1,2,5-Thiadiazolidin-3-one 1,1 dioxide: a powerful scaffold for probing the S' subsites of (chymo)trypsin-like serine proteases

The 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold (I) embodies a motif that allows it to dock to the active site of (chymo)trypsin-like proteases in a predictable and substrate-like fashion. Consequently, inhibitors derived from this heterocyclic scaffold interact with both the S and S' subsites of an enzyme. Exploitation of binding interactions with both the S and S' subsites of a target enzyme may lead to compounds with greatly enhanced enzyme selectivity and inhibitory potency. This preliminary report describes the use of a series of compounds having the heterocyclic scaffold linked to various amino acids to probe the S' subsites of human leukocyte elastase (HLE), proteinase 3 (PR 3), and cathepsin G (Cat G). For comparative purposes, a series of compounds derived from a related scaffold, isothiazolidin-3-one 1,1 dioxide (II), was also generated. Several of the compounds were found to be highly potent and selective time-dependent inhibitors of HLE, PR 3, and Cat G.     

Inhibition of serine proteases by functionalized sulfonamides coupled to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold.

A challenge associated with drug design is the development of selective inhibitors of proteases (serine or cysteine) that exhibit the same primary substrate specificity, that is, show a preference for the same P(1) residue. While these proteases have similar active sites, nevertheless there are subtle differences in their S and S' subsites which can be exploited. We describe herein for the first time the use of functionalized sulfonamides as a design and diversity element which, when coupled to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold yields potent, time-dependent inhibitors of the serine proteases human leukocyte elastase (HLE), proteinase 3 (PR 3) and cathepsin G(Cat G). Our preliminary findings suggest that (a) appending to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold recognition and diversity elements that interact with both the S and S' subsites of a target protease may result in optimal enzyme selectivity and potency and, (b) functionalized sulfonamides constitute a powerful design and diversity element with low intrinsic chemical reactivity and potentially wide applicability.     

Potent inhibition of human leukocyte elastase by 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamide derivatives

The design, synthesis, and in vitro biochemical evaluation of a class of mechanism-based inhibitors of human leukocyte elastase (HLE) that incorporate in their structure a 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold with appropriate recognition and reactivity elements appended to it is described. The synthesized compounds were found to be efficient, time-dependent inhibitors of HLE. The interaction of the inhibitors with HLE is postulated to lead to the formation of a highly reactive N-sulfonyl imine (a Michael acceptor) that arises from an enzyme-induced sulfonamide fragmentation cascade. Subsequent reaction ultimately leads to the formation of a relatively stable acyl enzyme. The results cited herein demonstrate convincingly the superiority of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold over other scaffolds (e.g., saccharin) in the design of inhibitors of (chymo)trypsin-like serine proteases.     

Utilization of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold in the design of potent inhibitors of serine proteases: SAR studies using carboxylates

A series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazolidin-3-one 1,1 dioxide scaffolds has been synthesized and the inhibitory profile of these compounds toward human leukocyte elastase (HLE), cathepsin G (Cat G) and proteinase 3 (PR 3) was then determined. Most of the compounds were found to be potent, time-dependent inhibitors of elastase, with some of the compounds exhibiting k(inact)/K1 values as high as 4,928,300 M(-1) s(-1). The inhibitory potency of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide platform was found to be influenced by both the pKa and the inherent structure of the leaving group. Proper selection of the primary specificity group (R(I)) was found to lead to selective inhibition of HLE over Cat G, however, those compounds that inhibited HLE also inhibited PR 3, albeit less efficiently. The predictable mode of binding of these compounds suggests that, among closely-related serine proteases, highly selective inhibitors of a particular serine protease can be fashioned by exploiting subtle differences in their S' subsites. This study has also demonstrated that the degradative action of elastase on elastin can be abrogated in the presence of inhibitor 17.     

