Factor VII is the coagulation protease responsible for starting a cascade of proteolytic events that lead to thrombin generation, fibrin deposition, and platelet activation. As such, FVII has ...attracted wide interest as a target for clinical anti-coagulant applications. Commensurate with the critical importance of maintaining balance between thrombosis and hemostasis and its place at the beginning of the coagulation process, FVII is subject to a variety of biological and biochemical control mechanisms, among them allosteric influences exerted by cofactors, substrates, and inhibitors. Sites on FVIIa where allosteric influences are exerted and manifested have been identified and characterized in considerable detail. In recent years, a three-dimensional context for the interpretation of these results has become available from structural studies. New X-ray structures have augmented specific aspects of our understanding, in particular the X-ray structure of a fragment of the FVII zymogen. This review summarizes general allosteric behaviors of FVIIa and recapitulates structural findings since 1996, with particular emphasis on the recently determined zymogen structure.
Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an integral membrane protein expressed on epithelial cells and contains two extracellular Kunitz domains (N-terminal KD1 and C-terminal KD2) ...known to inhibit trypsin-like serine proteases. In tumorigenesis and tissue regeneration, HAI-1 regulates the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form. By screening a placental cDNA library, we discovered a new splice variant of HAI-1 designated HAI-1B that contains an extra 16 amino acids adjacent to the C terminus of KD1. To investigate possible consequences on Kunitz domain function, a soluble form of HAI-1B (sHAI-1B) comprising the entire extracellular domain was produced. First, we found that sHAI-1B displayed remarkable enzyme specificity by potently inhibiting only HGFA (IC50 = 30.5 nm), matriptase (IC50 = 16.5 nm), and trypsin (IC50 = 2.4 nm) among 16 serine proteases examined, including plasminogen activators (urokinase- and tissue-type plasminogen activators), coagulation enzymes thrombin, factors VIIa, Xa, XIa, and XIIa, and activated protein C. Relatively weak inhibition was found for plasmin (IC50 = 399 nm) and plasma kallikrein (IC50 = 686 nm). Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P1 residue-directed mutagenesis to show that inhibition of HGFA, matriptase, trypsin, and plasmin was due to KD1 and not KD2. Furthermore, analysis by reverse transcription-PCR demonstrated that HAI-1B and HAI-1 were co-expressed in normal tissues and various epithelial-derived cancer cell lines. Both isoforms were up-regulated in eight examined ovarian carcinoma specimens, three of which had higher levels of HAI-1B RNA than of HAI-1 RNA. Therefore, previously demonstrated roles of HAI-1 in various physiological and pathological processes likely involve both HAI-1B and HAI-1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Factor VIIa (FVIIa) is a key serine protease involved in the initiation of the coagulation cascade. It is a glycosylated disulfide-linked heterodimer comprised of an amino-terminal ?-carboxyglutamic ...acid-rich (Gla) domain and two epidermal growth factor (EGF)-like domains in the light chain, and a chymotrypsin-like serine protease domain in the heavy chain. FVIIa requires tissue factor (TF), a membrane bound protein, as an essential cofactor for maximal activity towards its biological substrates Factor X, Factor IX and Factor VII (FVII). Inhibition of TF • FVIIa activity may prevent the formation of fibrin clots and thus be useful in the management of thrombotic disease. The development of TF • FVIIa inhibitors to validate this target has been of great interest. A wide array of strategic approaches to inhibiting the biochemical and biological functions of the TF • FVIIa complex has been pursued. This has been greatly aided from our understanding of the structures for TF, FVII, FVIIa, and the TF • FVIIa complex. These approaches have resulted in inhibitors directed specifically towards either FVIIa or TF. Antagonists include active site inhibited FVIIa, TF mutants, anti-TF antibodies, anti-FVII / FVIIa antibodies, naturally-occurring protein inhibitors, peptide exosite inhibitors, and protein and small molecule active site inhibitors. These antagonists can inhibit catalysis directly at the active site as well as impair function by binding to exosites that may interfere with substrate, membrane, or cofactor binding. The rationale of TF • FVIIa as a target and the development, characteristics and biological uses of TF • FVIIa inhibitors are discussed.
