Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability

The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2?μM to as low as 110?nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5?brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.     

Deficiency for the Cysteine Protease Cathepsin L Impairs Myc-Induced Tumorigenesis in a Mouse Model of Pancreatic Neuroendocrine Cancer

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycER^TAM;Bcl-x_L model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycER^TAM;Bcl-x_L tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.     

Recent advances in the synthesis,design and selection of cysteine protease inhibitors

Inhibition of cysteine proteases is emerging as an important strategy for the treatment of a variety of human diseases. Intense efforts involving structure-based inhibitor design have been directed toward several cysteine proteases, including cathepsin K, calpain, human rhinovirus 3C protease and several parasitic cysteine protease targets. Other successful recent efforts have involved combinatorial synthesis and screening for identification of new inhibitor templates.     

Identification of Semicarbazones,Thiosemicarbazones and Triazine Nitriles as Inhibitors of Leishmania mexicana Cysteine Protease CPB

Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.     

Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer's disease

Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Peptidomimetic 2-cyanopyrrolidines as potent selective cathepsin L inhibitors

Cathepsins have been found to have important physiological roles. The implication of cathepsin L in various types of cancers is well established. In a search for selective cathepsin L inhibitors as anticancer agents, a series of 2-cyanoprrolidine peptidomimetics, carrying a nitrile group as warhead, were designed. Two series of compounds, one with a benzyl moiety and a second with an isobutyl moiety at P₂ position of the enzyme were synthesized. The synthesized compounds were evaluated for inhibitory activity against human cathepsin L and cathepsin B. Although, none of the compounds showed promising inhibitory activity, (E)N-{(S)1-[(S)2-cyano-1-pyrrolidinecarbonyl]-3-methylbutyl}-2,3-diphenylacrylamide (24) with an isobutyl moiety at P₂ was found to show selectivity as a cathepsin L inhibitor (Ki 5.3 μM for cathepsin L and Ki > 100 μM for cathepsin B). This compound could act as a new lead for the further development of improved inhibitors within this inhibitor type.     

SAR studies of some acetophenone phenylhydrazone based pyrazole derivatives as anticathepsin agents

Cathepsins have emerged as promising molecular targets in a number of diseases such as Alzeimer’s, inflammation and cancer. Elevated cathepsin’s levels and decreased cellular inhibitor concentrations have emphasized the search for novel inhibitors of cathepsins. The present work is focused on the design and synthesis of some acetophenone phenylhydrazone based pyrazole derivatives as novel non peptidyl inhibitors of cathepsins B, H and L. The synthesized compounds after characterization have been explored for their inhibitory potency against cathepsins B, H and L. The results show that some of the synthesized compounds exhibit anti-catheptic activity with K_i value of the order of 10⁻¹⁰ M. Differential inhibitory effects have been observed for cathepsins B, H and L. Cathepsin L is inhibited more pronounced than cathepsin B and cathepsin H in that order.     

Functional in vivo imaging of cysteine cathepsin activity in murine model of inflammation

Near-infrared fluorophore (NIRF)-labeled imaging probes are becoming increasingly important in bio-molecular imaging applications, that is, in animal models for tumor imaging or inflammation studies. In this study we showed that the previously introduced chemical concept of ‘Reverse Design’ represents an efficient strategy for the generation of selective probes for cysteine proteases from chemically optimized protease inhibitors for investigations in proteomic lysates as well as for in vivo molecular imaging studies. The newly developed activity-based probe AW-091 was demonstrated to be highly selective for cathepsin S in vitro and proved useful in monitoring cysteine cathepsin activity in vivo, that is, in zymosan-induced mouse model of inflammation. AW-091 showed higher signal-to-background ratios at earlier time points than the commercially available polymer-based ProSense680 (VisEn Medical) and thus represents an efficient new tool for studying early proteolytic processes leading to various diseases, including inflammation, cancer, and rheumatoid arthritis. In addition, the fluorescent signal originating from the cleaved AW-091 was shown to be reduced by the administration of an anti-inflammatory drug, dexamethasone and by the cathepsin inhibitor E-64, providing a valuable system for the evaluation of small-molecule inhibitors of cathepsins.     

Macrobrachium rosenbergii cathepsin L: Molecular characterization and gene expression in response to viral and bacterial infections

Cathepsin L (MrCathL) was identified from a constructed cDNA library of freshwater prawn Macrobrachium rosenbergii. MrCathL full-length cDNA is 1161 base pairs (bp) with an ORF of 1026 bp which encodes a polypeptide of 342 amino acid (aa) long. The eukaryotic cysteine proteases, histidine and asparagine active site residues were identified in the aa sequence of MrCathL at 143–154, 286–296 and 304–323, respectively. The pair wise clustalW analysis of MrCathL showed the highest similarity (97%) with the homologous cathepsin L from Macrobrachium nipponense and the lowest similarity (70%) from human. Phylogenetic analysis revealed two distinct clusters of the invertebrates and vertebrates cathepsin L in the phylogenetic tree. MrCathL and cathepsin L from M. nipponense were clustered together, formed a sister group to cathepsin L of Penaeus monodon, and finally clustered to Lepeophtheirus salmonis. High level of (P < 0.05) MrCathL gene expression was noticed in haemocyte and lowest in eyestalk. Furthermore, the MrCathL gene expression in M. rosenbergii was up-regulated in haemocyte by virus [M. rosenbergii nodovirus (MrNV) and white spot syndrome baculovirus (WSBV)] and bacteria (Vibrio harveyi and Aeromonas hydrophila). The recombinant MrCathL exhibited a wide range of activity in various pH between 3 and 10 and highest at pH 7.5. Cysteine proteinase (stefin A, stefin B and antipain) showed significant influence (100%) on recombinant MrCathL enzyme activity. The relative activity and residual activity of recombinant MrCathL against various metal ions or salts and detergent tested at different concentrations. These results indicated that the metal ions, salts and detergent had an influence on the proteinase activity of recombinant MrCathL. Conclusively, the results of this study imply that MrCathL has high pH stability and is fascinating object for further research on the function of cathepsin L in prawn innate immune system.     

