The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.Proteases hydrolyze amide bonds in proteins and peptides and represent one of the largest and most important protein families known. They comprise over 2% of the human genome and play diverse physiological roles (merops.sanger.ac.uk) (1). The substrate specificity of a protease enables the enzyme to preferentially cleave its substrates in the presence of other peptides or proteins. Therefore, specificity information can provide clues about the biological function of the protease and aid in the design of efficient substrates and potent, selective inhibitors. Various methods including both biological and chemicalbased approaches to study protease specificity have been developed and were recently reviewed (2).Positional scanning synthetic combinatorial libraries (PS-SCLs) 2 of fluorogenic substrates have emerged as useful reagents for the rapid and exhaustive determination of protease specificity (3). A peptide-based PS-SCL is composed of sublibraries in which one peptide position is fixed with an amino acid, whereas the remaining positions contain an equimolar mixture of amino acids. Assaying proteases with these sublibraries rapidly establishes the amino acid preferences at the defined position. Initially, the substrate specificities of caspases and granzyme B were profiled using PSSCLs with the P1 position fixed as an aspartic acid.The limitations of the original P1 fixed libraries were overcome through the development of a modified coumarin, 7-amino-...

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.T he ability of an enzyme to discriminate among many potential substrates is an important factor in maintaining the fidelity of most biological functions. While substrate selection can be regulated on many levels in a biological context, such as spatial and temporal localization of enzyme and substrate, concentrations of enzyme and substrate, and requirement of cofactors, the substrate specificity at the enzyme active site is the overriding principle that determines the turnover of a substrate. Characterization of the substrate specificity of an enzyme clearly provides invaluable information for the dissection of complex biological pathways. Definition of substrate specificity also provides the basis for the design of selective substrates and inhibitors to study enzyme activity.Of the genomes that have been completely sequenced, 2% of the gene products encode proteases (1). This family of enzymes is crucial to every aspect of life and death of an organism. With the identification of new proteases, there is a need for the development of rapid and general methods to determine protease substrate specificity. While several biological methods, such as peptides displayed on filamentous phage (2, 3), and chemical methods, such as support-bound combinatorial libraries (4), have been developed to identify proteolytic substrate specificity, few offer the ability to rapidly and continuously monitor proteolytic activity against complex mixt...

Many naturally occurring substances, traditionally used in popular medicines around the world, contain the coumarin moiety. Coumarin represents a privileged scaffold for medicinal chemists, because of its peculiar physicochemical features, and the versatile and easy synthetic transformation into a large variety of functionalized coumarins. As a consequence, a huge number of coumarin derivatives have been designed, synthesized, and tested to address many pharmacological targets in a selective way, e.g., selective enzyme inhibitors, and more recently, a number of selected targets (multitarget ligands) involved in multifactorial diseases, such as Alzheimer’s and Parkinson’s diseases. In this review an overview of the most recent synthetic pathways leading to mono- and polyfunctionalized coumarins will be presented, along with the main biological pathways of their biosynthesis and metabolic transformations. The many existing and recent reviews in the field prompted us to make some drastic selections, and therefore, the review is focused on monoamine oxidase, cholinesterase, and aromatase inhibitors, and on multitarget coumarins acting on selected targets of neurodegenerative diseases.

We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmin's physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.

A large series of coumarin derivatives (71 compounds) were tested for their monoamine oxidase A and B (MAO-A and MAO-B) inhibitory activity. Most of the compounds acted preferentially on MAO-B with IC(50) values in the micromolar to low-nanomolar range; high inhibitory activities toward MAO-A were also measured for sulfonic acid esters. The most active compound was 7-[(3, 4-difluorobenzyl)oxy]-3,4-dimethylcoumarin, with an IC(50) value toward MAO-B of 1.14 nM. A QSAR study of 7-X-benzyloxy meta-substituted 3,4-dimethylcoumarin derivatives acting on MAO-B yielded good statistical results (q(2)() = 0.72, r(2)() = 0.86), revealing the importance of lipophilic interactions in modulating the inhibition and excluding any dependence on electronic properties. CoMFA was performed on two data sets of MAO-A and MAO-B inhibitors. The GOLPE procedure, with variable selection criteria, was applied to improve the predictivity of the models and to facilitate the graphical interpretation of results.