Optimization of peptidyl allyl sulfones as clan CA cysteine protease inhibitors

This research investigates the synthesis and inhibitory potency of a series of novel dipeptidyl allyl sulfones as clan CA cysteine protease inhibitors. The structure of the inhibitors consists of a R₁-Phe-R₂-AS-Ph scaffold (AS?=?allyl sulfone). R₁ was varied with benzyloxycarbonyl, morpholinocarbonyl, or N-methylpiperazinocarbonyl substituents. R₂ was varied with either Phe of Hfe residues. Synthesis involved preparation of vinyl sulfone analogues followed by isomerization to allyl sulfones using n-butyl lithium and t-butyl hydroperoxide. Sterics, temperature and base strength were all factors that affected the formation and stereochemistry of the allyl sulfone moiety. The inhibitors were assayed with three clan CA cysteine proteases (cruzain, cathepsin B and calpain I) as well as one serine protease (trypsin). The most potent inhibitor, (E)-Mu-Phe-Hfe-AS-Ph, displayed at least 10-fold selectivity for cruzain over clan CA cysteine proteases cathepsin B and calpain I with a k_obs/[I] of 6080?±?1390?M^?1s^?1.     

Dipeptidyl alpha-fluorovinyl Michael acceptors: synthesis and activity against cysteine proteases

The synthesis of novel dipeptidyl alpha-fluorovinyl sulfones using a Horner-Wadsworth-Emmons approach on N-Boc-l-phenylalaninal is described. Inhibitory assays against a Leishmania mexicana cysteine protease (CPB2.8DeltaCTE) revealed low biological activity. Relative rates of Michael additions of 2'-(phenethyl)thiol with vinyl sulfone and alpha-fluorovinyl sulfone were determined, and ab initio calculations on several Michael acceptor model structures were performed; both were in agreement with the biological testing results.     

Glyceraldehyde-3-phosphate dehydrogenase as a biochemical marker of cytotoxicity by vinyl sulfones in cultured murine spleen lymphocytes

Recently, vinyl sulfones have been observed to selectively inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an important ATP-generating enzyme in glycolysis. The possibility of using GAPDH as a biochemical parameter of cytotoxicity by vinyl sulfones was investigated using mouse lymphocytes. Incubation of lymphocyte GAPDH with ethylvinyl sulfone resulted in a pseudo-first-order loss of enzyme activity. The exposure of lymphocytes to ethylvinyl sulfone resulted in the decrease of GAPDH activity followed by ATP depletion and cell death, which were both dependent on the concentration of ethylvinyl sulfone. A further study on the time-dependent change indicated that cell death was preceded by ATP loss. Compared to ethylvinyl sulfone, divinyl sulfone was more than 8 times more potent in causing either ATP depletion or cell death.Abbreviations DTT dithiothreitol - GAPDH glyceraldehyde-3-phosphate dehydrogenase - NAD nicotinamide adenine dinucleotide     

Design and evaluation of inhibitors for dipeptidyl peptidase I (Cathepsin C)

Dipeptidyl peptidase I (DPPI, cathepsin C) is a lysosomal cysteine protease that can activate zymogens of several different serine proteases by one step or sequential removal of dipeptides from the N-termini of the pro-protease protein substrates. To find DPPI inhibitors more suitable for cellular applications than diazomethyl ketones, we synthesized three types of inhibitors: dipeptide acyloxymethyl ketones, fluoromethyl ketones, and vinyl sulfones (VS). The acyloxymethyl ketones inhibited DPPI slowly and are moderate inhibitors of cellular DPPI. The fluoromethyl ketones were potent, but the inhibited DPPI regained activity quickly. The dipeptide vinyl sulfones were effective inhibitors for DPPI, but they also inhibited cathepsins B, H, and L weakly. The best inhibitor, Ala-Hph-VS-Ph, had a k2/K(I) of 2,000,000M(-1)s(-1). The vinyl sulfones also inhibited intracellular DPPI, and for this application the more stable inhibitors exhibit better potency. We conclude that vinyl sulfones are promising inhibitors to study the intracellular functions of DPPI.     

