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.

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.

Potent inhibition of serine proteases by heterocyclic sulfide derivatives of 1,2,5-thiadiazolidin-3-one 1,1 dioxide.

The existence of subtle differences in the Sn' subsites of closely-related (chymo)trypsin-like serine proteases, and the fact that the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold docks to the active site of (chymo)trypsin-like enzymes in a substrate-like fashion, suggested that the introduction of recognition elements that can potentially interact with the Sn' subsites of these proteases might provide an effective means for optimizing enzyme potency and selectivity. Accordingly, a series of heterocyclic sulfide derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold (I) was synthesized and the inhibitory activity and selectivity of these compounds toward human leukocyte elastase (HLE), proteinase 3 (PR 3) and cathepsin G (Cat G) were then determined. Compounds with P1 = isobutyl were found to be potent, time-dependent inhibitors of HLE and, to a lesser extent PR 3, while those with P1 = benzyl inactivated Cat G rapidly and irreversibly. This study has demonstrated that 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based heterocyclic sulfides are effective 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.

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.

Potent and specific inhibition of human leukocyte elastase, cathepsin G and proteinase 3 by sulfone derivatives employing the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold.

This paper describes the results of structure-activity relationship studies in a series of heterocyclic mechanism-based inhibitors based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold I and capable of interacting with the Sn and Sn' subsites of a serine proteinase. Sulfone derivatives of I were found to be highly effective, time-dependent inhibitors of human leukocyte elastase (HLE), cathepsin G (Cat G) and proteinase 3 (PR 3). The judicious selection of an R1 group (accommodated at the primary specificity site S1) that is based on the known substrate specificity of a target serine proteinase, was found to yield highly selective inhibitors. The presence of a benzyl group (R2 = benzyl) at the S2 subsite was found to lead to a pronounced enhancement in inhibitory potency. Furthermore, the effective use of computer graphics and modeling has led to the design of potent, water-soluble inhibitors. The results of these studies demonstrate that the 1,2,5-thiadiazolidin-3-one 1,1, dioxide platform provides an effective means for appending recognition elements in a well-defined vector relationship, and in fashioning highly-selective and potent inhibitors of serine proteinases.

Use of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazolidin-3-one 1,1 dioxide scaffolds in the design of potent inhibitors of serine proteinases.

The attachment of a phosphate leaving group to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazolidin-3-one 1,1 dioxide scaffolds was found to yield highly potent, time-dependent inhibitors of human leukocyte elastase (HLE).

Inhibition of human leukocyte proteinase 3 by a novel recombinant serine proteinase inhibitor (LEX032).

The interaction of a bioengineered serpin (LEX032) with human leukocyte proteinase 3 (PR 3) has been investigated. LEX032 was found to be a time-dependent inhibitor of PR 3, forming a highly-stable enzyme-inhibitor complex (Ki 12 nM).

Structure-based design of a general class of mechanism-based inhibitors of the serine proteinases employing a novel amino acid-derived heterocyclic scaffold.

We describe in this paper the structure-based design of a general class of heterocyclic mechanism-based inhibitors of the serine proteinases that embody in their structure a novel peptidomimetic scaffold (1,2,5-thiadiazolidin-3-one 1,1-dioxide). Sulfone derivatives of this class (I) were found to be time-dependent, potent, and highly efficient irreversible inhibitors of human leukocyte elastase, cathepsin G, and proteinase 3. The partition ratios for a select number of inhibitors were found to range between 0 and 1. We furthermore demonstrate that these inhibitors exhibit remarkable enzyme selectivity that is dictated by the nature of the P1 residue and is consistent with the known substrate specificity reported for these enzymes. Thus, inhibitors with small hydrophobic side chains were found to be effective inhibitors of elastase, those with aromatic side chains of cathepsin G, and those with a basic side chain of bovine trypsin. Taken together, the findings cited herein reveal the emergence of a general class of stable mechanism-based inhibitors of the serine proteinases which can be readily synthesized using amino acid precursors. Biochemical and high-field NMR studies show that the interaction of this class of inhibitors with a serine proteinase results in the formation of a stable acyl complex(es) and the release of benzenesulfinate, formaldehyde, and a low molecular weight heterocycle. The data are consistent with initial formation of a Michaelis-Menten complex, acylation of Ser195, and tandem loss of the leaving group. The initial HLE-inhibitor complex reacts with water generating formaldehyde and a stable HLE-inhibitor complex. Whether the initial HLE-inhibitor complex also reacts with His57 to form a third complex is not known at this point. The desirable salient parameters associated with this class of inhibitors, including the expeditious generation of structurally diverse libraries of inhibitors based on I, suggest that this class of mechanism-based inhibitors is of general applicability and can be used in the development of inhibitors of human and viral serine proteinases of clinical relevance.

