Lawrence A. Reiter

Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation

We report how rotational variations in transmembrane (TM) helix interactions participate in the activity states of the thrombopoietin receptor (TpoR), a type 1 cytokine receptor that controls the production of blood platelets. We also explore the mechanism of small‐molecule agonists that do not mimic the natural ligand. We show, by a combination of cysteine cross‐linking, alanine‐scanning mutagenesis, and computational simulations, that the TpoR TM dimerizes strongly and can adopt 3 different stable, rotationally related conformations, which may correspond to specific states of the full‐length receptor (active, inactive, and partially active). Thus, our data suggest that signaling and inactive states of the receptor are related by receptor subunit rotations, rather than a simple monomer‐dimer transition. Moreover, results from experiments with and without agonists in vitro and in cells allow us to propose a novel allosteric mechanism of action for a class of small molecules, in which they activate TpoR by binding to the TM region and by exploiting the rotational states of the dimeric receptor. Overall, our results support the emerging view of the participation of mutual rotations of the TM domains in cytokine receptor activation.—Matthews, E. E., Thévenin, D., Rogers, J. M., Gotow, L., Lira, P. D., Reiter, L. A., Brissette, W. H., Engelman, D. M. Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation. FASEB J. 25, 2234–2244 (2011). www.fasebj.org

An MMP13-Selective Inhibitor Delays Primary Tumor Growth and the Onset of Tumor-Associated Osteolytic Lesions in Experimental Models of Breast Cancer

We investigated the effects of the matrix metalloproteinase 13 (MMP13)-selective inhibitor, 5-(4-{4-[4-(4-fluorophenyl)-1,3-oxazol-2-yl]phenoxy}phenoxy)-5-(2-methoxyethyl) pyrimidine-2,4,6(1H,3H,5H)-trione (Cmpd-1), on the primary tumor growth and breast cancer-associated bone remodeling using xenograft and syngeneic mouse models. We used human breast cancer MDA-MB-231 cells inoculated into the mammary fat pad and left ventricle of BALB/c Nu/Nu mice, respectively, and spontaneously metastasizing 4T1.2-Luc mouse mammary cells inoculated into mammary fat pad of BALB/c mice. In a prevention setting, treatment with Cmpd-1 markedly delayed the growth of primary tumors in both models, and reduced the onset and severity of osteolytic lesions in the MDA-MB-231 intracardiac model. Intervention treatment with Cmpd-1 on established MDA-MB-231 primary tumors also significantly inhibited subsequent growth. In contrast, no effects of Cmpd-1 were observed on soft organ metastatic burden following intracardiac or mammary fat pad inoculations of MDA-MB-231 and 4T1.2-Luc cells respectively. MMP13 immunostaining of clinical primary breast tumors and experimental mice tumors revealed intra-tumoral and stromal expression in most tumors, and vasculature expression in all. MMP13 was also detected in osteoblasts in clinical samples of breast-to-bone metastases. The data suggest that MMP13-selective inhibitors, which lack musculoskeletal side effects, may have therapeutic potential both in primary breast cancer and cancer-induced bone osteolysis.

Structure and Property Guided Design in the Identification of PRMT5 Tool Compound EPZ015666

The recent publication of a potent and selective inhibitor of protein methyltransferase 5 (PRMT5) provides the scientific community with in vivo-active tool compound EPZ015666 (GSK3235025) to probe the underlying pharmacology of this key enzyme. Herein, we report the design and optimization strategies employed on an initial hit compound with poor in vitro clearance to yield in vivo tool compound EPZ015666 and an additional potent in vitro tool molecule EPZ015866 (GSK3203591).

Difluoroketones as inhibitors of matrix metalloprotease-13

Substrate-like difluoroketones have been prepared as potential inhibitors of MMP-13. Weak inhibition was seen with the key target 2. This and the more potent activity of intermediate 7b illustrates that hydrated ketones can be used to inhibit MMP-13 and perhaps other members of this class of enzymes.

Molecular features crucial to the activity of pyrimidine benzamide-based thrombopoietin receptor agonists

The identification of small molecule modulators of biological processes mediated via protein-protein interactions has generally proved to be a challenging endeavor. In the case of the thrombopoietin receptor (TPOr), however, a number of small molecule types have been reported to display biological activity similar to that of the agonist protein TPO. Through a detailed analysis of structure-activity relationships, X-ray crystal structures, NMR coupling constants, nuclear Overhauser effects, and computational data, we have determined the agonism-inducing conformation of one series of small molecule TPOr agonists. The relationship of this agonism-inducing conformation to that of other series of TPO receptor agonists is discussed.

CP-66,948: An antisecretory histamine H2-receptor antagonist with mucosal protective properties

CP-66,948 is a histamine H2-receptor antagonist with gastric antisecretory activity and mucosal protective properties. The affinity of CP-66,948 for the guinea pig atria histamine H2-receptor is 15 times greater than that of cimetidine and seven times greater than that of ranitidine.In vivo, the ED50 value for inhibition of gastric acid secretion in pylorusligated rats is 2 mg/kg intraduodenally, and in histamine or pentagastrin-stimulated Heidenhain pouch dogs the antisecretory ED50 values are 0.3 mg/kgper os and 1.0 mg/kgper os, respectively. CP-66,948 also inhibits ethanol-induced gastric hemorrhagic lesions in rats following either oral or systemic administration (ED50 values of 12 mg/kgper os and 6 mg/kg subcutaneously). In addition, the mucosal protective activity is independent of prostaglandin synthesis. CP-66,948 inhibits gastric acid secretion in man, and its mucosal protective activity may provide additional benefits in peptic ulcer therapy.

A general synthesis of 4(5)-acylimidazoles from 4-acylaminoisoxazoles.

Abstract 2-Substituted-4(5)-acyl, 1,2-disubstituted 4-acyl and 1,2-disubstituted-5-acylimidazoles can be specifically prepared from 4-aminoisoxazoles.

Structure-activity relationships and hepatic safety risks of thiazole agonists of the thrombopoietin receptor.

5-F substitution of an aminothiazole moiety within a series of thrombopoietin receptor agonists leads to potent agents with an improved hepatic safety profile in rodent toxicology studies.

The synthesis of spirocyclic [1,2,4] triazolo [4,3-a] quinolines as potential ligands for the benzodiazepine receptor

Two spirocyclic [1,2,4] triazolo [4,3-a] quinolines (1a and b) have been prepared as potential ligands for the benzodiazepine receptor. Both compounds were prepared using the strategy of doubly deprotonating an acetanilide derivative followed by reaction with α-tetralone. The resulting alcohols were converted to the corresponding spirocyclic 3,4-dihydroquinolin-2-one or -2-thione by treatment with neat HF. Triazole formation was effected by standard procedures. An alternate strategy starting with an intact triazole was unsuccessful but this route led to the first example of a lateral metallation of a 4-substituted 1,2,4-triazole

The identification of orally bioavailable thrombopoietin agonists.

Recently, we disclosed a series of potent pyrimidine benzamide-based thrombopoietin receptor agonists. Unfortunately, the structural features required for the desired activity conferred physicochemical properties that were not favorable for the development of an oral agent. The physical properties of the series were improved by replacing the aminopyrimidinyl group with a piperidine-4-carboxylic acid moiety. The resulting compounds possessed favorable in vivo pharmacokinetic properties, including good bioavailability.