Structural basis for inhibition of cathepsin B drug target from the human blood fluke, Schistosoma mansoni

Schistosomiasis caused by a parasitic blood fluke of the genus Schistosoma afflicts over 200 million people worldwide. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated peptidase that digests host blood proteins as a source of nutrients. It is under investigation as a drug target. To further this goal, we report three crystal structures of SmCB1 complexed with peptidomimetic inhibitors as follows: the epoxide CA074 at 1.3 Å resolution and the vinyl sulfones K11017 and K11777 at 1.8 and 2.5 Å resolutions, respectively. Interactions of the inhibitors with the subsites of the active-site cleft were evaluated by quantum chemical calculations. These data and inhibition profiling with a panel of vinyl sulfone derivatives identify key binding interactions and provide insight into the specificity of SmCB1 inhibition. Furthermore, hydrolysis profiling of SmCB1 using synthetic peptides and the natural substrate hemoglobin revealed that carboxydipeptidase activity predominates over endopeptidolysis, thereby demonstrating the contribution of the occluding loop that restricts access to the active-site cleft. Critically, the severity of phenotypes induced in the parasite by vinyl sulfone inhibitors correlated with enzyme inhibition, providing support that SmCB1 is a valuable drug target. The present structure and inhibitor interaction data provide a footing for the rational design of anti-schistosomal inhibitors.     

Triply-convergent synthesis of a homochiral 3,3-dimethyl, 15-cyclohexyl prostacyclin analog

Allylic bromocuprate reagent undergoes synfacial SN2′ addition to homochiral allyl ammonium salt to provide vinyl sulfone as a single stereoisomer. Addition of homochiral acetylenic anion to vinyl sulfone smoothly provides the bicyclic sulfone which is further transformed to prostacyclin analog . Analog was only a weak inhibitor of platelet aggregation having an IC₅₀ of 0.48 μM.     

Antimalarial effects in mice of orally administered peptidyl cysteine protease inhibitors.

The Plasmodium falciparum cysteine protease falcipain is required for the degradation of hemoglobin by erythrocytic malaria parasites. In prior studies, peptidyl inhibitors of falcipain blocked hemoglobin degradation and development by cultured parasites and one of these compounds, when administered parenterally, cured Plasmodium vinckei-infected mice. We now report an evaluation of orally administered peptidyl inhibitors of falcipain in a mouse malaria model. In studies with a fluoromethyl ketone, orally administered morpholine urea-phenylalanine-homophenylalanine-fluoromethyl ketone delayed the progression of murine malaria. In studies of a new series of vinyl sulfones, a set of related compounds demonstrated marked inhibition of falcipain and of parasite biological activities in vitro. One of these compounds, N-methyl piperazine urea-leucine-homophenylalanine-2-naphthalene vinyl sulfone, cured about 40% of mice when administered orally twice-a-day for four days. Our results suggest that peptidyl inhibitors of falcipain have promise as antimalarial chemotherapeutic agents.     

Fasciola gigantica: evaluation of the effect of phenyl vinyl sulfone in vitro

Phenyl vinyl sulfone is a synthetic inhibitor of cysteine protease and has antihelminthic and antiprotozoal properties. Phenyl vinyl sulfone was assayed in vitro for antifasciola activity against adult Fasciola gigantica worms using a well-established culture medium. Worms were treated with phenyl vinyl sulfone for incubation periods ranging from 0 to 12h and its activity was assessed in terms of viability, motility and death of worms. Phenyl vinyl sulfone exhibited a minimum effective concentration of 50 ppm after 12h. Three hundred parts per million concentrations were most potent causing immediate death of adult flukes in vitro. Histopathological studies showed that there was tegumental flattening, rupture of vesicles, and spine loss. Marked reduction in size and number of ova and sperms in the convoluted tubules of the reproductive organs was observed in comparison to the untreated control group. In conclusion, phenyl vinyl sulfone shows potent activity against F. gigantica in vitro, and the authors recommend carrying out more studies to detect its efficacy in vivo.     

Localization of rat cathepsin K in osteoclasts and resorption pits: inhibition of bone resorption and cathepsin K-activity by peptidyl vinyl sulfones.

We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.     

Allyl and propargyl substituted penam sulfones as versatile intermediates toward the syntheses of new beta-lactamase inhibitors

Several alkenyl derivatives were prepared using allyl penam sulfone as the key intermediate. Isomers of these derivatives having beta configuration at C-6 showed potent activity against CcrA enzyme. A new method was developed to prepare propargyl penam sulfone. The majority of the triazoles prepared by this route exhibited good activity against all three representative enzymes used for the inhibition assay.     