Inhibition of neurotoxic esterase in vitro by novel carbamates.

Carbamyl sulfonate (CS) compounds are a novel class of carbamates derived from amino acid methyl esters. They have the general structure RCH(COOCH3)NH(CO)SO-3K+, where R is the sidechain of the parent amino acid. These compounds were developed as active site-directed inhibitors of human leukocyte elastase (HLE). The purpose of this study was to characterize the inhibition of hen brain neurotoxic esterase (neuropathy target esterase, NTE), horse serum butyrylcholinesterase (BuChE), and bovine erythrocyte acetylcholinesterase (AChE) by CS analogs derived from the methyl esters of L-ala, D-norval, L-norval, L-phe, L-val, L-norleu, D-met, and L-met. Bimolecular rate constants of inhibition (ki) for NTE ranged from 0.571 for L-ala-CS to 17.7 mM-1 min-1 for L-norleu-CS (10-min I50 values of 123 and 3.92 microM, respectively). Potency against NTE increased with chain length for straight-chain R-groups of L-CS compounds. Unlike HLE, NTE was only weakly stereoselective for CS compound enantiomers. The L-isomers were weaker inhibitors of BuChE than NTE (10-min I50 range of 742 to 35.6 microM). In contrast to the L-enantiomers, the I50 plots of D-met-CS and D-norval-CS were not linear for BuChE, suggesting a possible stereospecific mechanistic shift for inhibition of this enzyme, AChE was not effectively inhibited by any of the CS compounds (I50 values > 750 microM). The specificity and charged nature of CS compounds give these unusual NTE inhibitors potential advantages for mechanistic studies of organophosphorus compound-induced delayed neurotoxicity (OPIDN) and its protection or potentiation.

Design, synthesis, and in vitro inhibitory activity toward human leukocyte elastase, cathepsin G, and proteinase 3 of saccharin-derived sulfones and congeners.

The inhibitory activity toward human leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR 3) of a series of saccharin derivatives having a sulfinate leaving group was investigated. The results of this study revealed that (a) inhibitory activity is dependent on the nature and pKa of the leaving group, and (b) the synthesized saccharin derivatives exhibit selective inhibition toward HLE and PR 3, with low or no activity toward cathepsin G. The results of exploratory biochemical, HPLC and high-field 13C NMR studies are also described.

Amino acid-derived phthalimide and saccharin derivatives as inhibitors of human leukocyte elastase, cathepsin G, and proteinase 3.

Amino acid-derived phthalimide and saccharin derivatives have been investigated for their inhibitory activity toward the serine proteinases human leukocyte elastase, cathepsin G, and proteinase 3. The saccharin derivatives were found to be effective time-dependent inhibitors of elastase and proteinase 3 (kobs/[I] values ranged between 180 and 3620 M-1 S-1) and showed weak or no inhibition toward cathepsin G. The corresponding phthalimide derivatives were found to be inactive.

Design, synthesis and biological evaluation of succinimide derivatives as potential mechanism-based inhibitors of human leukocyte elastase, cathepsin G and proteinase 3.

