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JAK2-V617F is the most frequent somatic mutation causing myeloproliferative neoplasm (MPN). However,JAK2-V617F can also be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the prevalence of MPN. The factors controlling the conversion ofJAK2-V617F CHIP to MPN are largely unknown. We hypothesized that IL-1β mediated inflammation is one of the factors that favors this progression. We examined mono- or oligoclonal evolution of MPN by performing bone marrow transplantations at limiting dilutions with only 1-3JAK2-mutant HSCs per recipient. Genetic loss ofIL-1βinJAK2-mutant hematopoietic cells or inhibition by a neutralizing anti-IL-1β antibody restricted the early clonal expansion of theseJAK2-mutant HSCs resulting in a reduced frequency of a CHIP-like state and a lower rate of conversion to MPN. The MPN disease-promoting effects of IL-1β were associated with damage to sympathetic innervation leading to loss of nestin-positive mesenchymal stromal cells and required the presence ofIL-1R1on bone marrow stromal cells. The anti-IL-1β antibody protected these mesenchymal stromal cells from IL-1β mediated damage and limited the expansion of theJAK2-mutant clone. Our results identify IL-1β as a potential therapeutic target for preventing the transition fromJAK2-V617F CHIP to MPN.Brief summaryIn a mouse model of oligo-clonal myeloproliferative neoplasm (MPN), IL-1β produced byJAK2-mutant cells favored expansion of sub-clinicalJAK2-V617F clones and initiation of MPN disease.

Supplementary Figure 4 from Tumor Detection by Imaging Proteolytic Activity

Supplementary Figure 2 from Tumor Detection by Imaging Proteolytic Activity

Supplementary Figure 1 from Tumor Detection by Imaging Proteolytic Activity

The cell surface protease membrane-type serine protease-1 (MT-SP1), also known as matriptase, is often upregulated in epithelial cancers. We hypothesized that dysregulation of MT-SP1 with regard to its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1), a situation that increases proteolytic activity, might be exploited for imaging purposes to differentiate malignant from normal tissue. In this study, we show that MT-SP1 is active on cancer cells and that its activity may be targeted in vivo for tumor detection. A proteolytic activity assay with several MT-SP1–positive human cancer cell lines showed that MT-SP1 antibodies that inhibit recombinant enzyme activity in vitro also bind and inhibit the full-length enzyme expressed on cells. In contrast, in the same assay, MT-SP1–negative cancer cell lines were inactive. Fluorescence microscopy confirmed the cell surface localization of labeled antibodies bound to MT-SP1–positive cells. To evaluate in vivo targeting capability, 0.7 to 2 nmoles of fluorescently labeled antibodies were administered to mice bearing tumors that were positive or negative for MT-SP1. Antibodies localized to MT-SP1–positive tumors (n = 3), permitting visualization of MT-SP1 activity, whereas MT-SP1–negative tumors (n = 2) were not visualized. Our findings define MT-SP1 activity as a useful biomarker to visualize epithelial cancers using a noninvasive antibody-based method. Cancer Res; 70(4); 1505–12

Inflammasomes are intracellular protein complexes that promote an inflammatory host defense in response to pathogens and damaged or neoplastic tissues and are implicated in inflammatory disorders and therapeutic-induced toxicity. We investigated the mechanisms of activation for inflammasomes nucleated by NOD-like receptor (NLR) proteins. A screen of a small-molecule library revealed that several tyrosine kinase inhibitors (TKIs)—including those that are clinically approved (such as imatinib and crizotinib) or are in clinical trials (such as masitinib)—activated the NLRP3 inflammasome. Furthermore, imatinib and masitinib caused lysosomal swelling and damage independently of their kinase target, leading to cathepsin-mediated destabilization of myeloid cell membranes and, ultimately, cell lysis that was accompanied by potassium (K + ) efflux, which activated NLRP3. This effect was specific to primary myeloid cells (such as peripheral blood mononuclear cells and mouse bone marrow–derived dendritic cells) and did not occur in other primary cell types or various cell lines. TKI-induced lytic cell death and NLRP3 activation, but not lysosomal damage, were prevented by stabilizing cell membranes. Our findings reveal a potential immunological off-target of some TKIs that may contribute to their clinical efficacy or to their adverse effects.