Synthesis and β-lactamase inhibitory activity of 6-fluoropenicillanic acids

The benzyl 6-fluoro-penicillanate sulfides 4a, 6a, 7a; and sulfones 6c, 7d were synthesized. The conversion to their free acids 4b, 4b, 6d, 7b, 7e and potassium salts 7c, 7f are described. These acids and salt 7c were evaluated as β- lactamase inhibitors using β-lactamase I from Bacillus cereus. The data indicate that substitution of the 6-hydrogen by a 6- fluorine atom on 6β-bromopenicillanic acid (1), leads to loss of β-lactamase inhibitory activity. In the case of the isomers 6β- and 6-fluoropenicillanic acids the 6β-enantiomer proved to be considerably more potent. Potassium salts of 6β- fluoropenicillanate sulfide and sulfone were unstable in solid state and in water solution. The fragmentation of the sulfone in two parts in water solution is consistent with the hydrolytic behavior of the penicillanic acid sulfone (2) with 0.5 N NaOH.     

Inhibitors of the eukaryotic 20S proteasome core particle: a structural approach

The ubiquitin-proteasome pathway is particularly important for the regulated degradation of various proteins which control a vast array of biological processes. Therefore, proteasome inhibitors are promising candidates for anti-tumoral or anti-inflammatory drugs. N-Acetyl-Leu-Leu-Norleucinal (Ac-LLN-al, also termed calpain inhibitor I) was one of the first proteasome inhibitors discovered and has been widely used to study the 20S proteasome core particle (CP) function in vivo, despite its lack of specificity. Vinyl sulfones, like Ac-PRLN-vs, show covalent binding of the beta-carbon atom of the vinyl sulfone group to the Thr1Ogamma only of subunit beta2. However, vinyl sulfones have similar limitations as peptide aldehydes as they have been reported also to bind and block intracellular cysteine proteases. A more specific proteasome inhibitor is the natural product lactacystin, which can be isolated from Streptomyces. It was found that this compound forms an ester bond only to the Thr1Ogamma of the chymotrypsin-like active subunit beta5 due to specific P1 interactions. In contrast to most other proteasome inhibitors, the natural alpha',beta'-epoxyketone peptide epoxomicin binds specifically to the small class of N-terminal nucleophilic (Ntn) hydrolases (CPs belong to this protease family) with the formation of a morpholino adduct. All previously described proteasome inhibitors bind covalently to the proteolytic active sites. However, as the proteasome is involved in a variety of biological important functions, it is of particular interest to block the CP only for limited time in order to reduce cytotoxic effects. Recently, the binding mode of the natural specific proteasome inhibitor TMC-95 obtained from Apiospora montagnei was investigated. The crystal structure revealed that the TMC-95 blocks the active sites of the CP noncovalently in the low nanomolar range. This review summarizes the current structural knowledge of inhibitory compounds bound to the CP, showing the proteasome as a potential target for drug development in medical research.     

Biodegradation of benzothiophene sulfones by a filamentous bacterium.

Previous studies showed that benzothiophene and 3- and 5-methylbenzothiophenes are oxidized by some bacteria to yield their corresponding sulfones, which were not subsequently degraded. In this study, a filamentous bacterium was isolated, which grew on each of these three sulfones as its sole carbon, sulfur, and energy source. Based on 16S rRNA gene sequencing and scanning electron microscopy, the isolate was found to belong to the genus Pseudonocardia and assigned the strain designation DB1. Benzothiophene sulfone and 3-methylbenzothiophene sulfone were more readily biodegraded than 5-methylbenzothiophene sulfone, and growth on these three compounds resulted in the release of 57, 62, and 28% of the substrate carbon as CO2, respectively. The thiophene ring was also cleaved, and between 44 and 88% of the sulfur from the consumed substrate was found as sulfate and (or) sulfite. Strain DB1 grew on benzoate, dibenzothiophene sulfone, and hexadecanoic acid, but it could not grow on benzofuran, dibenzothiophene, dibenzothiophene sulfoxide, hexadecane, indole, naphthalene, phenol, 2-sulfobenzoic acid, sulfolane, benzothiophene, or 3- or 5-methylbenzothiophenes. In addition, it did not oxidize the latter three compounds to their sulfones.     

