ABSTRACT
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.
Subject(s)
Chymotrypsin/chemistry , Cyclic S-Oxides/chemistry , Molecular Probes/chemistry , Serine Endopeptidases/chemistry , Thiazoles/chemistry , Cathepsin G , Cathepsins/chemistry , Cathepsins/metabolism , Humans , Kinetics , Leukocyte Elastase/chemistry , Leukocyte Elastase/metabolism , Models, Chemical , Myeloblastin , Protein Binding , Serine Endopeptidases/metabolism , Temperature , Time FactorsABSTRACT
We developed a convenient colorimetric assay for monitoring RNA synthesis from DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp). ATP and GTP with a p-nitrophenyl moiety attached to the gamma-phosphate were synthesized (PNP-NTPs). These PNP-NTPs can be used for RNA synthesis by several RNA polymerases, including the RdRps from brome mosaic virus and bovine viral diarrhea virus and the DdRps from bacteriophage T7 and SP6. When the polymerase reactions were performed in the presence of alkaline phosphatase, which digests the p-nitrophenylpyrophosphate side-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was observed. These nucleotide analogues were used in continuous colorimetric monitoring of polymerase activity. Furthermore, the PNP-NTPs were found to be stable and utilized by RNA polymerases in the presence of human plasma. This simple colorimetric polymerase assay can be performed in a standard laboratory spectrophotometer and will be useful in screens for inhibitors of viral RNA synthesis.
Subject(s)
Colorimetry/methods , DNA-Directed RNA Polymerases/metabolism , RNA-Dependent RNA Polymerase/metabolism , Viruses/enzymology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Alkaline Phosphatase/metabolism , Bacteriophage T7/enzymology , Base Sequence , Bromovirus/enzymology , Coloring Agents , Diarrhea Viruses, Bovine Viral/enzymology , Drug Evaluation, Preclinical/methods , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/blood , Guanosine Triphosphate/metabolism , Hordeum/virology , Humans , Kinetics , Nitrophenols/blood , Nitrophenols/metabolism , Plasmids/genetics , RNA/biosynthesis , RNA/genetics , Spectrophotometry , Substrate Specificity , Templates, Genetic , Viral ProteinsABSTRACT
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.
Subject(s)
Cyclic S-Oxides/chemistry , Serine Proteinase Inhibitors/chemistry , Thiadiazoles/chemistry , Drug Design , Elastin/metabolism , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Serine Proteinase Inhibitors/metabolism , Serine Proteinase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Cathepsins/antagonists & inhibitors , Leukocyte Elastase/antagonists & inhibitors , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Thiadiazoles/pharmacology , Cathepsin G , Humans , Kinetics , Models, Molecular , Myeloblastin , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/chemistry , Structure-Activity Relationship , Thiadiazoles/chemical synthesis , Thiadiazoles/chemistryABSTRACT
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.
Subject(s)
Drug Design , Heterocyclic Compounds/chemistry , Serine Proteinase Inhibitors/chemical synthesis , Amino Acids , Autoantigens/metabolism , Cathepsin G , Cathepsins/antagonists & inhibitors , Humans , Leukocyte Elastase/antagonists & inhibitors , Magnetic Resonance Spectroscopy , Models, Molecular , Myeloblastin , Serine Endopeptidases/metabolism , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Cathepsins/antagonists & inhibitors , Leukocyte Elastase/antagonists & inhibitors , Serine Endopeptidases/metabolism , Sulfones/antagonists & inhibitors , Autoantigens/immunology , Cathepsin G , Chromatography, High Pressure Liquid , Humans , Kinetics , Magnetic Resonance Spectroscopy , Models, Molecular , Myeloblastin , Protease Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Cathepsins/antagonists & inhibitors , Leukocyte Elastase/antagonists & inhibitors , Phthalimides/pharmacology , Saccharin/analogs & derivatives , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Amino Acid Sequence , Animals , Binding Sites , Cathepsin G , Drug Design , Humans , Hydrogen Bonding , In Vitro Techniques , Leukocyte Elastase/chemistry , Leukocyte Elastase/genetics , Leukocytes/enzymology , Models, Molecular , Molecular Sequence Data , Myeloblastin , Phthalimides/chemistry , Protein Conformation , Saccharin/chemistry , Saccharin/pharmacology , Serine Proteinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Cathepsins/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Pancreatic Elastase/antagonists & inhibitors , Serine Endopeptidases/drug effects , Serine Proteinase Inhibitors/pharmacology , Succinimides/chemistry , Succinimides/pharmacology , Amino Acids/chemistry , Cathepsin G , Drug Design , Enzyme Inhibitors/chemical synthesis , Humans , Leukocyte Elastase , Myeloblastin , Structure-Activity Relationship , Succinimides/chemical synthesis , Time FactorsSubject(s)
Cathepsins/antagonists & inhibitors , Isoxazoles/pharmacology , Pancreatic Elastase/antagonists & inhibitors , Protease Inhibitors/pharmacology , Serine Endopeptidases/drug effects , Cathepsin G , Humans , Isoxazoles/chemical synthesis , Isoxazoles/chemistry , Kinetics , Leukocyte Elastase , Magnetic Resonance Spectroscopy , Molecular Structure , Myeloblastin , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Cathepsins/antagonists & inhibitors , Pancreatic Elastase/antagonists & inhibitors , Phthalimides/chemical synthesis , Saccharin/analogs & derivatives , Serine Endopeptidases/drug effects , Cathepsin G , Humans , Leukocyte Elastase , Magnetic Resonance Spectroscopy , Myeloblastin , Phthalimides/chemistry , Phthalimides/pharmacology , Saccharin/chemical synthesis , Saccharin/chemistry , Saccharin/pharmacology , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Cathepsins/antagonists & inhibitors , Leukocyte Elastase/antagonists & inhibitors , Leukocytes/enzymology , Pancreatic Elastase/antagonists & inhibitors , Uracil/analogs & derivatives , Cathepsin G , Drug Design , Humans , Leukocytes/drug effects , Magnetic Resonance Spectroscopy , Models, Molecular , Serine Endopeptidases , Structure-Activity Relationship , Uracil/chemical synthesis , Uracil/chemistry , Uracil/pharmacologyABSTRACT
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.
