Sushil K. Sharma

Depletion of methionine aminopeptidase 2 does not alter cell response to fumagillin or bengamides.

Inhibition of endothelial cell growth by fumagillin has been assumed to be mediated by inhibition of the molecular target methionine aminopeptidase 2 (MetAp2). New data show that depletion of MetAp2 by siRNA does not inhibit endothelial cell growth. Moreover, MetAp2-depleted endothelial cells remain responsive to inhibition by either fumagillin or a newly identified MetAp2 enzyme inhibitor. These data suggest that MetAp2 function is not required for endothelial cell proliferation.

Development of Peptidomimetics Targeting IAPs

Inhibitor of apoptosis proteins (IAPs) such as XIAP subvert apoptosis by binding and inhibiting caspases. Because occupation of the XIAP BIR3 peptide binding pocket by Smac abolishes the XIAP–caspase 9 interaction, it is a proapoptotic event of great therapeutic interest. An assay for pocket binding was developed based on the displacement of Smac 7-mer from BIR3. Through the physical and biochemical analysis of a variety of peptides, we have determined the minimum sequence required for inhibition of the Smac–BIR3 interaction and detailed the dimensions and topology of the BIR3 peptide binding pocket. This work describes the structure–activity relationship (SAR) for peptide inhibitors of Smac-IAP binding.

A new Antennapedia-derived vector for intracellular delivery of exogenous compounds.

We describe the design, synthesis and cell translocation capacity of a peptide derived from the third alpha-helix of the homeodomain of Antennapedia. The new sequence appears to be an efficient and nontoxic means to deliver a covalently linked peptide cargo into cells.

Identification of E2F-1/Cyclin A antagonists

A simple method for the synthesis of a rationally designed (S,S)-[Pro-Leu]-spirolactam scaffold is described. This was expanded to a small biased library of compounds mimicking the ZRXL motif in order to identify E2F-1/Cyclin A antagonists. The synthesized compounds were evaluated in an E2F-1/Cyclin A binding assay and moderately active analogues were identified. In addition, the critical roles of Phe, Leu, Lys, and Arg residues of the identified motif were determined.

On-resin macrocyclization of peptides via intramolecular SNAr reactions

On-resin macrocyclization via an SNAr reaction is employed in the synthesis of tocinoic acid analogs. Specifically, an N-terminal nitrofluorobenzene is attacked by a nucleophilic C-terminal sidechain. The remaining nitro group can be reduced and acylated. NMR is used to compare the conformation of the new macrocyclic peptides to tocinoic acid.

Abstract 2775: A phase I study of LCL161, an oral IAP inhibitor, in patients with advanced cancer

Introduction: LCL161 is a potent small molecule mimetic of the Smac mitochondrial protein. Like Smac, it binds to inhibitor of apoptosis proteins (IAPs) and prevents their interaction with active caspases. In addition, LCL1619s binding to cIAP1 and cIAP2 leads to their proteasomal degradation, with subsequent NFκB activation and induction of IL-8 and MCP-1. LCL161 has single agent activity in tumor models with high basal TNF-α levels and broad synergistic anti-tumor activity in combination with taxanes. This first-in-human study of single agent LCL161 assesses its safety, pharmacokinetics (PK), pharmacodynamics (PD) and evidence of anti-tumor activity. Methods: Patients with advanced treatment-refractory solid tumors receive LCL161 as an oral solution once weekly. The dose of LCL161 is increased in successive cohorts of patients, with dose escalation guided by a Bayesian logistic regression model. PK and PD are assessed primarily during the first cycle (3 weeks) of treatment and disease status is assessed after the completion of every 2 cycles. Therapy continues until disease progression or unacceptable toxicity occurs. PK parameters are derived from non-compartmental analysis of plasma concentration versus time profiles. cIAP1 is detected in tissue by immunohistochemistry (IHC) and in peripheral blood mononuclear cells (PMBCs) by western blot. Circulating markers of cell death (e.g., M65) and a panel of cytokines are measured in plasma by ELISA. Results: As of November 1, 2009, 27 patients with an ECOG PS 0-1 have been enrolled and treated with LCL161 doses ranging from 10-1800 mg/week. In patients receiving doses ≤ 900 mg the median number of cycles is 2 (range 1-5). There have been no dose-limiting toxicities, although grade 1/2 nausea and vomiting have occurred at the 1800 mg dose. LCL161 exhibits dose-proportional PK through 1800 mg, demonstrates rapid absorption with a median Tmax of 1 h (range 0.5-2 h), and has a median half-life of 7 h (range 3-11 h). Doses >500 mg achieve an AUC0–24 equal to or greater than those needed for either single agent or combination activity in preclinical models. At LCL161 doses ≥ 320 mg cIAP1 levels are reduced consistently in skin punch biopsies 8 h after the first dose, and in a tumor biopsy at 24 h. cIAP1 levels in PMBCs are decreased 2 h post-dose and recover by the following week. Circulating markers of cell death peak on day 2 following doses ≥ 320 mg, and circulating cytokines, including MCP-1 and IL-8, increase 4 h post-doses ≥ 900 mg. No objective responses have been observed. At the 900 mg dose one patient (with rectal cancer) remains on study with stable disease for > 5 cycles. Conclusions: Oral LCL161 is well tolerated, has dose-proportional PK, and demonstrates target inhibition in patients, as demonstrated by cIAP degradation and cytokine induction. A solid oral formulation of LCL161 is being evaluated in this study, with plans to continue dose escalation and evaluate consecutive days of treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2775.