The NLRP3 inflammasome is a critical component of the innate immune response to sterile inflammation. Its regulation involves a priming step, required for up-regulation of inflammasome protagonists and an activation step leading to NLRP3 inflammasome complex assembly, which triggers caspase-1 activity. The IκKβ kinase regulates canonical NF-κB, a key pathway involved in transcriptional priming. We found that IκKβ also regulates the activation and function of the NLRP3 inflammasome beyond the priming step. Two unrelated IκKβ inhibitors, AFN700 and TPCA-1, when applied after priming, fully blocked IL-1β secretion triggered by nigericin in THP-1 cells. Both inhibitors prevented neither inflammasome assembly, as monitored by measuring the formation of ASC specks, nor the generation of caspase-1 p20, a hallmark of caspase-1 activity, but they impaired the initial cleavage and activation of procaspase-1. These data thus indicate that IκKβ activity is required for efficient activation of NLRP3, suggesting that IκKβ may fulfill a dual role in coupling priming and activation of the NLRP3 inflammasome.

Inflammasomes are intracellular protein complexes that control proteolytic maturation and secretion of inflammatory interleukin-1 (IL-1) family cytokines and are thus important in host defense. While some inflammasomes are activated simply by binding to pathogen-derived molecules, others, including those nucleated by NLRP3 and NLRP1, have more complex activation mechanisms that are not fully understood. We screened a library of small molecules to identify new inflammasome activators that might shed light on activation mechanisms. In addition to validating dipeptidyl peptidase (DPP) inhibitors as NLRP1 activators, we find that clinical tyrosine kinase inhibitors (TKIs) including imatinib and masitinib activate the NLRP3 inflammasome. Mechanistically, these TKIs cause lysosomal swelling and damage, leading to cathepsin-mediated destabilization of myeloid cell membranes and cell lysis. This is accompanied by potassium (K+) efflux, which activates NLRP3. Both lytic cell death and NLRP3 activation but not lysosomal damage induced by TKIs are prevented by the cytoprotectant high molecular weight polyethylene glycol (PEG). Our study establishes a screening method that can be expanded for inflammasome research and immunostimulatory drug development, and provides new insight into immunological off-targets that may contribute to efficacy or adverse effects of TKIs.One Sentence SummaryA functional small molecule screen identifies imatinib, masitinib and other tyrosine kinase inhibitors that destabilize myeloid cell lysosomes, leading to cell lysis and K+ efflux-dependent NLRP3 inflammasome activation.

The NLRP3 inflammasome assembles in response to a variety of pathogenic and sterile danger signals, resulting in the production of interleukin-1β and interleukin-18. NLRP3 is a key component of the innate immune system and has been implicated as a driver of a number of acute and chronic diseases. We report the 2.8 A crystal structure of the NLRP3 NACHT domain in complex with an inhibitor. The structure defines a binding pocket formed by the four subdomains of the NACHT domain, and shows the inhibitor acts as an intramolecular glue, which locks the protein in an inactive conformation. It provides further molecular insight into our understanding of NLRP3 activation, helps to detail the residues involved in subdomain coordination within the NLRP3 NACHT domain, and gives molecular insights into how gain-of-function mutations de-stabilize the inactive conformation of NLRP3. Finally, it suggests stabilizing the auto-inhibited form of the NACHT domain is an effective way to inhibit NLRP3, and will aid the structure-based development of NLRP3 inhibitors for a range of inflammatory diseases.

An increasing number of nonantibody format proteins are entering clinical development. However, one of the major hurdles for the production of nonantibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics, and genetic knockdowns, we have identified, out of the >700 known proteases in rodents, matriptase-1 as the major protease involved in the degradation of recombinant proteins expressed in CHO-K1 cells. Subsequently, matriptase-1 was deleted in CHO-K1 cells using "transcription activator-like effector nucleases" (TALENs) as well as zinc-finger nucleases (ZFNs). This resulted in a superior CHO-K1 matriptase (KO) cell line with strongly reduced or no proteolytic degradation activity toward a panel of recombinantly expressed proteins. The matriptase KO cell line was evaluated in spike-in experiments and showed little or no degradation of proteins incubated in culture supernatant derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line. In summary, the combination of novel cell line engineering tools, next-generation sequencing screening methods, and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins.

