Potent and selective small-molecule MCL-1 inhibitors demonstrate on-target cancer cell killing activity as single agents and in combination with ABT-263 (navitoclax).

The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.

3-(2-(3-Pyridinyl)thiazolidin-4-oyl)indoles, a novel series of platelet activating factor antagonists.

(2RS,4R)-3-(2-(3-Pyridinyl)thiazolidin-4-oyl)indoles represent a new class of potent, orally active antagonists of platelet activating factor (PAF). The compounds were prepared by acylation of the magnesium or zinc salts of substituted indoles with (2RS,4R)-2-(3-pyridinyl)-3-(tert-butoxycarbonyl)thiazolidin-4-oyl chloride. The 3-acylindole moiety functions as a hydrolytically stabilized and conformationally restricted anilide replacement, which imparts a considerable boost in potency to the series. Structure-activity relationships observed for substitution on the indole ring system are discussed. Members of the series compare favorably with other reported PAF antagonists.

Discovery and evaluation of a series of 3-acylindole imidazopyridine platelet-activating factor antagonists.

Studies conducted with the goal of discovering a second-generation platelet-activating factor (PAF) antagonist have identified a novel class of potent and orally active antagonists which have high aqueous solubility and long duration of action in animal models. The compounds arose from the combination of the lipophilic indole portion of Abbott's first-generation PAF antagonist ABT-299 (2) with the methylimidazopyridine heterocycle moiety of British Biotechnology's BB-882 (1) and possess the positive attributes of both of these clinical candidates. Structure-activity relationship (SAR) studies indicated that modification of the indole and benzoyl spacer of lead compound 7b gave analogues that were more potent, longer-lived, and bioavailable and resulted in the identification of 1-(N, N-dimethylcarbamoyl)-4-ethynyl-3-[3-fluoro-4-[(1H-2-methylimidazo[4,5-c] pyrid-1-yl)methyl]benzoyl]indole hydrochloride (ABT-491, 22 m.HCl) which has been evaluated extensively and is currently in clinical development.

Pharmacology of ABT-491, a highly potent platelet-activating factor receptor antagonist.

ABT-491 (4-ethynyl-N, N-dimethyl-3-[3-fluoro-4-[(2-methyl-1H-imidazo-[4,5-c]pyridin-1-yl)methy l]benzoyl]-1H- indole-1-carboxamide hydrochloride) is a novel PAF (platelet-activating factor) receptor antagonist with a K(i) for inhibiting PAF binding to human platelets of 0.6 nM. Binding kinetics of ABT-491 to the PAF receptor is consistent with a relatively slow off-rate of the antagonist when compared to PAF. Inhibition of PAF binding is selective and is correlated with functional antagonism of PAF-mediated cellular responses (Ca2+ mobilization, priming, and degranulation). Administration of ABT-491 in vivo leads to potent inhibition of PAF-induced inflammatory responses (increased vascular permeability, hypotension, and edema) and PAF-induced lethality. Oral potency (ED50) was between 0.03 and 0.4 mg/kg in rat, mouse, and guinea-pig. When administered intravenously in these species, ABT-491 exhibited ED50 values between 0.005 and 0.016 mg/kg. An oral dose of 0.5 mg/kg in rat provided > 50% protection for 8 h against cutaneous PAF challenge. ABT-491 administered orally was also effective in inhibiting lipopolysaccharide-induced hypotension (ED50 = 0.04 mg/kg), gastrointestinal damage (0.05 mg/kg, 79% inhibition), and lethality (1 mg/kg, 85% vs. 57% survival). The potency of this novel antagonist suggests that ABT-491 will be useful in the treatment of PAF-mediated diseases.

Ectopic expression of methionine aminopeptidase-2 causes cell transformation and stimulates proliferation.

