Solid‐Phase Synthesis of Double‐Headed Epoxysuccinyl Activity‐Based Probes for Selective Targeting of Papain Family Cysteine Proteases

Verhelst, Steven H. L.; Bogyo, Matthew

doi:10.1002/cbic.200400377

Cited by 26 publications

(24 citation statements)

References 25 publications

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“…The position of the bands that correspond to individual cathepsins was the same as reported in previous studies ( Fig. 1C) (LennonDumenil et al, 2002;Verhelst and Bogyo, 2005;Sadaghiani et al, 2007). Cathepsin X activity was 2.3-fold higher in overexpressing than in wildtype cells when comparing the medium density of the bands (Fig.…”

Section: Overexpression Of Cathepsin X In Stably Transfected T Lymphosupporting

confidence: 80%

The role of cathepsin X in the migration and invasiveness of T lymphocytes

Jevnikar

Obermajer

Bogyo

et al. 2008

Journal of Cell Science

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Cathepsin X is a lysosomal cysteine protease exhibiting carboxypeptidase activity. Its expression is high in the cells of immune system and its function has been related to the processes of inflammatory and immune responses. It regulates processes such as adhesion, T lymphocyte activation and phagocytosis through its interaction with β2 integrins. To investigate the role of cathepsin X in the migration of T lymphocytes, Jurkat T lymphocytes were stably transfected with a pcDNA3 expression vector containing cathepsin X cDNA. The cathepsin-X-overexpressing T lymphocytes exhibited polarised migration-associated morphology, enhanced migration on 2D and 3D models using intercellular adhesion molecule 1 (ICAM1)- and Matrigel-coated surfaces, and increased homotypic aggregation. The increased invasiveness of cathepsin-X-overexpressing cells does not involve proteolytic degradation of extracellular matrix. Confocal microscopy showed that the active mature form of cathepsin X was colocalised in migrating cells together with lymphocyte-function-associated antigen 1 (LFA-1). The colocalisation was particularly evident at the trailing edge protrusion, the uropod, that has an important role in T lymphocyte migration and cell-cell interactions. We propose that cathepsin X causes cytoskeletal rearrangements and stimulates migration of T lymphocytes by modulating the activity of the β2 integrin receptor LFA-1.

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Section: Overexpression Of Cathepsin X In Stably Transfected T Lymphosupporting

confidence: 80%

The role of cathepsin X in the migration and invasiveness of T lymphocytes

Jevnikar

Obermajer

Bogyo

et al. 2008

Journal of Cell Science

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“…Many researchers have tailored the structure of mechanism-based probes to specifically address enzymes or certain enzyme classes of their interest. To date mechanistic probes have been designed for different enzyme classes, namely serine hydrolases [45][46][47] including serine proteases [48][49][50][51], lipases [52][53][54][55][56][57][58] and PAF-acetylhydrolases [59], cysteine proteases including caspases [60,61], papains [62][63][64][65][66][67][68] and ubiquitin-and ubiquitin-like specific proteases [69,70], threonine proteases [71][72][73], tyrosine phosphatases [74,75] and glycosidases [76][77][78][79].…”

Section: Activity-based Proteomics For Target and Drug Discoverymentioning

confidence: 99%

Activity-Based Proteomics of Lipolytic Enzymes

Birner‐Gruenberger¹,

Hermetter

2007

CDDT

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Lipases play fundamental roles in biological processes since hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals. Perturbations in the metabolism and the cellular retention of lipids result in common diseases such as obesity, type 2 diabetes, and atherosclerosis. The introduction of active site-directed chemical probes for enzymatic activity profiling in complex mixtures, known as activity-based proteomics, has greatly facilitated and accelerated global analysis and functional annotation of lipolytic proteins. Here we review probe design and application for discovery and discrimination of lipolytic and esterolytic enzymes. These probes are usually detected by their fluorescent or affinity tags and their protein targets are analyzed using established proteomics techniques. Moreover, microarray technologies can be applied for higher throughput screenings of enzyme or probe specificity.