Antibodies display great versatility in protein interactions and have become important therapeutic agents for a variety of human diseases. Their ability to discriminate between highly conserved ...sequences could be of great use for therapeutic approaches that target proteases, for which structural features are conserved among family members. Recent crystal structures of antibody-protease complexes provide exciting insight into the variety of ways antibodies can interfere with the catalytic machinery of serine proteases. The studies revealed the molecular details of two fundamental mechanisms by which antibodies inhibit catalysis of trypsin-like serine proteases, exemplified by hepatocyte growth factor activator and MT-SP1 (matriptase). Enzyme kinetics defines both mechanisms as competitive inhibition systems, yet, on the molecular level, they involve distinct structural elements of the active-site region. In the steric hindrance mechanism, the antibody binds to protruding surface loops and inserts one or two CDR (complementarity-determining region) loops into the enzyme's substrate-binding cleft, which results in obstruction of substrate access. In the allosteric inhibition mechanism the antibody binds outside the active site at the periphery of the substrate-binding cleft and, mediated through a conformational change of a surface loop, imposes structural changes at important substrate interaction sites resulting in impaired catalysis. At the centre of this allosteric mechanism is the 99-loop, which is sandwiched between the substrate and the antibody-binding sites and serves as a mobile conduit between these sites. These findings provide comprehensive structural and functional insight into the molecular versatility of antibodies for interfering with the catalytic machinery of proteases.
Limitations of current anticoagulant therapies have led us to develop two distinct classes of exosite peptide inhibitors for the initiator of the clotting process, the tissue factor-factor VIIa ...(TF·FVIIa) complex (Roberge, M., Santell, L., Dennis, M. S., Eigenbrot, C., Dwyer, M. A., and Lazarus, R. A. (2001) Biochemistry 40, 9522–9531). Although both peptide classes are potent and selective inhibitors of TF·FVIIa, neither showed 100% inhibition at saturating concentrations. Crystal structures of these peptides in complex with the FVII/FVIIa protease domain revealed their distinct binding sites and close proximity to the active site. The favorable orientation of the 15-mer A-site peptide A-183 (EEWEVLCWTWETCER) suggested that a C-terminal extension into the FVIIa active site could yield a chimeric inhibitor that was not only potent and selective but complete as well. A novel two-step “protease switch” approach using substrate phage display was developed by first binding all phage containing A-183 and C-terminal extension libraries to immobilized and inactive FVIIa. Upon altering pH and adding TF to switch on FVIIa enzymatic activity, only those phage released by proteolytic cleavage within the extension were propagated. This process selected for both preferred sequence and length in the extension, leading to a 27-mer peptide A-183X (EEWEVLCWTWETCERGEGVEEELWEWR) with a C-terminal 12-mer extension containing an Arg in the P1 position. A-183X was a more potent and complete inhibitor of FX activation, having a maximal extent of inhibition of ∼99% with an IC50 of 230 pmversus A-183 which maximally inhibited to 74% with an IC50 of 1.5 nm. A-183X also had a maximal prolongation of the prothrombin time of 7.6- versus 1.9-fold for A-183, making it a more effective anticoagulant.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Thousands of genetic variants have been identified, which contribute to the development of complex diseases, but determining how to elucidate their biological consequences for translation into ...clinical benefit is challenging. Conflicting evidence regarding the functional impact of genetic variants in the tyrosine kinase 2 (TYK2) gene, which is differentially associated with common autoimmune diseases, currently obscures the potential of TYK2 as a therapeutic target. We aimed to resolve this conflict by performing genetic meta-analysis across disorders; subsequent molecular, cellular, in vivo, and structural functional follow-up; and epidemiological studies. Our data revealed a protective homozygous effect that defined a signaling optimum between autoimmunity and immunodeficiency and identified TYK2 as a potential drug target for certain common autoimmune disorders.
PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong ...LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 μm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å2 largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 μm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the β-strand and a discontinuous short α-helix, and it engaged in the same β-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9.
Therapeutic inhibition of circulating PCSK9 reduces LDL-c levels.
A synthetic PCSK9-binding peptide, which restores cellular LDL receptors, was identified.
Pep2-8 is the smallest PCSK9 inhibitor with a defined inhibitory mechanism described to date and structurally mimics the EGF(A) domain of the receptor.
This work demonstrates the feasibility of developing a peptide-based inhibitor of PCSK9.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 ...somatic mutations across ∼1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for g subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a G subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF·FVIIa is also implicated in thrombosis-related disorders ...and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF·FVIIa (Ki = 0.35 ± 0.11 nm). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF·FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa·G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217–Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a “flipped” Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by ∼70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a ...therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
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