Methionine aminopeptidase-2 (MetAP2) processes N-terminal methionine from nascent cellular proteins. Inhibition of MetAP2 has been shown to block angiogenesis and suppress tumor growth in preclinical tumor models. However, the biological role of MetAP2 in cancer is not well understood. We examined the effect of three distinct chemical classes of MetAP2 inhibitors on the growth of a panel of human cancer cells in vitro. All MetAP2 inhibitors caused inhibition of tumor cell growth in both anchorage-dependent and, particularly, in anchorage-independent manner. These data prompted us to examine the possible roles of MetAP2 in cancers. Ectopic expression of MetAP2 in NIH-3T3 cells caused transformation, evidenced by the formation of foci in monolayer culture and growth of large colonies in soft agar. Overexpression of MetAP2 in an immortalized bronchial epithelial cell line NL20 accelerated growth. These phenotypes induced by the overexpression of MetAP2 were reversed by the treatment with MetAP2 inhibitors, indicating that the catalytic function of MetAP2 was essential. Accordingly, overexpression of a catalytically inactive MetAP2 resulted in growth retardation of HT1080 tumor cells, suggesting a dominant-negative role of the inactive MetAP2 mutant. Finally, we analysed the expression of MetAP2 in patient cancer samples by immunohistochemistry. Moderate-to-high staining was identified in the majority of breast, colon, lung, ovarian and prostate carcinomas examined. These data suggest that MetAP2 plays an important role in tumor cell growth and may contribute to tumorigenesis.

The role of platelet-activating factor (PAF) and the efficacy of ABT-491, a highly potent and selective PAF antagonist, in experimental allergic rhinitis.

Platelet-activating factor (PAF) may be an important mediator of allergic rhinitis. In the present study we evaluated the effectiveness of a recently described PAF antagonist (ABT-491) in rat and guinea pig models of allergic rhinitis. PAF, when perfused through the nasal passages of anesthetized Brown Norway rats, provoked an acute increase, measured as dye leakage, in nasal vascular permeability evident within 15 min after exposure to PAF. ABT-491, given orally 1 hr before PAF challenge, inhibited the response in a dose-related manner (ED50 = 0.3 mg/kg). Intranasal perfusion with ovalbumin in rats sensitized to the antigen 18 to 21 days before challenge also induced an increase in vascular permeability. The antigen-induced leakage was inhibited a maximum of 74% (P < or = .001) by pretreatment with ABT-491 (3 mg/kg p.o.). An antihistamine (mepyramine, 10 mg/kg i.p.), a serotonin antagonist (methysergide) and a 5-lipoxygenase inhibitor (A-79175) also exhibited efficacy in this model (56%, 87% and 65% inhibition, respectively). Nearly complete inhibition (93%, P < or = .001) of the response was achieved by coadministration of ABT-491 and methysergide. In guinea pigs intranasal administration of PAF resulted in increased airway resistance that was inhibited in a dose-dependent manner by oral administration of ABT-491 (ED50 = 1 mg/kg). Antigen-induced nasal airway resistance, triggered by exposure of sensitized animals to aerosolized ovalbumin, was also inhibited by ABT-491 (maximum inhibition 64%, P < or = .05, 10 mg/kg p.o.). The effectiveness of the antagonist was increased to 80% protection by coadministration with either an antihistamine or a 5-lipoxygenase inhibitor, agents which were separately insignificant in blocking the response to antigen. These results suggest a therapeutic utility for ABT-491, perhaps in combination with other anti-inflammatory agents, in the treatment of allergic rhinitis.

Aryl ketones as novel replacements for the C-terminal amide bond of succinyl hydroxamate MMP inhibitors.

A series of succinyl hydroxamate MMP inhibitors were prepared incorporating an aryl amino ketone moiety in place of the more typical C-terminal amino acid amides. Compounds of the C-terminal ketone series displayed potent inhibition of MMPs. Several compounds of the series were shown to be orally bioavailable.

Evaluation of the inhibition of other metalloproteinases by matrix metalloproteinase inhibitors.

Two series of compounds synthesized as specific matrix metalloproteinase (MMP) inhibitors have been evaluated for their inhibition of non-MMPs. In a series of substituted succinyl hydroxamic acids, some were found to be significant (IC50 < 1 microM) inhibitors of leucine (microsomal) aminopeptidase, neprilysin (3.4.24.11), and thermolysin. Macrocyclic compounds in which the alpha carbon of the succinyl hydroxamate is linked to the side chain of the P2' amino acid were found to be good inhibitors of aminopeptidase, but not of neprilysin or thermolysin. Compounds of neither series were found to be significant inhibitors of angiotensin converting enzyme or carboxypeptidase A.