1,2,5-Thiadiazolidin-3-one 1,1-dioxide-based heterocyclic sulfides are potent inhibitors of human tryptase

We describe herein the design, synthesis, and in vitro biochemical evaluation of a series of potent, time-dependent inhibitors of the mast cell-derived serine protease tryptase. The inhibitors were readily obtained by attaching various heterocyclic thiols, as well as a basic primary specificity residue P₁, to the 1,2,5-thiadiazolidin-3-one 1,1-dioxide scaffold. The inhibitors were found to be devoid of any inhibitory activity toward a neutral (elastase) or cysteine (papain) protease, however they were also fairly efficient inhibitors of bovine trypsin. The differential inhibition observed with trypsin suggests that enzyme selectivity can be optimized by exploiting differences in the S′ subsites of the two enzymes. The results described herein demonstrate the versatility of the heterocyclic scaffold in fashioning mechanism-based inhibitors of neutral, basic, and acidic (chymo)trypsin-like serine proteases.     

Inactivation of human neutrophil elastase by 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamides

The interaction of a series of 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamides with neutrophil-derived serine proteases was investigated. The nature of the amino acid component, believed to be oriented toward the S' subsites, had a profound effect on enzyme selectivity. This series of compounds were found to be potent, time-dependent inhibitors of human neutrophil elastase (HNE) and were devoid of any inhibitory activity toward neutrophil proteinase 3 (PR 3) and cathepsin G (Cat G). The results of these studies demonstrate that exploitation of differences in the S' subsites of HNE and PR 3 can lead to highly selective inhibitors of HNE.     

Design, synthesis, and in vitro evaluation of inhibitors of human leukocyte elastase based on a functionalized cyclic sulfamide scaffold

The design of novel functionalized templates capable of binding to the active site of serine proteases could potentially lead to the development of potent and highly selective non-covalent inhibitors of these enzymes. Using the elastase-turkey ovomucoid inhibitor complex and insights gained from earlier work based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold (I), a surrogate cyclosulfamide scaffold (II) was used for the first time in the design of reversible inhibitors of human leukocyte elastase. Compounds 7 and 8 were found to be micromolar reversible inhibitors of the enzyme.     

Human chymase inhibitors based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold.

A series of compounds that utilize the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold was synthesized and shown to be highly effective inhibitors of recombinant human skin chymase.     

Mechanism-based inactivation of human leukocyte elastase via an enzyme-induced sulfonamide fragmentation process

We describe herein the design and in vitro biochemical evaluation of a novel class of mechanism-based inhibitors of human leukocyte elastase (HLE) that inactivate the enzyme via an unprecedented enzyme-induced sulfonamide fragmentation cascade. The inhibitors incorporate in their structure an appropriately functionalized saccharin scaffold. Furthermore, the inactivation of the enzyme by these inhibitors was found to be time-dependent and to involve the active site. Biochemical, HPLC, and mass spectrometric studies show that the interaction of these inhibitors with HLE results in the formation of a stable acyl complex and is accompanied by the release of (L) phenylalanine methyl ester. The data are consistent with initial formation of a Michaelis-Menten complex and subsequent formation of a tetrahedral intermediate with the active site serine (Ser(195)). Collapse of the tetrahedral intermediate with tandem fragmentation results in the formation of a highly reactive conjugated sulfonyl imine which can either react with water to form a stable acyl enzyme and/or undergo a Michael addition reaction with an active site nucleophilic residue (His(57)). It is also demonstrated herein that this class of compounds can be used in the design of inhibitors of serine proteases having either a neutral or basic primary substrate specificity. Thus, the results suggest that these inhibitors constitute a potential general class of mechanism-based inhibitors of (chymo)trypsin-like serine proteases.     

Utilization of the 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide scaffold in the design of potential inhibitors of human neutrophil proteinase 3

The S′ subsites of human neutrophil proteinase 3 (Pr 3) were probed by constructing diverse libraries of compounds based on the 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide using combinational and click chemistry methods. The multiple points of diversity embodied in the heterocyclic scaffold render it well-suited to the exploration of the S′ subsites of Pr 3. Molecular modeling studies suggest that further exploration of the S′ subsites of Pr 3 using the aforementioned heterocyclic scaffold may lead to the identification of highly selective, reversible competitive inhibitors of Pr 3.     

Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through protease-activated receptor 2

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.     

