Comprehensive characterization of the Published Kinase Inhibitor Set.

Despite the success of protein kinase inhibitors as approved therapeutics, drug discovery has focused on a small subset of kinase targets. Here we provide a thorough characterization of the Published Kinase Inhibitor Set (PKIS), a set of 367 small-molecule ATP-competitive kinase inhibitors that was recently made freely available with the aim of expanding research in this field and as an experiment in open-source target validation. We screen the set in activity assays with 224 recombinant kinases and 24 G protein-coupled receptors and in cellular assays of cancer cell proliferation and angiogenesis. We identify chemical starting points for designing new chemical probes of orphan kinases and illustrate the utility of these leads by developing a selective inhibitor for the previously untargeted kinases LOK and SLK. Our cellular screens reveal compounds that modulate cancer cell growth and angiogenesis in vitro. These reagents and associated data illustrate an efficient way forward to increasing understanding of the historically untargeted kinome.

Activity, specificity, and probe design for the smallpox virus protease K7L.

The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

Functional characterization of a SUMO deconjugating protease of Plasmodium falciparum using newly identified small molecule inhibitors.

Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum.

Distribution and paralogue specificity of mammalian deSUMOylating enzymes.

The covalent attachment of SUMO (small ubiquitin-like protein modifier) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalysed by SENPs (SUMO-specific processing proteases). Mammals contain four SUMO paralogues and six SENP enzymes. In the present paper, we describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal cSENPs (SENP catalytic domains). We first characterized the reaction of each endogenous SENP and cSENPs with HA-SUMO-VS [HA (haemagglutinin)-tagged SUMO-vinyl sulfones], active-site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogues, whereas all other endogenous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1- or SUMO2-AMC (7-amino-4-methylcoumarin) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by Km and kcat, whereas cSENP7 works only on SUMO2. Compared with cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity, indicating that paralogue specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggest that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.

Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes).

DUBs (deubiquitinating enzymes) are a family of proteases responsible for the specific removal of ubiquitin attached to target proteins and thus control the free cellular pools of this molecule. DUB activity is usually assayed using full-length ubiquitin, and these enzymes generally show low activity towards small substrates that constitute the P4-P1 LRGG (Lys-Arg-Gly-Gly) C-terminal motif of ubiquitin. To gain insight into the C-terminal recognition region of ubiquitin by DUBs, we synthesized positional scanning libraries of fluorigenic tetrapeptides and tested them on three examples of human DUBs [OTU-1 (ovarian tumour 1), Iso-T (isopeptidase T) and UCH-L3 (ubiquitin C-terminal hydrolase L3)] and one viral ubiquitin-specific protease, namely PLpro (papain-like protease) from SARS (severe acute respiratory syndrome) virus. In most cases the results show flexibility in the P4 position, very high specificity for arginine in the P3 position and glycine in the P2 position, in accord with the sequence of the natural substrate, ubiquitin. Surprisingly, screening of the P2 position revealed that UCH-L3, in contrast with all the other tested DUBs, demonstrates substantial tolerance of alanine and valine at P2, and a parallel analysis using the appropriate mutation of the full-length ubiquitin confirms this. We have also used an optimal tetrapeptide substrate, acetyl-Lys-Arg-Gly-Gly-7-amino-4-methylcoumarin, to investigate the activation mechanism of DUBs by ubiquitin and elevated salt concentration. Together, our results reveal the importance of the dual features of (1) substrate specificity and (2) the mechanism of ubiquitin binding in determining deubiquitination by this group of proteases.

Activity profiling of human deSUMOylating enzymes (SENPs) with synthetic substrates suggests an unexpected specificity of two newly characterized members of the family.

SENPs [Sentrin/SUMO (small ubiquitin-related modifier)-specific proteases] include proteases that activate the precursors of SUMOs, or deconjugate SUMOs attached to target proteins. SENPs are usually assayed on protein substrates, and for the first time we demonstrate that synthetic substrates can be convenient tools in determining activity and specificity of these proteases. We synthesized a group of short synthetic peptide fluorogenic molecules based on the cleavage site within SUMOs. We demonstrate the activity of human SENP1, 2, 5, 6, 7 and 8 on these substrates. A parallel positional scanning approach using a fluorogenic tetrapeptide library established preferences of SENPs in the P3 and P4 positions that allowed us to design optimal peptidyl reporter substrates. We show that the specificity of SENP1, 2, 5 and 8 on the optimal peptidyl substrates matches their natural protein substrates, and that the presence of the SUMO domain enhances catalysis by 2-3 orders of magnitude. We also show that SENP6 and 7 have an unexpected specificity that distinguishes them from other members of the family, implying that, in contrast to previous predictions, their natural substrate(s) may not be SUMO conjugates.

Small ubiquitin-related modifier (SUMO)-specific proteases: profiling the specificities and activities of human SENPs.