Subject(s)
Leukocyte Elastase/antagonists & inhibitors , Leukocytes/enzymology , Pancreatic Elastase/antagonists & inhibitors , Succinimides/chemistry , Chemical Phenomena , Chemistry, Physical , Crystallography, X-Ray , Humans , Leukocyte Elastase/chemistry , Magnetic Resonance Spectroscopy , Pancreatic Elastase/chemistry , Structure-Activity Relationship , Succinimides/pharmacologyABSTRACT
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.
Subject(s)
Cathepsins/antagonists & inhibitors , Pancreatic Elastase/antagonists & inhibitors , Saccharin/analogs & derivatives , Cathepsin G , Humans , Leukocyte Elastase , Serine Endopeptidases , Structure-Activity RelationshipSubject(s)
Cystic Fibrosis/drug therapy , Pancreatic Elastase/antagonists & inhibitors , Protease Inhibitors/pharmacology , Amino Acid Sequence , Aspirin/analogs & derivatives , Aspirin/chemical synthesis , Aspirin/pharmacology , Cystic Fibrosis/enzymology , Drug Design , Humans , In Vitro Techniques , Ketoprofen/analogs & derivatives , Ketoprofen/chemical synthesis , Ketoprofen/pharmacology , Kinetics , Leukocytes/enzymology , Molecular Sequence Data , Molecular Structure , Oligopeptides/chemistry , Pancreatic Elastase/blood , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Saccharin/analogs & derivatives , Saccharin/chemical synthesis , Saccharin/pharmacology , Structure-Activity RelationshipABSTRACT
A series of 3-(alkylthio)-N-hydroxysuccinimide derivatives was synthesized and their inhibitory activity towards human leukocyte elastase (HLE) was investigated. The interaction of the compounds having a 3-alkylthioether side chain (compounds 1 and 2) with HLE was found to involve rapid acylation of the enzyme, followed by total regain of enzymatic activity within 3 h. Interestingly, compounds 3-8, having an oxidized thioether side chain, were found to be highly effective, time-dependent, irreversible inhibitors of the enzyme. The k(obs)/I values for compounds 3-8 ranged between 890 and 24,000 M-1 s-1. These findings demonstrate that, unlike the physiological inhibitor of HLE (alpha-1-proteinase inhibitor), which is inactivated upon oxidation, low-molecular-weight compounds retain and/or show enhanced inhibitory activity towards HLE upon oxidation of the thioether side chain and lay the groundwork for the development of compounds that embody proteinase inhibitory and antioxidant activity.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Leukocytes/enzymology , Pancreatic Elastase/antagonists & inhibitors , Succinimides/chemical synthesis , Sulfides/chemical synthesis , Acylation , Humans , Models, Molecular , Structure-Activity Relationship , Succinimides/chemistry , Succinimides/pharmacology , Sulfides/chemistry , Sulfides/pharmacology , alpha 1-Antitrypsin/pharmacologyABSTRACT
Neutrophil-derived mediators such as, for example, the serine proteinase elastase, cathepsin G and proteinase 3, play a critical role in inflammatory lung disease. This report describes the design, synthesis and in vitro inhibitory activity of some novel mechanism-based inhibitors of human leukocyte elastase and cathepsin G. The design of the inhibitors is based on the Gabriel-Colman rearrangement. The behavior of the synthesized compounds toward elastase and cathepsin G with respect to inhibitory prowess, mode of interaction, specificity, etc., has been found to be dependent on the recognition and reactivity elements present in each inhibitor.
Subject(s)
Cathepsins/antagonists & inhibitors , Leukocytes/enzymology , Pancreatic Elastase/antagonists & inhibitors , Amino Acid Sequence , Cathepsin G , Drug Design , Humans , Molecular Sequence Data , Serine EndopeptidasesABSTRACT
The interaction of a series of sulfonate and phosphate esters derived from N-hydroxysuccinimide with human leukocyte cathepsin G was investigated. The synthesized compounds were found to be time-dependent inhibitors of the enzyme. The composite interplay of steric and electronic effects leads to the formation of acyl enzymes of variable stability, ultimately resulting in partial or full recovery of enzymatic activity. Compounds acting via phosphorylation of the active site serine inactivated the enzyme rapidly and irreversibly.