A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with Ki values of 28, 14, 17 and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Compound 1 inhibits tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human clinical trials and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance in humans was 9 ± 3 mL/min/kg and volume of distribution was 0.6 ± 0.2 L/kg.

Antibody-drug conjugate therapy entails targeted killing of cancer cells with cytotoxic compounds covalently linked to tumor-specific antibodies and shows promise in the treatment of several human cancers. Current antibody-drug conjugate designs that incorporate a disulfide linker between the antibody and cytotoxic drug are inspired by indirect evidence suggesting that the redox potential within the endosomal system is reducing. It is presumed that antigen-dependent endocytosis leads to disulfide linker reduction and intracellular release of free drug, but direct demonstration of such a mechanism is lacking. To determine whether the disulfide N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) linker would be reduced during endocytic recycling of the anti-HER2 antibody trastuzumab (Herceptin, Genentech), we synthesized a trastuzumab-SPP-Rhodamine red conjugate and developed a linker cleavage assay by using the self-quenching property of this fluorophore. In breast carcinoma SKBr3 cells, no SPP linker cleavage was observed, as detected by fluorescence dequenching upon internalization. By contrast, the conjugate did display fluorescence dequenching when diverted to the lysosomal pathway by geldanamycin, an effect partly due to proteolytic degradation rather than disulfide reduction. To understand why linker reduction was inefficient, we measured redox potentials of endocytic compartments by expressing a redox-sensitive variant of GFP fused to various endocytic proteins. Unexpectedly, we found that recycling endosomes, late endosomes, and lysosomes are not reducing, but oxidizing and comparable with conditions in the endoplasmic reticulum. These results suggest that intracellular reduction is unlikely to account for the potency of disulfide-linked antibodydrug conjugates.disulfide linker ͉ redox potential ͉ endocytosis ͉ HER2 ͉ Herceptin O ne approach to the treatment of cancer is to specifically target cytotoxic drugs to tumor cells by linking them via a cleavable linker to antibodies that recognize a tumor-restricted antigen. Such linkers include hydrazone linkers, designed to hydrolyze upon internalization into acidic endosomes and lysosomes (1-3); peptide linkers optimized for cleavage by certain lysosomal proteases (3-5); and disulfide linkers, thought to be cleaved by the reducing environment within the endocytic pathway (6-10). In the latter category, the monoclonal antibody C242 against CanAg (a glycotope on the mucin1 (MUC1) colorectal tumor antigen) has shown efficacy against colorectal xenograft models in vivo when disulfide-linked to the ribosomal inhibitor ricin A chain via a 4-succinimdyloxycarbonyl-methyl-␣-[2-pyridyldithio]-toluene (SMPT) linker (11) and to the maytansinoid-derived microtubule active drug DM1 via an N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) linker (12). The latter conjugate is now humanized and in clinical trials as cantuzumab mertansine (9, 13). An ideal linker should be cleaved only upon internalization of the antibody-drug conjugate into the tumor cell, thereby specifical...

Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pK a and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.

p21-activated kinase 1 (PAK1) has an important role in transducing signals in several oncogenic pathways. The concept of inhibiting this kinase has garnered significant interest over the past decade, particularly for targeting cancers associated with PAK1 amplification. Animal studies with the selective group I PAK (pan-PAK1, 2, 3) inhibitor G-5555 from the pyrido[2,3-d]pyrimidin-7-one class uncovered acute toxicity with a narrow therapeutic window. To attempt mitigating the toxicity, we introduced significant structural changes, culminating in the discovery of the potent pyridone side chain analogue G-9791. Mouse tolerability studies with this compound, other members of this series, and compounds from two structurally distinct classes revealed persistent toxicity and a correlation of minimum toxic concentrations and PAK1/2 mediated cellular potencies. Broad screening of selected PAK inhibitors revealed PAK1, 2, and 3 as the only overlapping targets. Our data suggest acute cardiovascular toxicity resulting from the inhibition of PAK2, which may be enhanced by PAK1 inhibition, and cautions against continued pursuit of pan-group I PAK inhibitors in drug discovery.

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Tryptophan 2,3-dioxygenase 2 (TDO2) catalyzes the conversion of tryptophan to the immunosuppressive metabolite kynurenine. TDO2 overexpression has been observed in a number of cancers; therefore, TDO inhibition may be a useful therapeutic intervention for cancers. We identified an aminoisoxazole series as potent TDO2 inhibitors from a high-throughput screen (HTS). An extensive medicinal chemistry effort revealed that both the amino group and the isoxazole moiety are important for TDO2 inhibitory activity. Computational modeling yielded a binding hypothesis and provided insight into the observed structure-activity relationships. The optimized compound is a potent TDO2 inhibitor with modest selectivity over indolamine 2,3-dioxygenase 1 (IDO1) and with improved human whole blood stability.

Checkpoint kinase 1 (ChK1) is a serine/threonine kinase that functions as a central mediator of the intra-S and G 2 -M cell-cycle checkpoints. Following DNA damage or replication stress, ChK1-mediated phosphorylation of downstream effectors delays cell-cycle progression so that the damaged genome can be repaired. As a therapeutic strategy, inhibition of ChK1 should potentiate the antitumor effect of chemotherapeutic agents by inactivating the postreplication checkpoint, causing premature entry into mitosis with damaged DNA resulting in mitotic catastrophe. Here, we describe the characterization of GNE-900, an ATP-competitive, selective, and orally bioavailable ChK1 inhibitor. In combination with chemotherapeutic agents, GNE-900 sustains ATR/ATM signaling, enhances DNA damage, and induces apoptotic cell death. The kinetics of checkpoint abrogation seems to be more rapid in p53-mutant cells, resulting in premature mitotic entry and/or accelerated cell death. Importantly, we show that GNE-900 has little single-agent activity in the absence of chemotherapy and does not grossly potentiate the cytotoxicity of gemcitabine in normal bone marrow cells. In vivo scheduling studies show that optimal administration of the ChK1 inhibitor requires a defined lag between gemcitabine and GNE-900 administration. On the refined combination treatment schedule, gemcitabine's antitumor activity against chemotolerant xenografts is significantly enhanced and dose-dependent exacerbation of DNA damage correlates with extent of tumor growth inhibition. In summary, we show that in vivo potentiation of gemcitabine activity is mechanism based, with optimal efficacy observed when S-phase arrest and release is followed by checkpoint abrogation with a ChK1 inhibitor. Mol Cancer Ther; 12(10); 1968-80. Ó2013 AACR.