Effects of structure on inhibitory activity in a series of mechanism-based inhibitors of human neutrophil elastase

A structurally-diverse series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-one 1,1 dioxide scaffold were synthesized and used to probe the S′ subsites of human neutrophil elastase (HNE) and neutrophil proteinase 3 (Pr 3). Several compounds are potent inhibitors of HNE but devoid of inhibitory activity toward Pr 3, suggesting that the S′ subsites of HNE exhibit significant plasticity and can, unlike Pr 3, tolerate various large hydrophobic groups. The results provide a promising framework for the design of highly selective inhibitors of the two enzymes.     

Structure-based design of protein tyrosine phosphatase-1B inhibitors

Using structure-based design, a new class of inhibitors of protein tyrosine phosphatase-1B (PTP1B) has been identified, which incorporate the 1,2,5-thiadiazolidin-3-one-1,1-dioxide template.     

Mechanism-based inhibitors of serine proteases with high selectivity through optimization of S′ subsite binding

A series of mechanism-based inhibitors designed to interact with the S′ subsites of serine proteases was synthesized and their inhibitory activity toward the closely-related serine proteases human neutrophil elastase (HNE) and proteinase 3 (PR 3) was investigated. The compounds were found to be time-dependent inhibitors of HNE and were devoid of any inhibitory activity toward PR 3. The results suggest that highly selective inhibitors of serine proteases whose primary substrate specificity and active sites are similar can be identified by exploiting differences in their S′ subsites. The best inhibitor (compound 16) had a k_inact/K_I value of 4580 M^?1 s^?1.     

Potential of pyrazolooxadiazinone derivatives as serine protease inhibitors

As a part of an investigation on molecular hybrids as new serine protease inhibitors, the pyrazolo [4,3-c][1,2,5]oxadiazin-3(5H)-one ring system was selected as a model of potential mechanism-based inhibitors. Due to the inherent reactivity of this system an optimal balance between susceptibility to nucleophilic attack and stability in solvents was sought prior to development as therapeutic agents. Substitutions on N5 and C7 of the supporting pyrazole ring with either aliphatic or aromatic groups (compounds 2 a-m) and the replacement of the carbonyl oxygen on the reactive oxadiazinone ring with sulfur (compounds 3a,i) were explored. Two members (2i and 2k) of this class of inhibitors displayed time-dependent inhibition of HLE suggesting mechanism-based inhibition. The observation that HLE generated a product(s) from compound 2i which displayed an identical UV-Visible spectrum to that observed during non-enzymatic hydrolysis further supports this proposal. FlexX-based docking of these compounds into a model of the human leukocyte elastase (HLE) active site produced a molecular model of the inhibitor-enzyme interaction.     

Structure-activity relationships for the selectivity of hepatitis C virus NS3 protease inhibitors

The selectivity of hepatitis C virus (HCV) non-structural protein 3 (NS3) protease inhibitors was determined by evaluating their inhibitory effect on other serine proteases (human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine pancreatic chymotrypsin (BPC)) and a cysteine protease (cathepsin B). For these peptide inhibitors, the P1-side chain and the C-terminal group were the major determinants of selectivity. Inhibitors with electrophilic C-terminal residues were generally non-selective while compounds with non-electrophilic C-terminal residues were more selective. Furthermore, compounds with P1 aminobutyric acid residues were non-selective, while 1-aminocyclopropane-1-carboxylic acid (ACPC) and norvaline-based inhibitors were generally selective. The most potent and selective inhibitors of NS3 protease tested contained a non-electrophilic phenyl acyl sulfonamide C-terminal residue. HLE was most likely to be inhibited by the HCV protease inhibitors, in agreement with similar substrate specificities for these enzymes. The identified structure-activity relationships for selectivity are of significance for design of selective HCV NS3 protease inhibitors.     

