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.

Optimization of Substrate-Analogue Furin Inhibitors.

The proprotein convertase furin is a potential target for drug design, especially for the inhibition of furin-dependent virus replication. All effective synthetic furin inhibitors identified thus far are multibasic compounds; the highest potency was found for our previously developed inhibitor 4-(guanidinomethyl)phenylacetyl-Arg-Tle-Arg-4-amidinobenzylamide (MI-1148). An initial study in mice revealed a narrow therapeutic range for this tetrabasic compound, while significantly reduced toxicity was observed for some tribasic analogues. This suggests that the toxicity depends at least to some extent on the overall multibasic character of this inhibitor. Therefore, in a first approach, the C-terminal benzamidine of MI-1148 was replaced by less basic P1 residues. Despite decreased potency, a few compounds still inhibit furin in the low nanomolar range, but display negligible efficacy in cells. In a second approach, the P2 arginine was replaced by lysine; compared to MI-1148, this furin inhibitor has slightly decreased potency, but exhibits similar antiviral activity against West Nile and Dengue virus in cell culture and decreased toxicity in mice. These results provide a promising starting point for the development of efficacious and well-tolerated furin inhibitors.

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.

Small-molecule inhibitors of lethal factor protease activity protect against anthrax infection.

Bacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.

Virtual medicinal chemistry: in silico pre-docking functional group transformation for discovery of novel inhibitors of botulinum toxin serotype A light chain.

A novel method for applying high-throughput docking to challenging metalloenzyme targets is described. The method utilizes information-based virtual transformation of library carboxylates to hydroxamic acids prior to docking, followed by compound acquisition, one-pot (two steps) chemical synthesis and in vitro screening. In two experiments targeting the botulinum neurotoxin serotype A metalloprotease light chain, hit rates of 32% and 18% were observed.

Antidotes to anthrax lethal factor intoxication. Part 3: Evaluation of core structures and further modifications to the C2-side chain.

Four core structures capable of providing sub-nanomolar inhibitors of anthrax lethal factor (LF) were evaluated by comparing the potential for toxicity, physicochemical properties, in vitro ADME profiles, and relative efficacy in a rat lethal toxin (LT) model of LF intoxication. Poor efficacy in the rat LT model exhibited by the phenoxyacetic acid series (3) correlated with low rat microsome and plasma stability. Specific molecular interactions contributing to the high affinity of inhibitors with a secondary amine in the C2-side chain were revealed by X-ray crystallography.

Inhibition of prohormone convertases PC1/3 and PC2 by 2,5-dideoxystreptamine derivatives.

The prohormone convertases PC1/3 and PC2 are eukaryotic serine proteases involved in the proteolytic maturation of peptide hormone precursors and are implicated in a variety of pathological conditions, including obesity, diabetes, and neurodegenerative diseases. In this work, we screened 45 compounds obtained by derivatization of a 2,5-dideoxystreptamine scaffold with guanidinyl and aryl substitutions for convertase inhibition. We identified four promising PC1/3 competitive inhibitors and three PC2 inhibitors that exhibited various inhibition mechanisms (competitive, noncompetitive, and mixed), with sub- and low micromolar inhibitory potency against a fluorogenic substrate. Low micromolar concentrations of certain compounds blocked the processing of the physiological substrate proglucagon. The best PC2 inhibitor effectively inhibited glucagon synthesis, a known PC2-mediated process, in a pancreatic cell line; no cytotoxicity was observed. We also identified compounds that were able to stimulate both 87 kDa PC1/3 and PC2 activity, behavior related to the presence of aryl groups on the dideoxystreptamine scaffold. By contrast, inhibitory activity was associated with the presence of guanidinyl groups. Molecular modeling revealed interactions of the PC1/3 inhibitors with the active site that suggest structural modifications to further enhance potency. In support of kinetic data suggesting that PC2 inhibition probably occurs via an allosteric mechanism, we identified several possible allosteric binding sites using computational searches. It is noteworthy that one compound was found to both inhibit PC2 and stimulate PC1/3. Because glucagon acts in functional opposition to insulin in blood glucose homeostasis, blocking glucagon formation and enhancing proinsulin cleavage with a single compound could represent an attractive therapeutic approach in diabetes.

Small molecule pan-dengue and West Nile virus NS3 protease inhibitors.

BACKGROUND: Dengue fever, dengue haemorrhagic fever, and dengue shock syndrome are caused by infections with any of the four serotypes of the dengue virus (DENV), and are an increasing global health risk. The related West Nile virus (WNV) causes significant morbidity and mortality as well, and continues to be a threat in endemic areas. Currently no FDA-approved vaccines or therapeutics are available to prevent or treat any of these infections. Like the other members of Flaviviridae, DENV and WNV encode a protease (NS3) which is essential for viral replication and therefore is a promising target for developing therapies to treat dengue and West Nile infections. METHODS: Flaviviral protease inhibitors were identified and biologically characterized for mechanism of inhibition and DENV antiviral activity. RESULTS: A guanidinylated 2,5-dideoxystreptamine class of compounds was identified that competitively inhibited the NS3 protease from DENV(1-4) and WNV with 50% inhibitory concentration values in the 1-70 µM range. Cytotoxicity was low; however, antiviral activity versus DENV-2 on VERO cells was not detectable. CONCLUSIONS: This class of compounds is the first to demonstrate competitive pan-dengue and WNV NS3 protease inhibition and, given the sequence conservation among flavivirus NS3 proteins, suggests that developing a pan-dengue or possibly pan-flavivirus therapeutic is feasible.

