Apoptosis resistance commonly occurs in cancers, preventing activation of Caspase family cell death proteases. XIAP is an endogenous inhibitor of Caspases overexpressed in many cancers. We developed an enzyme derepression assay, based on overcoming XIAP-mediated suppression of Caspase-3, and screened mixture-based combinatorial chemical libraries for compounds that reversed XIAP-mediated inhibition of Caspase-3, identifying a class of polyphenylureas with XIAP-inhibitory activity. These compounds, but not inactive structural analogs, stimulated increases in Caspase activity, directly induced apoptosis of many types of tumor cell lines in culture, and sensitized cancer cells to chemotherapeutic drugs. Active compounds also suppressed growth of established tumors in xenograft models in mice, while displaying little toxicity to normal tissues. These findings validate IAPs as targets for cancer drug discovery.

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Serine-, threonine-, and cysteine-derived cyclic building blocks (pseudo-prolines, ΨPro) serve as reversible protecting groups for Ser, Thr, and Cys and prove to be versatile tools for overcoming some intrinsic problems in the field of peptide chemistry. The presence of ΨPro within a peptide sequence results in the disruption of β-sheet structures considered as a source of intermolecular aggregation during chain elongation, thus increasing solvation and coupling kinetics in peptide assembly. Due to their easy synthetic access and variability in the chemical stability by modifications introduced in the C-2 position of the oxazolidine/thiazolidine ring system, this protection technique is adaptable to all common strategies in peptide synthesis. We describe new types of ΨPro building blocks suitable for standard Fmoc/tBu-based solid phase peptide synthesis, convergent strategies, and chemoselective ligation techniques as well as their use as a structure-disrupting, solubilizing protection technique for the example of peptides generally considered as “difficult sequences”.

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IntroductionAt a fundamental level, patients with acute leukemia do not respond to treatment because the malignant blasts are not eradicated by current chemotherapy. In part, this failure is due to defects in apoptosis pathways. 1 Therefore, agents that overcome roadblocks to apoptosis could be therapeutically useful for this disease.Classically, apoptosis is caused by the activation of caspases, a family of intracellular cysteine proteases that cleave substrates at aspartic acid residues. 2,3 Currently, at least 4 pathways for initiation of caspase activation exist: (1) the mitochondrial pathway with cytochrome c; (2) the death receptor pathway with the tumor necrosis factor (TNF) family of death receptors; (3) direct caspase activation by cytolytic T-cell protease Granzyme B; and (4) a pathway connected to the endoplasmic reticulum. These pathways launch a proteolytic cascade, in which upstream (initiator) caspases cleave and activate downstream (effector) caspases.The inhibitor of apoptosis proteins (IAPs) are a family of endogenous caspase inhibitors that share a common baculoviral IAP repeat (BIR) domain. To date, 8 IAP family members exist in humans. Of these, XIAP is probably the best characterized with respect to its structure and biochemical mechanisms. XIAP inhibits caspases 3, 7, and 9, but not caspases 1, 6, 8, or 10. 4 XIAP contains 3 tandem baculovirus IAP repeat (BIR) domains and a really interesting new gene (RING) domain. The second BIR domain of XIAP (BIR2) inhibits caspases 3 and 7, while the third BIR domain (BIR3) inhibits caspase 9. Through their ability to inhibit caspases, IAPs act as antiapoptotic proteins [5][6][7][8][9][10] and are promising therapeutic targets. Inhibition of XIAP by antisense strategies or peptides that bind and inhibit the BIR3 domain of XIAP sensitizes malignant cells to chemotherapy. [11][12][13][14][15][16][17][18] Based on the knowledge that XIAP directly inhibits active caspase 3, we devised an enzymatic derepression assay to screen for molecules that relieve protease inhibition. Using this assay, we screened combinatorial libraries of chemical compounds and identified active agents based on different pharmacophores. The initial report of these XIAP inhibitors described a series of compounds based on the polyphenylurea pharmacophore including the active compound N- -methyl-NЈ-phenylurea (1396-12) and structural analogues. 19 Corresponding to their activity in the enzymatic assay, active polyphenylurea-based inhibitors but not inactive controls, induced rapid apoptosis of several types of tumor cell lines. 19 We determined that active compounds inhibit XIAP by binding its BIR2 domain at a site distinct from the binding pocket of the endogenous XIAP inhibitor second modulator of apoptotic proteases (SMAC). 20 Given the potential therapeutic utility of IAP inhibition, we tested these chemical IAP inhibitors in cultured leukemia cell lines For personal use only. on June 19, 2019. by guest www.bloodjournal.org From and primary acute myelogenous leukemia (AML) patie...

Virtual screening is increasingly being used in drug discovery programs with a growing number of successful applications. Experimental methodologies developed to speed up the drug discovery processes include high-throughput screening and combinatorial chemistry. The complementarities between computational and experimental screenings have been recognized and reviewed in the literature. Computational methods have also been used in the combinatorial chemistry field, in particular in library design. However, the integration of computational and combinatorial chemistry screenings has been attempted only recently. Combinatorial libraries (experimental or virtual) represent a notable source of chemically related compounds. Advances in combinatorial chemistry and deconvolution strategies, have enabled the rapid exploration of novel and dense regions in the chemical space. The present review is focused on the integration of virtual and experimental screening of combinatorial libraries. Applications of virtual screening to discover novel anticancer agents and our ongoing efforts towards the integration of virtual screening and combinatorial chemistry are also discussed.

Mixture based synthetic combinatorial libraries offer a tremendous enhancement for the rate of drug discovery, allowing the activity of millions of compounds to be assessed through the testing of exponentially fewer samples. In this study, we used a scaffold-ranking library to screen 37 different libraries for antibacterial activity against the ESKAPE pathogens. Each library contained between 10000 and 750000 structural analogues for a total of >6 million compounds. From this, we identified a bis-cyclic guanidine library that displayed strong antibacterial activity. A positional scanning library for these compounds was developed and used to identify the most effective functional groups at each variant position. Individual compounds were synthesized that were broadly active against all ESKAPE organisms at concentrations <2 μM. In addition, these compounds were bactericidal, had antibiofilm effects, showed limited potential for the development of resistance, and displayed almost no toxicity when tested against human lung cells and erythrocytes. Using a murine model of peritonitis, we also demonstrate that these agents are highly efficacious in vivo.