Effects of structural modification on the DNA binding properties and photo-induced cleavage reactivity of propargylic sulfones conjugated with an anthraquinone structure

Propargylic sulfones are known as pH-dependent DNA cleaving agents. We have designed a novel propargylic sulfone conjugated with an anthraquinone structure and evaluated its DNA binding and cleavage characteristics. The propargylic sulfone 3 showed high intercalating ability attributable to anthraquinone chromophore, leading to the efficient alkylation of DNA. The anthraquinone chromophore in 3 also acted as a photosensitizer, and photoirradiation of 3 with DNA induced one-electron oxidation, resulting in the further DNA cleavage. Evaluation of the effect of 3 against EMT6/KU cells revealed that 3 exhibited potent cytotoxicity, even without photoirradiation.     

Modifications of the C6-substituent of penicillin sulfones with the goal of improving inhibitor recognition and efficacy

In order to evaluate the importance of a hydrogen-bond donating substituent in the design of β-lactamase inhibitors, a series of C6-substituted penicillin sulfones, lacking a C2′ substituent, and having an sp³ hybridized C6, was prepared and evaluated against a representative classes A and C β-lactamases. It was found that a C6 hydrogen-bond donor is necessary for good inhibitory activity, but that this feature alone is not sufficient in this series of C6β-substituted penicillin sulfones. Other factors which may impact the potency of the inhibitor include the steric bulk of the C6 substituent (e.g., methicillin sulfone) which may hinder recognition in the class A β-lactamases, and also high similarity to the natural substrates (e.g., penicillin G sulfone) which may render the prospective inhibitor a good substrate of both classes of enzyme. The best inhibitors had non-directional hydrogen-bonding substituents, such as hydroxymethyl, which may allow sufficient conformational flexibility of the acyl-enzyme for abstraction of the C6 proton by E166 (class A), thus promoting isomerization to the β-aminoacrylate as a stabilized acyl-enzyme.     

The enhancement of PCR amplification by low molecular-weight sulfones.

DNA amplification by polymerase chain reaction (PCR) is frequently complicated by the problems of low yield and specificity, especially when the GC content of the target sequence is high. A common approach to the optimization of such reactions is the addition of small quantities of certain organic chemicals, such as dimethylsulfoxide (DMSO), betaine, polyethylene glycol and formamide, to the reaction mixture. Even in the presence of such additives, however, the amplification of GC-rich templates is often ineffective. In this paper, we introduce a novel class of PCR-enhancing compounds, the low molecular-weight sulfones, that are effective in the optimization of high GC template amplification. We describe here the results of an extensive structure-activity investigation in which we studied the effects of a series of six different sulfones on PCR amplification. We identify two sulfones, sulfolane and methyl sulfone, that are especially potent enhancers of high GC template amplification, and show that these compounds often outperform DMSO and betaine, two of the most effective PCR enhancers currently used. We conclude with a brief discussion of the role that the sulfone functional group may play in such enhancement.     

Highly tunable thiosulfonates as a novel class of cysteine protease inhibitors with anti-parasitic activity against Schistosoma mansoni

The development of a new class of cysteine protease inhibitors utilising the thiosulfonate moiety as an SH specific electrophile is described. This moiety has been introduced into suitable amino acid derived building blocks, which were incorporated into peptidic sequences leading to very potent i.e. sub micromolar inhibitors of the cysteine protease papain in the same range as the vinyl sulfone based inhibitor K11777. Therefore, their inhibitory effect on Schistosoma mansoni, a human blood parasite, that expresses several cysteine proteases, was evaluated. The homophenylalanine side chain containing compounds 27–30 and especially 36 showed promising activities compared with K11777 and warrant further investigations of these peptidic thiosulfonate inhibitors as new potential anti-parasitic compounds.     

Kojyl thioether derivatives having both tyrosinase inhibitory and anti-inflammatory properties

This study was conducted to examine the tyrosinase inhibitory and anti-inflammatory activities of kojic acid derivatives. A series of kojic acid derivatives containing thioether, sulfoxide, and sulfone linkages were synthesized. In the tyrosinase assay, kojyl thioether derivatives containing appropriate lipophilic alkyl chains (pentane, hexane, and cyclohexane) showed potent inhibitory activity. However, sulfoxides and sulfones exhibited decreased activity. Similar experimental results were obtained with inhibitory activities of NO production being induced by LPS. The presence of thioether linkage and appropriated lipophilic acid moiety was critical for the tyrosinase inhibitory and anti-inflammatory activities.