Structure-activity relationship study and in vitro biochemical studies with human leukocyte elastase, cathepsin G and proteinase 3 were conducted using a series of succinimide derivatives.

The Gabriel-Colman rearrangement in biological systems: design, synthesis and biological evaluation of phthalimide and saccharin derivatives as potential mechanism-based inhibitors of human leukocyte elastase, cathepsin G and proteinase 3.

The results of a structure-activity relationship study focusing on the interaction of a series of phthalimide and saccharin derivatives with leukocyte elastase, cathepsin G and proteinase 3 are described. The phthalimide derivatives were found to be inactive while some of the saccharin derivatives were found to be fair inhibitors of these enzymes.

Mechanism-based inhibition of human leukocyte elastase and cathepsin G by substituted dihydrouracils.

A series of dihydrouracil derivatives has been synthesized and investigated for their in vitro inhibitory activity toward human leukocyte elastase (HLE) and cathepsin G (Cath G). Alkyl [sulfonyl(oxy)] uracils 1-2 were found to be efficient, time-dependent inhibitors of elastase (kobs/[I] M-1 s-1 values ranged between 480 and 8110). These compounds formed acyl enzymes that exhibited variable hydrolytic stability which appeared to be dependent on the nature of the R1 group (believed to be accommodated at the primary specificity site, S1). The acyl enzymes formed with cathepsin G deacylated rapidly, leading to a significant regain of enzymatic activity. In sharp contrast, the corresponding phosphorus compounds 3-4 were found to be potent, time-dependent irreversible inhibitors of HLE. Furthermore, the results of the structure-activity relationship studies suggest that the binding modes of compounds 1-2 and 3-4 may be different.

Derivatives of 3-alkyl-N-hydroxysuccinimide: probing the effect of structure on bioactivity toward human leukocyte elastase.

A structure-activity relationship study was conducted in order to probe the nature of the interaction between some 3-alkyl-N-hydroxysuccinimide derivatives and human leukocyte elastase. The structural features in substituent X (structure I) that lead to the manifestation and optimization of inhibitory activity have been examined. The data suggest that the presence of an alkyl or aryl(sulfonyloxy) group in the active compounds may serve a triple purpose, namely, it functions as a good leaving group as dictated by the established mechanism of action of this class of compounds, secondly, it may enhance binding by assuming a favorable spatial orientation and, thirdly, it may increase the chemical reactivity of the carbonyl carbon in the bioactive compounds.

Substituted 3-oxo-1,2,5-thiadiazolidine 1,1-dioxides: a new class of potential mechanism-based inhibitors of human leukocyte elastase and cathepsin G.

A series of substituted 3-oxo-1,2,5-thiadiazolidine 1,1-dioxides has been synthesized and their in vitro inhibitory activity toward human leukocyte elastase and cathepsin G was investigated. These compounds were found to inactivate the two enzymes efficiently and in a time-dependent fashion.

Novel potential mechanism-based inhibitors of human leukocyte elastase and cathepsin G: derivatives of isothiazolidin-3-one.

A series of heterocyclic compounds designed to function as mechanism-based inhibitors of human leukocyte elastase and cathepsin G has been synthesized and their inhibitory activity was investigated. These isothiazolidin-3-one derivatives were found to be effective inhibitors of cathepsin G.

Efficient inhibition of human leukocyte elastase and cathepsin G by saccharin derivatives.

A series of saccharin derivatives I has been synthesized and evaluated for their inhibitory activity toward human leukocyte elastase and cathepsin G. Most of the compounds were found to be efficient and time-dependent inhibitors of elastase. Inactivated elastase was found to regain its activity almost fully after 24 h (80-90% activity) and the half-lives of reactivation ranged between 12-15 h. Addition of hydroxylamine to fully-inactivated enzyme led to rapid and complete recovery of enzymatic activity. A tentative mechanism of action is proposed on the basis of biochemical and model studies.