The design and synthesis of macrocyclic inhibitors of human rhinovirus 3C protease is described. A macrocyclic linkage of the P1 and P3 residues, and the subsequent structure-based optimization of the macrocycle conformation and size led to the identification of a potent biochemical inhibitor 10 with sub-micromolar antiviral activity.

Myeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD), donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer, resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen-induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival, indicating in vivo biologic effects, no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1�� (IL-1��), we expected that IL-1�� KO donor MDSCs would be superior in subverting GVHD, but such MDSCs proved inferior relative to WT. IL-1�� release and IL-1 receptor expression was required for optimal MDSC function, and exogenous IL-1�� added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1�� could diminish survival in GVHD. However, loss of inflammasome activation and IL-1�� release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1�� signaling, improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum, the ATP release site, synergized with WT MDSCs, as did regulatory T-cell infusion, which we showed reduced but did not eliminate MDSC inflammasome activation, as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments.

Pyroptosis is a form of lytic inflammatory cell death driven by inflammatory caspase-1, caspase-4, caspase-5 and caspase-11. These caspases cleave and activate the pore-forming protein gasdermin D (GSDMD) to induce membrane damage. By contrast, apoptosis is driven by apoptotic caspase-8 or caspase-9 and has traditionally been classified as an immunologically silent form of cell death. Emerging evidence suggests that therapeutics designed for cancer chemotherapy or inflammatory disorders such as SMAC mimetics, TAK1 inhibitors and BH3 mimetics promote caspase-8 or caspase-9-dependent inflammatory cell death and NLRP3 inflammasome activation. However, the mechanism by which caspase-8 or caspase-9 triggers cell lysis and NLRP3 activation is still undefined. Here, we demonstrate that during extrinsic apoptosis, caspase-1 and caspase-8 cleave GSDMD to promote lytic cell death. By engineering a novel Gsdmd D88A knock-in mouse, we further demonstrate that this proinflammatory function of caspase-8 is counteracted by caspase-3-dependent cleavage and inactivation of GSDMD at aspartate 88, and is essential to suppress GSDMD-dependent cell lysis during caspase-8-dependent apoptosis. Lastly, we provide evidence that channel-forming glycoprotein pannexin-1, but not GSDMD or GSDME promotes NLRP3 inflammasome activation during caspase-8 or caspase-9-dependent apoptosis.

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi-protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill-characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro. We show that the N-terminal fragment of caspase-1-cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N-terminal fragment of caspase-1-cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome-inserted GSDMD at nanometer resolution by cryo-electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death., The EMBO Journal, 35 (16), ISSN:0261-4189, ISSN:1460-2075

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces.