SENPs are proteases that participate in the regulation of SUMOylation by generating mature small ubiquitin-related modifiers (SUMO) for protein conjugation (endopeptidase activity) and removing conjugated SUMO from targets (isopeptidase activity). Using purified recombinant catalytic domains of 6 of the 7 human SENPs, we demonstrate the specificity of their respective activities on SUMO-1, -2, and -3. The primary mode of recognition of substrates is via the SUMO domain, and the C-terminal tails direct endopeptidase specificity. Broadly speaking, SENP1 is the most efficient endopeptidase, whereas SENP2 and -5-7 have substantially higher isopeptidase than endopeptidase activities. We developed fluorogenic tetrapeptide substrates that are cleaved by SENPs, enabling us to characterize the environmental profiles of each enzyme. Using these synthetic substrates we reveal that the SUMO domain enhances catalysis of SENP1, -2, -5, -6, and -7, demonstrating substrate-induced activation of SENPs by SUMOs.

Design, synthesis, and evaluation of aza-peptide Michael acceptors as selective and potent inhibitors of caspases-2, -3, -6, -7, -8, -9, and -10.

Aza-peptide Michael acceptors are a novel class of inhibitors that are potent and specific for caspases-2, -3, -6, -7, -8, -9, and -10. The second-order rate constants are in the order of 10(6) M(-1) s(-1). The aza-peptide Michael acceptor inhibitor 18t (Cbz-Asp-Glu-Val-AAsp-trans-CH=CH-CON(CH(2)-1-Naphth)(2) is the most potent compound and it inhibits caspase-3 with a k(2) value of 5620000 M(-1) s(-1). The inhibitor 18t is 13700, 190, 6.4, 594, 37500, and 173-fold more selective for caspase-3 over caspases-2, -6, -7, -8, -9, and -10, respectively. Aza-peptide Michael acceptors designed with caspase specific sequences are selective and do not show any cross reactivity with clan CA cysteine proteases such as papain, cathepsin B, and calpains. High-resolution crystal structures of caspase-3 and caspase-8 in complex with aza-peptide Michael acceptor inhibitors demonstrate the nucleophilic attack on C2 and provide insight into the selectivity and potency of the inhibitors with respect to the P1' moiety.

Tumor-associated alterations in caspase-14 expression in epithelial malignancies.

PURPOSE: Caspase-14 is unique among caspase family proteases in that its proteolytic processing has been principally associated with epithelial cell differentiation rather than apoptosis or inflammation. We investigated caspase-14 expression in several types of human epithelial malignancy by immunohistochemistry, correlating results with stage, histologic grade, and patient survival. EXPERIMENTAL DESIGN: Tumor-associated alterations in caspase-14 expression were observed for cervical, ovarian, breast, gastric, and colon cancers. RESULTS: In cervical (n = 445), ovarian (n = 91), and colon (n = 106) specimens, expression of caspase-14 was significantly reduced in cancers compared with normal epithelium. Decreases in caspase-14 immunopositivity correlated with the histologic progression of cervical cancer (P < 0.0001, ANOVA). In localized gastric cancers, caspase-14 immunostaining was significantly lower in poorly differentiated tumors compared with well-differentiated tumors (P = 0.02, Pearson's chi(2) analysis). Lower caspase-14 expression was associated with advanced clinical stage in ovarian cancer (P = 0.04, ANOVA) and with shorter overall survival among ovarian cancer patients with serous tumors (n = 62) in both univariate (P = 0.005) and multivariate (P = 0.03) analysis. Lower caspase-14 expression correlated with shorter overall survival among patients with T(3)N(0)M(0) stage gastric cancers (n = 94; P = 0.006, log-rank test). In contrast to cervical, ovarian, and colon cancers, caspase-14 expression was increased in ductal carcinoma in situ and invasive cancers compared with normal mammary epithelium (P = 0.001, t test). CONCLUSIONS: The findings reveal tumor-specific alterations in caspase-14 expression and suggest that differences in its expression may define subsets of epithelial cancers with distinct clinical behaviors.

Early processing of Bid and caspase-6, -8, -10, -14 in the canine brain during cardiac arrest and resuscitation.

A clinically relevant model of transient global brain ischemia involving cardiac arrest followed by resuscitation in dogs was utilized to study the expression and proteolytic processing of apoptosis-regulatory proteins. In the hippocampus, an increase in pro-apoptotic Bcl-2 family proteins Bcl-XS and Bak was detected, concomitant with proteolysis of Bcl-XL and Bcl-2, following ischemia-reperfusion injury. Also, biphasic cleavage of Bid was found in this region of the brain, with early generation of tBid-p11 within 10 min of cardiac arrest, followed by generation of tBid-p15 within 30-min reperfusion, consistent with activation of this pro-apoptotic protein. In addition, cardiac arrest and resuscitation induced early, reperfusion-dependent proteolytic processing of pro-caspase-6, -8, -10, and -14, which preceded caspase-3 activation. Immunohistochemical analysis using antibodies, which preferentially recognize processed caspase-3, -6, -8, and -10, provided evidence of time-dependent activation of these proteases in both neurons and glia in ischemia-sensitive regions of the brain. In conclusion, extremely rapid, cell-selective processing of apoptosis-regulatory proteins occurs in a clinically relevant model of ischemic brain injury caused by cardiac arrest and resuscitation. The early cleavage of Bid and rapid depletion of 32-kDa pro-caspase-14 from the canine hippocampus after induction of ischemia suggests the involvement of calpains in the processing of these proteins. Demonstration of in vitro cleavage of recombinant mouse caspase-14 by calpain I in the present study lends support to this hypothesis, further implicating cross-talk between different protease families in the pathophysiology of ischemic neural cell death.