Identification of a novel series of potent and selective CCR6 inhibitors as biological probes

CCR6 has been implicated in both autoimmune diseases and non-autoimmune diseases. Thus, inhibition of CCR6-dependent cell migration is an attractive strategy for their treatment. An orally available small molecule inhibitor of CCR6 could therefore be a useful biological probe for the pathophysiological studies. Initial SAR study of a hit compound provided potent N-benzenesulfonylpiperidine derivatives that suppressed CCL20-induced Gi signals. By subsequent scaffold morphing of the central ring and further optimization, we identified a novel series of 1,4-trans-1-benzenesulfonyl-4-aminocyclohexanes as potent and selective CCR6 inhibitors with good pharmacokinetic properties. Our compounds showed good correlation between Gi signal inhibitory activity and cell migration inhibitory activity in human CCR6-transfected CHO cells. In addition, representative compound 35 potently inhibited CCR6-dependent cell migration and the increase in ERK phosphorylation in human primary cells. Therefore, the compound could be used effectively as a biological probe against human CCR6.     

Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion

Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18-19, 8-9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides.     

Hypothetical cathepsin-like protein from Nematostella vectensis and its silicatein-like cathepsin mutant for biosilica production

Silicatein has high sequence identity and similarity with that of cathepsin L. In silicatein, serine replaces the active-site cysteine that is found in cathepsin L. Here, we obtained hypothetical cathepsin-like protein (CAT) from Nematostella vectensis which is 55% identical and 75% similar to mature silicatein alpha (SIL) of Suberites domuncula. When this protein was expressed in Escherichia coli, it displayed protease activity with both N-carbobenzoxy-l-phenylalanyl-l-arginine-4-methylcoumaryl-7-amide (Z-FR-AMC) and gelatin substrates, as well as silica-condensing activity using the tetraethoxy silane (TEOS) substrate. To increase its silica-forming activity and stability, some residues including the active site cysteine, were mutated into conserved silicatein residues, resulting in a mutant with 65% identity and 79% similarity to SIL. The mutant silicatein-like cathepsin (SLC) had increased expression levels in E. coli, and silica-forming activity comparable to that of SIL. In addition, SLC exhibited decreased protease activity as compared to that of CAT. Both CAT and SLC produced silica particles of sizes smaller than 50 nm, which increased to 200–300 nm in the presence of a structure-directing agent, such as Triton X-100. In conclusion, CAT was evolved to function as a biosilica-forming protein, and SLC was engineered by mutating CAT residues into conserved SIL residues to produce various silica-based materials.     

Cloning and characterization of a basic cysteine-like protease (cathepsin L1) expressed in the gut of larval Diaprepes abbreviatus L. (Coleoptera: Curculionidae)

Diaprepes abbreviatus is an important pest that causes extensive damage to citrus in the USA. Analysis of an expressed sequence tag (EST) library from the digestive tract of larvae and adult D. abbreviatus identified cathepsins as major putative digestive enzymes. One class, sharing amino acid sequence identity with cathepsin L’s, was the most abundant in the EST dataset representing 14.4% and 3.6% of the total sequences in feeding larvae and adults, respectively. The predominant cathepsin (Da-CTSL1) among this class was further studied. Three dimensional modeling of the protein sequence showed that the mature Da-CTSL1 protein folds into an expected cathepsin L structure producing a substrate binding pocket with appropriate positioning of conserved amino acid residues. A full-length cDNA was obtained and the proCTSL1 encoding sequence was expressed in Rosetta™ Escherichia coli cells engineered to express tRNAs specific for eukaryotic codon usage. The Da-CTSL1 was expressed as a fusion protein with GST and His₆ tags and purified in the presence of 1% Triton X-100 by Ni-NTA affinity and size exclusion chromatography. Recombinant mature Da-CTSL1 (23 KDa) exhibits optimal activity at pH 8, rather than at acidic pH that was shown of all previously characterized cathepsins L. Substrate specificity supports the hypothesis that Da-CTSL1 is a unique basic cathepsin L and protease inhibitor studies also suggest unique activity, unlike other characterized acidic cathepsin Ls. This paper describes for the first time a prokaryotic expression system for the production of a functional eukaryotic cathepsin L1 from larval gut of D. abbreviatus.     

Unique neuronal functions of cathepsin L and cathepsin B in secretory vesicles: biosynthesis of peptides in neurotransmission and neurodegenerative disease

Proteases are required for the production of peptide neurotransmitters and toxic peptides in neurodegenerative diseases. Unique roles of the cysteine proteases cathepsin L and cathepsin B in secretory vesicles for the production of biologically active peptides have been demonstrated in recent studies. Secretory vesicle cathepsin L participates in the proteolytic conversion of proenkephalin into the active enkephalin, an opioid peptide neurotransmitter that mediates pain relief. Moreover, recent findings provide evidence that cathepsin B in regulated secretory vesicles participates in the production of toxic beta-amyloid peptides that are known to accumulate extracellularly in Alzheimer's disease brains. The neurobiological functions of cathepsins L and B demonstrate that these secretory vesicle cysteine proteases produce biologically active peptides. These results demonstrate newly identified roles for cathepsins L and B in neurosecretory vesicles in the production of biologically active peptides.