Proteolytic enzymes are ubiquitous in all organisms and constitute 2–4% of the encoded gene products. They are critical for diverse biological processes such as digestion, blood clotting, host defense, pathogenic infection, viral replication, wound healing, and disease progression, to name a few. Because proteases trigger an irreversible event - the cleavage of a protein - their activity must be tightly controlled. Dysregulated proteolytic activity causes a disruption in the homeostatic balance of a biological system and can result in any number of poor biological outcomes. As a result, nature has developed a number of strategies to control proteolysis, including spatial and temporal regulation, zymogen activation and protease degradation, and through the inhibition of proteases by macromolecular inhibitors. Somewhat surprisingly, relatively few design principles underlie the mechanisms of inhibition of a myriad range of macromolecular protease inhibitors. Significant engineering efforts have gone into modifying and improving inhibitor potency and specificity, and to a large extent, the same design principles that work well for naturally occurring protease inhibitors have proved valuable for inhibitors developed in the laboratory. This review aims to survey the mechanisms by which macromolecular protease inhibitors function. To do this, inhibitors have been divided into categories based on their mechanism in order to illustrate that a relatively small number of design principles can be combined to develop new and effective protease inhibitors. These divisions are not strict, and many inhibitors could be grouped in a number of classes. The list of mechanisms presented here is not exhaustive in its treatment of all inhibitors, but aims to be illustrative of the many ways proteases can be inhibited. For more information on genome-wide protease mining,[1] protease mechanism,[2] pre-clinical inhibition,[3] and drug discovery efforts,[4] the reader is directed to excellent reviews that have been written in recent years. Figure 1 provides an overview of basic substrate and protease nomenclature that will be used in this review. Open in a separate window Figure 1 (A) Diagram of a protease active site. A protease cleaves a peptide at the scissile bond, and has a number of specificity subsites, which determine protease specificity. Substrates bind to a protease with their non-prime residues on the N-terminal side of the scissile bond and their prime-side residues C-terminal to the scissile bond. The catalytic residues determine the class of protease. Serine, cysteine, and threonine proteases hydrolyze a peptide bond via a covalent acyl-enzyme intermediate, and aspartic, glutamic and metalloproteases activate a water molecule to hydrolyze the peptide bond in a non-covalent manner. (B) A serine protease (matriptase/MT-SP1, 1EAX.pdb) with the catalytic triad in yellow and the surface loops that surround the active site colored in blue. While the catalytic architecture of proteases is remarkably conserved, the surface loops are areas of high sequential and structural diversity.

The cell surface protease membrane-type serine protease 1 [MT-SP1]/matriptase is often upregulated in epithelial cancers. A dysregulation in MT-SP1/matriptase levels with respect to its cognate inhibitor hepatocyte growth factor activator inhibitor-1 [HAI-1] suggests that it is an increase in proteolytic activity that significantly differentiates malignant from normal tissue. Here we use antibodies to demonstrate that MT-SP1 is active on cancer cells and that this activity may be targeted for tumor detection in vivo. A proteolytic activity assay with the MT-SP1-positive human cancer cell lines MCF-7, HT29, LNCaP, and MDA-MB-468 showed that the antibodies, which inhibit recombinant catalytic MT-SP1, are able to bind and inhibit the full-length enzyme. The same experiment with the MT-SP1-negative breast cancer cell lines MDA-MB-231, COLO 320DM and HT1080 showed no inhibition of proteolysis. Fluorescent microscopy then confirmed localization of labeled antibodies to the surface of MT-SP1-positive cells. To evaluate these antibodies as probes for targeting MT-SP1 activity in vivo, 0.7-2 nanomoles of fluorescently labeled antibodies were administered to xenograft mouse cancer models. The antibodies localized to the MT-SP1-positive MCF-7 and MCF-7/Luc+ tumors (n=3), permitting visualization of MT-SP1 activity. Fluorescence was not observed in MT-SP1-negative MDA-MD-231/Luc+ tumors (n=2), suggesting that MT-SP1 activity is a novel biomarker for epithelial cancer and these antibodies provide a non-invasive method for detecting this activity in vivo.

The high degree of specificity displayed by antibodies often results in varying potencies against antigen orthologs, which can affect the efficacy of these molecules in different animal models of disease. We have used a computational design strategy to improve the species cross-reactivity of an antibody-based inhibitor of the cancer-associated serine protease MT-SP1. In silico predictions were tested in vitro, and the most effective mutation, T98R, was shown to improve antibody affinity for the mouse ortholog of the enzyme 14-fold, resulting in an inhibitor with a K(I) of 340 pM. This improved affinity will be valuable when exploring the role of MT-SP1 in mouse models of cancer, and the strategy outlined here could be useful in fine-tuning antibody specificity.

The vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition, but due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be greatly aided by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 Å resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold.