6-Acylamino-2-1[(ethylsulfonyl)oxy]-1H-isoindole-1,3-diones mechanism-based inhibitors of human leukocyte elastase and cathepsin G: effect of chirality in the 6-acylamino substituent on inhibitory potency and selectivity

Inhibition of human leukocyte elastase(HLE) by a series of 6-acylamino-2-[(ethylsulfonyl)oxy)]-1H-isoindole-1,3-diones was determined and compared to their inhibition of ChT, PPE, and Cat G. The best inhibitor of the series was 6-((1'S)-camphanyl)amino-2-[(ethylsulfonyl) oxy]-1H-isoindole-1,3-dione 5b, with a k(obs)/[I] = 11,000 M(-1) s(-1). This study revealed that HLE shows a preference for the S stereochemistry and tolerates hydrophobic substituents in the Sn' binding sites. Molecular modeling of non-covalent HLE-inhibitor complexes was used as a tool to investigate our binding model. Buffer stability assays reveal that these compounds are susceptible to hydrolysis at physiological pH.     

Peptidic phosphonylating agents as irreversible inhibitors of serine proteases and models of the tetrahedral intermediates

Peptide analogues incorporating an electrophilic phosphorus moiety (2-6) have been synthesized and studied as inhibitors of a variety of serine proteases. Inhibition is irreversible and, for alpha-lytic protease (ALP), shown to result from covalent binding to the active site serine hydroxyl [Bone, R., Sampson, N. S., Bartlett, P.A., & Agard, D. A. (1991) Biochemistry (following paper in this issue)]. For reaction of human leukocyte elastase (HLE) with the thiophenyl esters 6s-V (Boc-AAPV psi [P = O(SPh)O]AA-OMe), 4s-V (BocAAPV psi [P = O(SPh)O]-Me), and 3s-V (Boc-V psi [P = O(SPh)O]AA-OMe), evidence is presented to suggest that the S4-S1 subsites, but not the S1' and S2' positions, are occupied by the inhibitors during the inactivation process. The selectivity that is observed between the proteases and the hexapeptide phosphonates 6o-V (Boc-AAPV psi [P = O(OPh)O]AA-OMe) and 6o-F (Boc-AAPF psi [P = O(OPh)O]AA-OMe) parallels that between these enzymes and their substrates: ALP and HLE are selectively inactivated by the ValP-containing analogue 6o-V, while subtilisin (SUB) shows a preference for the PheP derivative 6o-F. A detailed kinetic analysis of the enzyme-inhibitor interactions was complicated by the susceptibility of the inhibitors to enzymatic degradation. The configuration at phosphorus was found not to have a significant influence on the rate at which the inhibitors react with the peptidases. Moreover, in the case of inactivation of ALP by the hexapeptide 6o-V, the same covalent adduct is formed from both stereoisomers (Bone et al., 1991), indicating that one of these diastereomers undergoes substitution with retention of configuration.     

Proteinase 3 contributes to endothelial dysfunction in an experimental model of sepsis

In sepsis-induced inflammation, polymorphonuclear neutrophils (PMNs) contribute to vascular dysfunction. The serine proteases proteinase 3 (PR3) and human leukocyte elastase (HLE) are abundant in PMNs and are released upon degranulation. While HLE’s role in inflammation-induced endothelial dysfunction is well studied, PR3’s role is largely uninvestigated. We hypothesized that PR3, similarly to HLE, contributes to vascular barrier dysfunction in sepsis. Plasma PR3 and HLE concentrations and their leukocyte mRNA levels were measured by ELISA and qPCR, respectively, in sepsis patients and controls. Exogenous PR3 or HLE was applied to human umbilical vein endothelial cells (HUVECs) and HUVEC dysfunction was assessed by FITC-dextran permeability and electrical resistance. Both PR3 and HLE protein and mRNA levels were significantly increased in sepsis patients (P < 0.0001 and P < 0.05, respectively). Additionally, each enzyme independently increased HUVEC monolayer FITC-dextran permeability (P < 0.01), and decreased electrical resistance in a time- and dose-dependent manner (P < 0.001), an effect that could be ameliorated by novel treatment with carbon monoxide-releasing molecule 3 (CORM-3). The serine protease PR3, in addition to HLE, lead to vascular dysfunction and increased endothelial permeability, a hallmark pathological consequence of sepsis-induced inflammation. CORMs may offer a new strategy to reduce serine protease-induced vascular dysfunction.     

The action of neutrophil serine proteases on elastin and its precursor

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P₁, which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P₁ in tropoelastin, whereas Lys was not identified at P₁ in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P₂ and P₄′. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG.