Anne M. Fourie

HSP70 Binding Sites in the Tumor Suppressor Protein p53

Mutations within conserved regions of the tumor suppressor protein, p53, result in oncogenic forms of the protein with altered tertiary structures. In most cases, the mutant p53 proteins are selectively recognized and bound by members of the HSP70 family of molecular chaperones, but the binding site(s) in p53 for these chaperones have not been clearly defined. We have screened a library of overlapping biotinylated peptides, spanning the entire human p53 sequence, for binding to the HSP70 proteins, Hsc70 and DnaK. We show that most of the high affinity binding sites for these proteins map to secondary structure elements, particularly β-strands, in the hydrophobic core of the central DNA binding domain, where the majority of oncogenic p53 mutations are found. Although peptides corresponding to the C-terminal region of p53 also contain potential binding sites, p53 proteins with C-terminal deletions are capable of binding to Hsc70, indicating that this region is not required for complex formation. We propose that mutations in the p53 protein alter the tertiary structure of the central DNA binding domain, thus exposing high affinity HSP70 binding sites that are cryptic in the wild-type molecule.

Anti-Inflammatory Activity of a Potent, Selective Leukotriene A4 Hydrolase Inhibitor in Comparison with the 5-Lipoxygenase Inhibitor Zileuton

Leukotriene A4 hydrolase (LTA4H) catalyzes production of the proinflammatory lipid mediator, leukotriene (LT) B4, which is implicated in a number of inflammatory diseases. We have identified a potent and selective inhibitor of both the epoxide hydrolase and aminopeptidase activities of recombinant human LTA4H (IC50, approximately 10 nM). In a murine model of arachidonic acid-induced ear inflammation, the LTA4H inhibitor, JNJ-26993135 (1-[4-(benzothiazol-2-yloxy)-benzyl]-piperidine-4-carboxylic acid), dose-dependently inhibited ex vivo LTB4 production in blood, in parallel with dose-dependent inhibition of neutrophil influx (ED50, 1–3 mg/kg) and ear edema. In murine whole blood and in zymosan-induced peritonitis, JNJ-26993135 selectively inhibited LTB4 production, without affecting cysteinyl leukotriene production, while maintaining or increasing production of the anti-inflammatory mediator, lipoxin (LX) A4. The 5-lipoxygenase (5-LO) inhibitor zileuton showed inhibition of LTB4, LTC4, and LXA4 production. Although zileuton inhibited LTB4 production in the peritonitis model more effectively than the LTA4H inhibitor, the influx of neutrophils into the peritoneum after 1 and 2 h was significantly higher in zileuton- versus JNJ-26993135-treated animals. This difference may have been mediated by the increased LXA4 levels in the presence of the LTA4H inhibitor. The selective inhibition of LTB4 production by JNJ-26993135, while increasing levels of the anti-inflammatory mediator, LXA4, may translate to superior therapeutic efficacy versus 5-LO or 5-LO-activating protein inhibitors in LTB4-mediated inflammatory diseases.

Leukotriene A4 Hydrolase Inhibition Attenuates Allergic Airway Inflammation and Hyperresponsiveness

RATIONALE Allergic asthma is characterized by reversible airway obstruction, lung inflammation, and airway hyperresponsiveness (AHR). Previous studies using leukotriene B(4) (LTB(4)) receptor 1-deficient mice and adoptive transfer experiments have suggested that LTB(4) plays a role in lung inflammation and AHR. OBJECTIVES In this study, we used a leukotriene A(4) hydrolase (LTA(4)H) inhibitor as a pharmacological tool to directly examine the role of LTB(4) in a mast cell-dependent murine model of allergic airway inflammation. METHODS We used the forced oscillation technique to test the effects of an LTA(4)H inhibitor dosed during the challenge phase on AHR. Lung tissue and lavage were collected for analysis. MEASUREMENTS AND MAIN RESULTS Treatment with an LTA(4)H inhibitor improved multiple parameters encompassing AHR and lung function. Significant decreases in inflammatory leukocytes, cytokines, and mucin were observed in the lung lumen. Serum levels of antigen-specific IgE and IgG1 were also decreased. Labeled antigen uptake by lung dendritic cells and subsequent trafficking to draining lymph nodes and the lung were decreased on LTA(4)H inhibitor treatment. Provocatively, inhibition of LTA(4)H increased lipoxin A(4) levels in lung lavage fluid. CONCLUSIONS These data suggest that LTB(4) plays a key role in driving lung inflammation and AHR. Mechanistically, we provide evidence that inhibition of LTA(4)H, affects recruitment of both CD4(+) and CD8(+) T cells, as well as trafficking of dendritic cells to draining lymph nodes, and may beneficially modulate other pro- and antiinflammatory eicosanoids in the lung. Inhibition of LTA(4)H is thus a potential therapeutic strategy that could modulate key aspects of asthma.

Identification of a potent, selective, and orally active leukotriene a4 hydrolase inhibitor with anti-inflammatory activity.

LTA 4H is a ubiquitously distributed 69 kDa zinc-containing cytosolic enzyme with both hydrolase and aminopeptidase activity. As a hydrolase, LTA 4H stereospecifically catalyzes the transformation of the unstable epoxide LTA 4 to the diol LTB 4, a potent chemoattractant and activator of neutrophils and a chemoattractant of eosinophils, macrophages, mast cells, and T cells. Inhibiting the formation of LTB 4 is expected to be beneficial in the treatment of inflammatory diseases such as inflammatory bowel disease (IBD), asthma, and atherosclerosis. We developed a pharmacophore model using a known inhibitor manually docked into the active site of LTA 4H to identify a subset of compounds for screening. From this work we identified a series of benzoxazole, benzthiazole, and benzimidazole inhibitors. SAR studies resulted in the identification of several potent inhibitors with an appropriate cross-reactivity profile and excellent PK/PD properties. Our efforts focused on further profiling JNJ 27265732, which showed encouraging efficacy in a disease model relevant to IBD.

A novel B-RAF inhibitor blocks interleukin-8 (IL-8) synthesis in human melanoma xenografts, revealing IL-8 as a potential pharmacodynamic biomarker.

B-RAF mutations have been identified in the majority of melanoma and a large fraction of colorectal and papillary thyroid carcinoma. Drug discovery efforts targeting mutated B-RAF have yielded several interesting molecules, and currently, three compounds are undergoing clinical evaluation. Inhibition of B-RAF in animal models leads to a slowing of tumor growth and, in some cases, tumor reduction. Described within is a novel series of diaryl imidazoles with potent, single-digit nanomolar, anti-B-RAF activity. One compound from this series has been detailed here and has been shown to block B-RAFV600E-dependent extracellular signal-regulated kinase 1/2 phosphorylation in SK-MEL-28 melanoma cells as well as soft agar colony formation and proliferation. Importantly, interleukin-8 (IL-8) was identified by quantitative real-time PCR and ELISA as a product of the elevated mitogen-activated protein kinase signaling in these cells. Plasma concentrations of IL-8 in mice bearing melanoma xenografts were significantly reduced following exposure to B-RAF inhibitors. Taken together, these data suggest that IL-8 could serve as a tractable clinical biomarker. [Mol Cancer Ther 2008;7(3):492–9]

Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis

The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases.

RORγt and RORα signature genes in human Th17 cells

RORγt and RORα are transcription factors of the RAR-related orphan nuclear receptor (ROR) family. They are expressed in Th17 cells and have been suggested to play a role in Th17 differentiation. Although RORγt signature genes have been characterized in mouse Th17 cells, detailed information on its transcriptional control in human Th17 cells is limited and even less is known about RORα signature genes which have not been reported in either human or mouse T cells. In this study, global gene expression of human CD4 T cells activated under Th17 skewing conditions was profiled by RNA sequencing. RORγt and RORα signature genes were identified in these Th17 cells treated with specific siRNAs to knock down RORγt or RORα expression. We have generated selective small molecule RORγt modulators and they were also utilized as pharmacological tools in RORγt signature gene identification. Our results showed that RORγt controlled the expression of a very selective number of genes in Th17 cells and most of them were regulated by RORα as well albeit a weaker influence. Key Th17 genes including IL-17A, IL-17F, IL-23R, CCL20 and CCR6 were shown to be regulated by both RORγt and RORα. Our results demonstrated an overlapping role of RORγt and RORα in human Th17 cell differentiation through regulation of a defined common set of Th17 genes. RORγt as a drug target for treatment of Th17 mediated autoimmune diseases such as psoriasis has been demonstrated recently in clinical trials. Our results suggest that RORα could be involved in same disease mechanisms and gene signatures identified in this report could be valuable biomarkers for tracking the pharmacodynamic effects of compounds that modulate RORγt or RORα activities in patients.

CHAPTER 3:Leukotriene A4 Hydrolase: Biology, Inhibitors and Clinical Applications

Leukotriene A4 hydrolase is a zinc-containing cytosolic enzyme with both hydrolase and aminopeptidase activity. LTA4H stereospecifically catalyzes the transformation of the unstable epoxide LTA4 to the potent pro-inflammatory mediator LTB4. Variations in the lta4h gene have been linked to susceptibility to multiple diseases including myocardial infarction, stroke and asthma. Pre-clinical animal models and human biomarker data have implicated LTB4 in inflammatory diseases. Several groups have now identified selective inhibitors of LTA4H, many of which were influenced by the disclosure of a protein crystal structure a decade ago. Clinical validation of LTA4H remains elusive despite the progression of inhibitors into pre-clinical and clinical development.