A multidisciplinary method combining transcriptional data, specificity profiling, and biological characterization of an enzyme may be used to predict novel substrates. By integrating protease substrate profiling with microarray gene coexpression data from nearly 2,000 human normal and cancerous tissue samples, three fundamental components of a protease-activated signaling pathway were identified. We find that MT-SP1 mediates extracellular signaling by regulating the local activation of the prometastatic growth factor MSP-1. We demonstrate MT-SP1 expression in peritoneal macrophages, and biochemical methods confirm the ability of MT-SP1 to cleave and activate pro-MSP-1 in vitro and in a cellular context. MT-SP1 induced the ability of MSP-1 to inhibit nitric oxide production in bone marrow macrophages. Addition of HAI-1 or an MT-SP1-specific antibody inhibitor blocked the proteolytic activation of MSP-1 at the cell surface of peritoneal macrophages. Taken together, our work indicates that MT-SP1 is sufficient for MSP-1 activation and that MT-SP1, MSP-1, and the previously shown MSP-1 tyrosine kinase receptor RON are required for peritoneal macrophage activation. This work shows that this triad of growth factor, growth factor activator protease, and growth factor receptor is a protease-activated signaling pathway. Individually, MT-SP1 and RON overexpression have been implicated in cancer progression and metastasis. Transcriptional coexpression of these genes suggests that this signaling pathway may be involved in several human cancers.

The type II transmembrane serine protease family consists of 18 closely related serine proteases that are implicated in multiple functions. To identify selective, inhibitory antibodies against one particular type II transmembrane serine protease, matriptase [MT-SP1 (membrane-type serine protease 1)], a phage display library was created with a natural repertoire of Fabs [fragment antigen binding (Fab)] from human naive B cells. Fab A11 was identified with a 720 pM inhibition constant and high specificity for matriptase over other trypsin-fold serine proteases. A Trichoderma reesei system expressed A11 with a yield of ∼ 200 mg/L. The crystal structure of A11 in complex with matriptase has been determined and compared to the crystal structure of another antibody inhibitor (S4) in complex with matriptase. Previously discovered from a synthetic single-chain variable fragment library, S4 is also a highly selective and potent matriptase inhibitor. The crystal structures of the A11/matriptase and S4/matriptase complexes were solved to 2.1 A and 1.5 A, respectively. Although these antibodies, discovered from separate libraries, interact differently with the protease surface loops for their specificity, the structures reveal a similar novel mechanism of protease inhibition. Through the insertion of the H3 variable loop in a reverse orientation at the substrate-binding pocket, these antibodies bury a large surface area for potent inhibition and avoid proteolytic inactivation. This discovery highlights the critical role that the antibody scaffold plays in positioning loops to bind and inhibit protease function in a highly selective manner. Additionally, Fab A11 is a fully human antibody that specifically inhibits matriptase over other closely related proteases, suggesting that this approach could be useful for clinical applications.

Proteolytic enzymes are ubiquitous in all organisms and constitute 2–4% of the encoded gene products. They are critical for diverse biological processes such as digestion, blood clotting, host defense, pathogenic infection, viral replication, wound healing, and disease progression, to name a few. Because proteases trigger an irreversible event - the cleavage of a protein - their activity must be tightly controlled. Dysregulated proteolytic activity causes a disruption in the homeostatic balance of a biological system and can result in any number of poor biological outcomes. As a result, nature has developed a number of strategies to control proteolysis, including spatial and temporal regulation, zymogen activation and protease degradation, and through the inhibition of proteases by macromolecular inhibitors. Somewhat surprisingly, relatively few design principles underlie the mechanisms of inhibition of a myriad range of macromolecular protease inhibitors. Significant engineering efforts have gone into modifying and improving inhibitor potency and specificity, and to a large extent, the same design principles that work well for naturally occurring protease inhibitors have proved valuable for inhibitors developed in the laboratory. This review aims to survey the mechanisms by which macromolecular protease inhibitors function. To do this, inhibitors have been divided into categories based on their mechanism in order to illustrate that a relatively small number of design principles can be combined to develop new and effective protease inhibitors. These divisions are not strict, and many inhibitors could be grouped in a number of classes. The list of mechanisms presented here is not exhaustive in its treatment of all inhibitors, but aims to be illustrative of the many ways proteases can be inhibited. For more information on genome-wide protease mining,[1] protease mechanism,[2] pre-clinical inhibition,[3] and drug discovery efforts,[4] the reader is directed to excellent reviews that have been written in recent years. Figure 1 provides an overview of basic substrate and protease nomenclature that will be used in this review. Open in a separate window Figure 1 (A) Diagram of a protease active site. A protease cleaves a peptide at the scissile bond, and has a number of specificity subsites, which determine protease specificity. Substrates bind to a protease with their non-prime residues on the N-terminal side of the scissile bond and their prime-side residues C-terminal to the scissile bond. The catalytic residues determine the class of protease. Serine, cysteine, and threonine proteases hydrolyze a peptide bond via a covalent acyl-enzyme intermediate, and aspartic, glutamic and metalloproteases activate a water molecule to hydrolyze the peptide bond in a non-covalent manner. (B) A serine protease (matriptase/MT-SP1, 1EAX.pdb) with the catalytic triad in yellow and the surface loops that surround the active site colored in blue. While the catalytic architecture of proteases is remarkably conserved, the surface loops are areas of high sequential and structural diversity.