Structural characterization of three novel hydroxamate-based zinc chelating inhibitors of the Clostridium botulinum serotype A neurotoxin light chain metalloprotease reveals a compact binding site resulting from 60/70 loop flexibility.

Neurotoxins synthesized by Clostridium botulinum bacteria (BoNT), the etiological agent of human botulism, are extremely toxic proteins making them high-risk agents for bioterrorism. Small molecule inhibitor development has been focused on the light chain zinc-dependent metalloprotease domain of the neurotoxin, an effort that has been hampered by its relatively flexible active site. Developed in concert with structure--activity relationship studies, the X-ray crystal structures of the complex of BoNT serotype A light chain (BoNT/A LC) with three different micromolar-potency hydroxamate-based inhibitors are reported here. Comparison with an unliganded BoNT/A LC structure reveals significant changes in the active site as a result of binding by the unique inhibitor scaffolds. The 60/70 loop at the opening of the active site pocket undergoes the largest conformational change, presumably through an induced-fit mechanism, resulting in the most compact catalytic pocket observed in all known BoNT/A LC structures.

Antidotes to anthrax lethal factor intoxication. Part 2: structural modifications leading to improved in vivo efficacy.

New anthrax lethal factor inhibitors (LFIs) were designed based upon previously identified potent inhibitors 1a and 2. Combining the new core structures with modifications to the C2-side chain yielded analogs with improved efficacy in the rat lethal toxin model.

Antidotes to anthrax lethal factor intoxication. Part 1: Discovery of potent lethal factor inhibitors with in vivo efficacy.

Sub-nanomolar small molecule inhibitors of anthrax lethal factor have been identified using SAR and Merck L915 (4) as a model compound. One of these compounds (16) provided 100% protection in a rat lethal toxin model of anthrax disease.

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.

Syntheses, photophysical properties, and application of through-bond energy-transfer cassettes for biotechnology.

We have designed fluorescent "through-bond energy-transfer cassettes" that can harvest energy of a relatively short wavelength (e.g., 490 nm), and emit it at appreciably longer wavelengths without significant loss of intensity. Probes of this type could be particularly useful in biotechnology for multiplexing experiments in which several different outputs are to be observed from a single excitation source. Cassettes 1-4 were designed, prepared, and studied as model systems to achieve this end. They were synthesized through convergent routes that feature coupling of specially prepared fluorescein- and rhodamine-derived fragments. The four cassettes were shown to emit strongly, with highly efficient energy transfer. Their emission maxima cover a broad range of wavelengths (broader than the four dye cassettes currently used for most high-throughput DNA sequencing), and they exhibit faster energy-transfer rates than a similar through-space energy-transfer cassette. Specifically, energy-transfer rates in these cassettes is around 6-7 ps, in contrast to a similar through-space energy-transfer system shown to have a decay time of around 35 ps. Moreover, the cassettes are considerably more stable to photobleaching than fluorescein, even though they each contain fluorescein-derived donors. This was confirmed by bulk fluorescent measurements, and in single-molecule-detection studies. Modification of a commercial automated DNA-sequencing apparatus to detect the emissions of these four energy-transfer cassettes enabled single-color dye-primer sequencing.

Selectively guanidinylated derivatives of neamine. Syntheses and inhibition of anthrax lethal factor protease.

A series of mono-, di-, and tri-guanidinylated derivatives of neamine were prepared via selective guanidinylation of neamine. These molecules represent a novel scaffold as inhibitors of anthrax lethal factor zinc metalloprotease. Methods for the synthesis of these compounds are described, and structure-activity relationships among the series are analyzed. In addition, initial findings regarding the mechanism of LF inhibition for these molecules are presented.

Guanidinylated 2,5-dideoxystreptamine derivatives as anthrax lethal factor inhibitors.

Anthrax lethal factor is a Zn(2+)-dependent metalloprotease and the key virulence factor of tripartite anthrax toxin secreted by Bacillus anthracis, the causative agent of anthrax. A series of guanidinylated 2,5-dideoxystreptamine derivatives were designed and synthesized as inhibitors of lethal factor, some of which show strong inhibitory activity against lethal factor in an in vitro FRET assay. Preparation and structure-activity relationships of these compounds are presented.

On the stability of furanopyrimidin-2-one bases in oligonucleotides.

The fluoresceinated furanopyrimidin-2-one nucleobase incorporated into an oligonucleotide undergoes unexpectedly facile hydrolytic ring-opening in aqueous buffer at slightly elevated temperatures.

A blue-to-red energy-transfer thymidine analogue that functions in DNA.

The thymidine analogue 1 is a blue-to-red energy transfer cassette designed to absorb UV light in the 300 nm region and emit it as fluorescence at around 520 nm. When incorporated into DNA, the fluorescence intensity of this modified nucleobase is not significantly reduced by the surrounding structure in the oligonucleotides. [structure--see text]