Identification of Selective, Nonpeptidic Nitrile Inhibitors of Cathepsin S Using the Substrate Activity Screening Method

Patterson, Andrew W.; Wood, Warren J. L.; Hornsby, Michael; Lesley, Scott A.; Spraggon, Glen; Ellman, Jonathan A.

doi:10.1021/jm060701s

Cited by 85 publications

(98 citation statements)

References 32 publications

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“…1) can be performed before converting substrates into inhibitors because transition-state theory for enzyme-catalyzed reactions predicts that the inhibitory activity (K i ) of mechanism-based inhibitors can be correlated with the inverse of the catalytic efficiency of the corresponding substrates (K m /k cat ) 10 . Other researchers have confirmed this correlation for peptide substrates 11,12 , and we have observed this correlation for non-peptidic substrates and inhibitors for both cathepsin S and chymotrypsin [1][2][3] . To obtain accurate relative substrate cleavage efficiencies (k cat /K m ) in the substrate optimization step, assays should be conducted at substrate concentrations below the K m of the substrates (Michaelis-Menten equation:…”

Section: Introductionsupporting

confidence: 88%

“…We have developed the first substrate-based fragment identification method, called 'substrate activity screening' (SAS) [1][2][3] . This method addresses two key challenges in fragment-based screening: (i) the accurate and efficient identification of weak binding fragments and (ii) the rapid optimization of the initial weak binding fragments into higher-affinity compounds 4,5 .…”

Section: Introductionmentioning

confidence: 99%

“…We have successfully applied the SAS method to both Cys and Ser protease targets to identify novel, low-molecular-weight substrates and have subsequently optimized and converted the substrates into potent, non-peptidic inhibitors [1][2][3] . The SAS method was initially successfully applied to the identification of two distinct classes of novel, non-peptidic inhibitors with nanomolar affinity to cathepsin S, which is an enzyme implicated in a number of autoinflammatory diseases (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

2007

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Substrate activity screening (SAS) is a fragment-based method for the rapid development of novel substrates and their conversion into non-peptidic inhibitors of Cys and Ser proteases. The method consists of three steps: (i) a library of N-acyl aminocoumarins with diverse, low-molecular-weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay; (ii) the identified N-acyl aminocoumarin substrates are optimized by rapid analog synthesis and evaluation; and (iii) the optimized substrates are converted into inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. This protocol describes a general procedure for the solid-phase synthesis of a library of N-acyl aminocoumarin substrates and the screening procedure to identify weak binding substrates. INTRODUCTIONWe have developed the first substrate-based fragment identification method, called 'substrate activity screening' (SAS) 1-3 . This method addresses two key challenges in fragment-based screening: (i) the accurate and efficient identification of weak binding fragments and (ii) the rapid optimization of the initial weak binding fragments into higher-affinity compounds 4,5 . In this method (Fig. 1), a library of N-acyl aminocoumarins with diverse, low-molecular-weight N-acyl fragments is prepared and then, in a first step, the library is screened to identify protease substrates using a single-step, highthroughput fluorescence-based assay. The substrate-based screening method in Step 1 has important attributes in detecting weak binding fragments in addition to being high throughput and straightforward to perform. False positives as a result of aggregation, protein precipitation or non-specific binding seen in traditional inhibitor screens are not observed. In fact, in the present approach, both an active enzyme and productive active-site binding are required for the protease-catalyzed amide bond hydrolysis to occur and for the subsequent release of the fluorescent coumarin group to be observed. In addition, in contrast to direct binding assays and traditional inhibitor screens, catalytic substrate turnover results in signal amplification, and therefore even very weak substrates can be identified at concentrations where only minimal binding to the enzyme occurs. The second and third steps (Fig. 1), which are not covered in this protocol, provide a strategy for systematically and efficiently optimizing substrates incorporating weak binding fragments into high-affinity inhibitors. In the second step, the activity of the substrates is rapidly optimized by the straightforward solidphase synthesis and subsequent assay of focused libraries of substrate analogs. The

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Section: Introductionsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

2007

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“…10,12 Substrate analogues were therefore first evaluated and optimized before conversion to inhibitors. A triazole-based substrate library consisting of more than 150 substrates was screened against cruzain.…”

Section: Initial Screeningmentioning

confidence: 99%

Identification of a New Class of Nonpeptidic Inhibitors of Cruzain

et al. 2008

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Cruzain is the major cysteine protease of T. cruzi, which is the causative agent of Chagas' disease and is a promising target for the development of new chemotherapy. With the goal of developing potent nonpeptidic inhibitors of cruzain, the Substrate Activity Screening (SAS) method was used to screen a library of protease substrates initially designed to target the homologous human protease cathepsin S. Structure-based design was next used to further improve substrate cleavage efficiency by introducing additional binding interactions in the S3 pocket of cruzain. The optimized substrates were then converted to inhibitors by the introduction of cysteine protease mechanism-based pharmacophores. Inhibitor 38 was determined to be reversible even though it incorporated the vinyl sulfone pharmacophore that is well documented to give irreversible cruzain inhibition for peptidic inhibitors. The previously unexplored β-chloro vinyl sulfone pharmacophore provided mechanistic insight that led to the development of potent irreversible acyl-and aryl-oxymethyl ketone cruzain inhibitors. For these inhibitors, potency did not solely depend on leaving group pK a , with 2,3,5,6-tetrafluorophenoxymethyl ketone 54 identified as one of the most potent inhibitors with a second order inactivation constant of 147,000 s −1 M −1 . This inhibitor completely eradicated the T. cruzi parasite from mammalian cell cultures and consequently has the potential to lead to new chemotherapeutics for Chagas' disease.

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“…In the ''cysteine proteinases'' superfamily, the transferase, arylamine N-acetyltransferase, 40 uses a peripheral region to insulate the ligand molecule. The counterpart of the hydrolase, cathepsin S, 41 lacks the corresponding peripheral region [ Fig. 3(a)].…”

Section: Other Strategiesmentioning

confidence: 99%

Alteration of oligomeric state and domain architecture is essential for functional transformation between transferase and hydrolase with the same scaffold

2009

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Transferases and hydrolases catalyze different chemical reactions and express different dynamic responses upon ligand binding. To insulate the ligand molecule from the surrounding water, transferases bury it inside the protein by closing the cleft, while hydrolases undergo a small conformational change and leave the ligand molecule exposed to the solvent. Despite these distinct ligand-binding modes, some transferases and hydrolases are homologous. To clarify how such different catalytic modes are possible with the same scaffold, we examined the solvent accessibility of ligand molecules for 15 SCOP superfamilies, each containing both transferase and hydrolase catalytic domains. In contrast to hydrolases, we found that nine superfamilies of transferases use two major strategies, oligomerization and domain fusion, to insulate the ligand molecules. The subunits and domains that were recruited by the transferases often act as a cover for the ligand molecule. The other strategies adopted by transferases to insulate the ligand molecule are the relocation of catalytic sites, the rearrangement of secondary structure elements, and the insertion of peripheral regions. These findings provide insights into how proteins have evolved and acquired distinct functions with a limited number of scaffolds.

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Identification of Selective, Nonpeptidic Nitrile Inhibitors of Cathepsin S Using the Substrate Activity Screening Method

Cited by 85 publications

References 32 publications

Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

Identification of a New Class of Nonpeptidic Inhibitors of Cruzain

Alteration of oligomeric state and domain architecture is essential for functional transformation between transferase and hydrolase with the same scaffold

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