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.

Relatively few design principles underlie the mechanisms of inhibition of a myriad range of protease inhibitors. Protease inhibitors tend to be competitive and to compete with substrate binding, either through direct competition or deformation of the protease active site. Although protein inhibitors can gain potency through the burial of a large surface area and specificity through contacts with specific exosites, small-molecule inhibitors primarily gain potency through interactions with the catalytic machinery of the enzyme and specificity through interactions with the substrate binding sites. Incorporation of these design principles into chemical probes and drugs have improved greatly our ability to create potent and specific protease inhibitors.

Membrane-type serine protease 1 (MT-SP1) is a cancer-associated serine protease implicated in the tumorogenesis and metastasis of breast cancer. Inhibition of MT-SP1 activity has been shown to decrease metastatic potential. We have developed a number of potent and specific single-chain (scFv) antibody inhibitors to MT-SP1, and have begun to characterize their mechanism of inhibition. Through kinetic characterization and site-directed mutagenesis experiments, it has been determined that three potent inhibitors have separate and novel mechanisms of inhibition which do not mimic either biologically or pharmaceutically relevant protease inhibitors. These novel modes of binding and inhibition are the basis for their specificity, and suggest these inhibitors will have less cross-reactivity and toxicity problems when used in vivo to further dissect the role of MT-SP1 in breast cancer.

The mechanisms of inhibition of two novel scFv antibody inhibitors of the serine protease MT-SP1/matriptase reveal the basis of their potency and specificity. Kinetic experiments characterize the inhibitors as extremely potent inhibitors with K(I) values in the low picomolar range that compete with substrate binding in the S1 site. Alanine scanning of the loops surrounding the protease active site provides a rationale for inhibitor specificity. Each antibody binds to a number of residues flanking the active site, forming a unique three-dimensional binding epitope. Interestingly, one inhibitor binds in the active site cleft in a substrate-like manner, can be processed by MT-SP1 at low pH, and is a standard mechanism inhibitor of the protease. The mechanisms of inhibition provide a rationale for the effectiveness of these inhibitors, and suggest that the development of specific antibody-based inhibitors against individual members of closely related enzyme families is feasible, and an effective way to develop tools to tease apart complex biological processes.

Caspases, a family of apoptotic proteases, are increasingly recognized as being extensively phosphorylated, usually leading to inactivation. To date, no structural mechanism for phosphorylation-based caspase inactivation is available, although this information may be key to achieving caspase-specific inhibition. Caspase-6 has recently been implicated in neurodegenerative conditions including Huntington's and Alzheimer's diseases. A full understanding of caspase-6 regulation is crucial to caspase-6-specific inhibition. Caspase-6 is phosphorylated by ARK5 kinase at serine 257 leading to suppression of cell death via caspase-6 inhibition. Our structure of the fully inactive phosphomimetic S257D reveals that phosphorylation results in a steric clash with P201 in the L2′ loop. Removal of the proline side chain alleviates the clash resulting in nearly wild-type activity levels. This phosphomimetic-mediated steric clash causes misalignment of the substrate-binding groove, preventing substrate binding. Substrate-binding loop misalignment appears to be a widely used regulatory strategy among caspases and may present a new paradigm for caspase-specific control.