Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.

Expression, purification, characterization and homology modeling of active Akt/PKB, a key enzyme involved in cell survival signaling.

Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its activation has been linked to tumorigenesis. Up-regulation of Akt as well as its upstream regulator phosphatidylinositol-3 kinase (PI3K) has been found in many tumors and the negative regulator of this pathway PTEN/MMAC is a tumor suppressor. As a target for drug discovery, we have expressed and purified an active Akt1 enzyme from a recombinant baculovirus-infected Sf9 cell culture. Coexpression of Akt1 with the catalytic subunit of PI3K or treatment with okadaic acid during expression was found to generate an active enzyme in the insect cell culture system. We have optimized the kinase activity and developed a simple quantitative kinase assay using biotinylated peptide substrates. Using the purified active enzyme, we have characterized its physical, catalytic and kinetic properties. Since Akt is closely related to protein kinase C (PKC) and protein kinase A, the issue of obtaining selective inhibitors of this enzyme was addressed by comparison of the structures of catalytic domains of Akt and PKC, derived by homology modeling methods. A number of amino acid differences in the ATP binding regions of these kinases were identified, suggesting that selective inhibitors of Akt can be discovered. However, the ATP binding regions are highly conserved in the three isoforms of Akt implying that the discovery of isoform-selective inhibitors would be very challenging.

Preclinical Characterization of the Antiviral Activity of SCH 900518 (Narlaprevir), a Novel Mechanism-Based Inhibitor of Hepatitis C Virus NS3 Protease

ABSTRACT Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K*i) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC90) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5× EC90 of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.

Drugs targeted against protein kinases

Current treatments for cancer (surgery, radiation and chemotherapy) are successful for early stage localised disease but have severe side effects. New treatments are needed to increase the cure rate and life expectancy of patients. With the discovery of oncogenes, tumour suppressor genes and an understanding of their role in the development of the malignant disease, a new era of therapy has begun. Cancer is a manifestation of deregulated signalling pathways that mediate cell growth and programmed cell death. Protein kinases are essential elements in these signalling pathways. In the US, Novartis launched Gleevec™ (imantinib, STI-571) in May 2001 as the first anticancer drug whose mechanism of action is kinase inhibition. In Phase I trials, 23/24 patients with chronic myelogenous leukaemia (CML) had complete remissions and the drug is relatively non-toxic. Herceptin® (trastuzumab) is a monoclonal antibody (mAb) against a member of the growth factor receptor family (HER-2/neu) that was launched in 1998 by Genentech for the treatment of breast cancer. Trastuzumab has an excellent antitumour profile, particularly when used in combination with doxorubicin and paclitaxol. These drugs are pioneering the treatment of cancer based on the molecular understanding of the disease. Numerous drugs that target growth factor receptors and their signalling pathways are in advanced clinical trials. Herein, antibodies against receptors and small molecule inhibitors of kinases in signalling pathways will be summarised. Inter-disciplinary preclinical studies have identified chemicals that target specific kinases. We believe that clinical studies of these agents will yield new anticancer agents that target specific diseases and that are less toxic than current agents.

Induced-fit docking of mometasone furoate and further evidence for glucocorticoid receptor 17α pocket flexibility

An induced-fit docking method was used to characterize the interactions of the glucocorticoid receptor binding-site with mometasone furoate, a glucocorticoid with a lipophilic ester at the C17alpha position. Two validation studies demonstrated that the protocol can reproduce crystal structures of nuclear receptors, and is appropriate for modeling ligand binding to the glucocorticoid receptor. Key hydrogen bonding interactions between mometasone furoate and the glucocorticoid receptor, as well as favorable hydrophobic interactions between the furoate group and the 17alpha pocket, contribute to high affinity and specificity of this ligand for the receptor. Using the glucocorticoid des-ciclesonide, which has an even larger moiety at the 16,17alpha position, induced-fit docking demonstrates the ability of the 17alpha pocket of the receptor to expand even further to accommodate the ligand.