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“…11 Next, we introduced the epoxysuccinate warhead using the activated nitrophenyl ester 5, followed by hydrolysis of the ethyl ester to introduce a R 3 group at the prime side of the molecule. However, treatment of the resin under saponifying conditions, as previously described, 9 resulted in premature cleavage from the solid support and therefore proved incompatible with the safety-catch resin. To overcome this problem, we coupled epoxysuccinate 6, 12 having two free acid groups, with PyBOP/DIEA.…”

Section: Received July 21 2006mentioning

confidence: 89%

“…After Fmoc deprotection, activated epoxysuccinate 5 was coupled to the free amine, and the ethyl ester was hydrolyzed with KOH as previously described. 9 The resulting free acid was then used for coupling of the R 3 amine using standard PyBOP-coupling methods. The products were cleaved from the resin with TFA and purified by HPLC (12-42% yield, relative to resin 7; see Table 1).…”

Section: Received July 21 2006mentioning

confidence: 99%

Solid-Phase Methods for the Preparation of Epoxysuccinate-Based Inhibitors of Cysteine Proteases

2006

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The CA clan papain family is one of the largest and best studied subfamilies of the cysteine proteases. In addition to their association with physiological processes such as antigen presentation, 1 it has become clear that these enzymes are involved in several pathological processes including rheumatoid arthiritis, 2 Alzheimer's disease, 3 cancer invasion and metastasis, 4 and several parasitic diseases. 5 Selective inhibition of papain cysteine proteases will aid in our understanding of their role in these human diseases. More importantly, recent studies using papain family selective epoxysuccinyl inhibitors in mouse models of cancer suggest they may represent a viable new class of anticancer chemotherapeutic agents. 6 Currently, one such epoxysuccinate inhibitor is in preclinical trials at the National Cancer Institute. Thus, methods allowing the rapid synthesis of libraries of diverse epoxysuccinates that contain reduced peptide character will be critical for the identification of optimal lead compounds for use in human clinical trials.The natural product E-64 7 (1, Figure 1) and its closely related analog JPM-565 8 (2) are epoxysuccinyl-based covalent inhibitors of papain-like cysteine proteases. Their broadspectrum activity against this family of enzymes is derived from the leucine residue that mimics the P2 position of a substrate and interacts with the hydrophobic S2 pocket of most papain-like proteases.The ethyl ester of 2, JPM-OEt (3), has been used in in vivo studies using a mouse model for pancreatic cancer. 6 Treatment with JPM-OEt resulted in a significant reduction in angiogenesis, tumor volume, and tumor invasiveness. Combined with overall low toxicity, these results support the development of this class of inhibitors as anticancer chemotherapeutics. However, the very short half-life of JPMOEt in vivo and its overall limited bioavailability makes it less suitable as a drug candidate.We recently reported a solid-phase synthesis route that allows the incorporation of variable peptide elements on both sides of the epoxide function. 9 Synthesis of a number of "double-headed" epoxides using this method confirmed the importance of the interactions of inhibitors with regions of the active site on both sides of the reactive cysteine nucleophile. However, the current synthetic methods are only able to produce compounds that are highly peptidic in nature and therefore unlikely to have optimal pharmacological properties. Furthermore, the incorporation of diversity elements is limited by the need for suitably protected amino acid building blocks for attachment to the resin.We reasoned that the incorporation of non-natural elements would be an advantageous strategy for the design of a cathepsin inhibitor because it would allow for the introduction of a larger diversity of R groups within the inhibitor scaffold (4, Figure 1). In addition, the reduction of the peptide character would significantly increase the druglike properties of the resulting compounds.The use of diverse amines to generate the R 1 element on...

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Solid‐Phase Synthesis of Double‐Headed Epoxysuccinyl Activity‐Based Probes for Selective Targeting of Papain Family Cysteine Proteases

Cited by 26 publications

References 25 publications

The role of cathepsin X in the migration and invasiveness of T lymphocytes

The role of cathepsin X in the migration and invasiveness of T lymphocytes

Activity-Based Proteomics of Lipolytic Enzymes

Solid-Phase Methods for the Preparation of Epoxysuccinate-Based Inhibitors of Cysteine Proteases

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