Activation and substrate specificity of caspase-14.

Caspase-14 is a developmentally regulated and tissue restricted member of the caspase family present in mammals. It is mainly found in epidermal keratinocytes and has been hypothesized to be involved in a tissue-specific form of cell senescence, leading to the differentiation of keratinocytes that form the cornified cell layer. However, the substrate specificity, activation mechanism, and function of this caspase have yet to be revealed. We report that caspase-14, in contrast to other caspases, is not produced in active form following expression in Escherichia coli but can be activated by high concentrations of kosmotropic salts. Moreover, proteolytic cleavage is also required since the kosmotropic salts were only effective on the cleaved enzyme. We propose that caspase-14 requires proteolytic cleavage within the catalytic domain, followed by dimerization and ordering of mobile active site loops, to generate a competent enzyme. In the presence of kosmotropic salt, we were able to determine the substrate specificities of mouse and human caspase-14. Surprisingly, the substrate preferences for the human and mouse enzyme are dissimilar. The results obtained with human caspase-14 classify this enzyme as a cytokine activator, but the mouse enzyme shows preferences similar to apical apoptotic caspases.

Aza-peptide Michael acceptors: a new class of inhibitors specific for caspases and other clan CD cysteine proteases.

Aza-peptide Michael acceptors are a new class of irreversible inhibitors that are highly potent and specific for clan CD cysteine proteases. The aza-Asp derivatives were specific for caspases, while aza-Asn derivatives were effective legumain inhibitors. Aza-Lys and aza-Orn derivatives were potent inhibitors of gingipain K and clostripain. Aza-peptide Michael acceptors showed no cross reactivity toward papain, cathepsin B, and calpain.

Design, synthesis, and evaluation of aza-peptide epoxides as selective and potent inhibitors of caspases-1, -3, -6, and -8.

Aza-peptide epoxides, a novel class of irreversible protease inhibitors, are specific for the clan CD cysteine proteases. Aza-peptide epoxides with an aza-Asp residue at P1 are excellent irreversible inhibitors of caspases-1, -3, -6, and -8 with second-order inhibition rates up to 1 910 000 M(-1) s(-1). In general, the order of reactivity of aza-peptide epoxides is S,S > R,R > trans > cis. Interestingly, some of the R,R epoxides while being less potent are actually more selective than the S,S epoxides. Our aza-peptide epoxides designed for caspases are stable, potent, and specific inhibitors, as they show little to no inhibition of other proteases such as the aspartyl proteases porcine pepsin, human cathepsin D, plasmepsin 2 from P. falciparum, HIV-1 protease, and the secreted aspartic proteinase 2 (SAP-2) from Candida albicans; the serine proteases granzyme B and alpha-chymotrypsin; and the cysteine proteases cathepsin B and papain (clan CA), and legumain (clan CD).

Sequential autolytic processing activates the zymogen of Arg-gingipain.

Most proteases are synthesized as inactive precursors to protect the synthetic machinery of the cell and allow timing of activation. The mechanisms used to render latency are varied but tend to be conserved within protease families. Proteases belonging to the caspase family have a unique mechanism mediated by transitions of two surface loops, and on the basis of conservation of mechanism one would expect this to be preserved by caspase relatives. We have been able to express the full-length precursor of the Arg-specific caspase relative from the bacterium Porphyromonas gingivalis, Arg-gingipain-B, and we show that it contains N- and C-terminal extensions that render a low amount of latency, meaning that the zymogen is substantially active. Three sequential autolytic processing steps at the N and C terminus are required for full activity, and the N-propeptide may serve as an intramolecular chaperone rather than an inhibitory peptide. Each step in activation requires the previous step, and an affinity probe reveals that incremental activity enhancements are achieved in a stepwise manner.

Aza-peptide epoxides: a new class of inhibitors selective for clan CD cysteine proteases.

Aza-peptide epoxides, a new class of irreversible protease inhibitors, are specific for the clan CD cysteine proteases. The inhibitors have second-order rate constants up to 10(5) M(-1) s(-1), with the most potent epoxides having the S,S stereochemistry. The aza-Asn derivatives are effective legumain inhibitors, while the aza-Asp epoxides were specific for caspases. The inhibitors have little or no inhibition with other proteases such as chymotrypsin, papain, or cathepsin B.