Identification of a novel Mcl-1 protein binding motif.

Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins.

Design, synthesis, and structure-activity relationship studies of thiophene-3-carboxamide derivatives as dual inhibitors of the c-Jun N-terminal kinase.

We report comprehensive structure-activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. Intriguingly, the compounds have a dual inhibitory activity by functioning as both ATP and JIP mimetics, possibly by binding to both the ATP binding site and to the docking site of the kinase. Several of such novel compounds display potent JNK inhibitory profiles both in vitro and in cell.

Identification of a new JNK inhibitor targeting the JNK-JIP interaction site.

JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics.

Synthesis and optimization of thiadiazole derivatives as a novel class of substrate competitive c-Jun N-terminal kinase inhibitors.

A series of thiadiazole derivatives has been designed as potential allosteric, substrate competitive inhibitors of the protein kinase JNK. We report on the synthesis, characterization and evaluation of a series of compounds that resulted in the identification of potent and selective JNK inhibitors targeting its JIP-1 docking site.

A TR3/Nur77 peptide-based high-throughput fluorescence polarization screen for small molecule Bcl-B inhibitors.

Nuclear receptor TR3/Nur77/NR4A1 binds several antiapoptotic Bcl-2-family proteins (Bcl-B, Bcl-2, Bfl-1) in a non-BH3-dependent manner. A 9-amino-acid peptide derived from full-length TR3 with polyarginine tail (TR3-r8) recapitulates TR3's binding specificity, displaying high affinity for Bcl-B. TR3-r8 peptide was used to screen for small molecule Bcl-B inhibitors. A fluorescence polarization assay (FPA) employing fluorescein isothiocyanate (FITC)-labeled TR3-r8 peptide (FITC-TR3-r8) and Bcl-B protein was optimized, with nonfluorescent TR3-r8 serving to demonstrate reversible, competitive binding. Approximately 50,000 compounds were screened at 3.75 mg/L, yielding 145 reproducible hits with > or =50% FITC-TR3-r8 displacement (a confirmed hit rate of 0.29%). After dose-response analyses and counterscreening with an unrelated FITC-based FPA, 6 candidate compounds remained. Nuclear magnetic resonance (NMR) showed that 2 of these compounds bound Bcl-B, but not glutathione S-transferase (GST) control protein. One Bcl-B-binding compound was unable to displace FITClabeled BH3 peptides from Bcl-B, confirming a unique binding mechanism compared with traditional antagonists of antiapoptotic Bcl-2-family proteins. This compound bound Bcl-B with Kd 1.94 +/- 0.38 microM, as determined by isothermal titration calorimetry. Experiments using Bcl-B overexpressing HeLa cells demonstrated that this compound induced Bcl-B-dependent cell death. The current FPA represents a screen that can identify noncanonical inhibitors of Bcl-2-family proteins.

SAR by interligand nuclear overhauser effects (ILOEs) based discovery of acylsulfonamide compounds active against Bcl-x(L) and Mcl-1.

Overexpression of antiapoptotic members of the Bcl-2 family proteins, such as Bcl-x(L) and Mfl-1, has been shown to be involved in resistance to chemotherapeutic drugs in many forms of cancers. Recent efforts from the Abbott Laboratories resulted in the development of the acylsulfonamide compound and clinical candidate that targets selectively Bcl-2, Bcl-x(L), and Bcl-w while it is not active against Mcl-1 and Bfl-1. However, early clinical and preclinical studies suggest that pan-Bcl-2 antagonists, targeting simultaneously Mcl-1, Bcl-xL, and possibly all other four antiapoptotic Bcl-2 proteins, may result in more efficacious drugs. Here, following an NMR fragment-based approach, SAR by ILOEs, we report on compounds that exhibit nanomolar affinities for both Bcl-x(L) and Mcl-1 in vitro. We believe that these molecules can be used as useful starting point for the development of novel Bcl-2 antagonists, in particular targeting Mcl-1.

Screening multicomponent reactions for X-linked inhibitor of apoptosis-baculoviral inhibitor of apoptosis protein repeats domain binder.

We report a second example of a general reaction screening approach to discover low molecular weight inhibitors of protein protein interactions. On the basis of the known pharmacophore model of SMAC mimetics, we predicted several inhibitors based on four different multicomponent reactions. The predicted inhibitors were subsequently synthesized, tested, and found to bind to the antiapoptotic protein X-linked inhibitor of apoptosis protein (XIAP) and showed cellular activity. Also the compounds are currently not highly potent. They could form a starting point for future medicinal chemistry optimization.

Fragment-based design of small molecule X-linked inhibitor of apoptosis protein inhibitors.

We report on a general structure- and NMR-based approach to derive druglike small molecule inhibitors of protein-protein interactions in a rapid and efficient manner. We demonstrate the utility of the approach by deriving novel and effective SMAC mimetics targeting the antiapoptotic protein X-linked inhibitor of apoptosis protein (XIAP). The XIAP baculovirus IAP repeat 3 (Bir3) domain binds directly to the N-terminal of caspase-9, thus inhibiting programmed cell death. It has been shown that in the cell this interaction can be displaced by the protein second mitochondrial activator of caspases (SMAC) and that its N-terminal tetrapeptide region (NH2-AVPI, Ala-Val-Pro-Ile) is responsible for this activity. However, because of their limited cell permeability, synthetic SMAC peptides are inefficient when tested in cultured cells, limiting their use as potential chemical tools or drug candidates against cancer cells. Hence, as an application, we report on the derivation of novel, selective, druglike, cell permeable SMAC mimics with cellular activity.