Small molecule inhibitors of anthrax edema factor.

Anthrax is a highly lethal disease caused by the Gram-(+) bacteria Bacillus anthracis. Edema toxin (ET) is a major contributor to the pathogenesis of disease in humans exposed to B. anthracis. ET is a bipartite toxin composed of two proteins secreted by the vegetative bacteria, edema factor (EF) and protective antigen (PA). Our work towards identifying a small molecule inhibitor of anthrax edema factor is the subject of this letter. First we demonstrate that the small molecule probe 5'-Fluorosulfonylbenzoyl 5'-adenosine (FSBA) reacts irreversibly with EF and blocks enzymatic activity. We then show that the adenosine portion of FSBA can be replaced to provide more drug-like molecules which are up to 1000-fold more potent against EF relative to FSBA, display low cross reactivity when tested against a panel of kinases, and are nanomolar inhibitors of EF in a cell-based assay of cAMP production.

Structural Studies Revealed Active Site Distortions of Human Furin by a Small Molecule Inhibitor.

Proprotein convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of several pathologies including cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. Here, we present the first experimental complex of furin with a non-substrate-like small molecule inhibitor, and the X-ray structure of the enzyme complexed to the small molecule inhibitor 1 at 1.9 Å resolution. Two molecules of inhibitor 1 were found to interact with furin. One is anchored at the S4 pocket of the enzyme and interferes directly with the conformation and function of the catalytic triade; the other molecule shows weaker binding and interacts with a distant, less conserved region of furin. The observed binding modes represent a new inhibition strategy of furin and imply the possibility to attain specificity among the PCs providing an innovative starting point of structure guided inhibitor development for furin.

Identification of potent and compartment-selective small molecule furin inhibitors using cell-based assays.

The proprotein convertase furin is implicated in a variety of pathogenic processes such as bacterial toxin activation, viral propagation, and cancer. Several groups have identified non-peptide compounds with high inhibitory potency against furin in vitro, although their efficacy in various cell-based assays is largely unknown. In this study we show that certain guanidinylated 2,5-dideoxystreptamine derivatives exhibit interesting ex vivo properties. Compound 1b (1,1'-(4-((2,4-diguanidino-5-(4-guanidinophenoxy)cyclohexyl)oxy)-1,3-phenylene)diguanidine) is a potent and cell-permeable inhibitor of cellular furin, since it was able to retard tumor cell migration, block release of a Golgi reporter, and protect cells against Bacillus anthracis (anthrax) and Pseudomonas aeruginosa intoxication, with no evident cell toxicity. Other compounds based on the 2,5-dideoxystreptamine scaffold, such as compound 1g (1,1'-(4,6-bis(4-guanidinophenoxy)cyclohexane-1,3-diyl)diguanidine) also efficiently protected cells against anthrax, but displayed only moderate protection against Pseudomonas exotoxin A and did not inhibit cell migration, suggesting poor cell permeability. Certain bis-guanidinophenyl ether derivatives such as 2f (1,3-bis(2,4-diguanidinophenoxy) benzene) exhibited micromolar potency against furin in vitro, low cell toxicity, and highly efficient protection against anthrax toxin; this compound only slightly inhibited intracellular furin. Thus, compounds 1g and 2f both represent potent furin inhibitors at the cell surface with low intracellular inhibitory action, and these particular compounds might therefore be of preferred therapeutic interest in the treatment of certain bacterial and viral infections.

Small molecule proprotein convertase inhibitors for inhibition of embryo implantation.

Uterine proprotein convertase (PC) 6 plays a critical role in embryo implantation and is pivotal for pregnancy establishment. Inhibition of PC6 may provide a novel approach for the development of non-hormonal and female-controlled contraceptives. We investigated a class of five synthetic non-peptidic small molecule compounds that were previously reported as potent inhibitors of furin, another PC member. We examined (i) the potency of these compounds in inhibiting PC6 activity in vitro; (ii) their binding modes in the PC6 active site in silico; (iii) their efficacy in inhibiting PC6-dependent cellular processes essential for embryo implantation using human cell-based models. All five compounds showed potent inhibition of PC6 activity in vitro, and in silico docking demonstrated that these inhibitors could adopt a similar binding mode in the PC6 active site. However, when these compounds were tested for their inhibition of decidualization of primary human endometrial stromal cells, a PC6-dependent cellular process critical for embryo implantation, only one (compound 1o) showed potent inhibition. The lack of activity in the cell-based assay may reflect the inability of the compounds to penetrate the cell membrane. Because compound's lipophilicity is linked to cell penetration, a measurement of lipophilicity (logP) was calculated for each compound. Compound 1o is unique as it appears the most lipophilic among the five compounds. Compound 1o also inhibited another crucial PC6-dependent process, the attachment of human trophoblast spheroids to endometrial epithelial cells (a model for human embryo attachment). We thus identified compound 1o as a potent small molecule PC6 inhibitor with pharmaceutical potential to inhibit embryo implantation. Our findings also highlight that human cell-based functional models are vital to complement the biochemical and in silico analyses in the selection of promising drug candidates. Further investigations for compound 1o are warranted in animal models to test its utility as an implantation-inhibiting contraceptive drug.

Synthetic small molecule furin inhibitors derived from 2,5-dideoxystreptamine.

Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of furin may provide a feasible and promising approach for therapeutic intervention of furin-mediated disease mechanisms. Here, we report on a class of small molecule furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addition of guanidinylated aryl groups yielded a set of furin inhibitors with nanomolar range potency against furin when assayed in a biochemical cleavage assay. Moreover, a subset of these furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of furin and to be relatively specific for furin. Molecular modeling revealed that these inhibitors may target the active site of furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-Lys-Arg-CH(2)Cl. The compounds presented here are bona fide synthetic small molecule furin inhibitors that exhibit potency in the nanomolar range, suggesting that they may serve as valuable tools for studying furin action and potential therapeutics agents for furin-dependent diseases.