Dengue protease activity: the structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target

2011 ◽  
Vol 31 (5) ◽  
pp. 399-409 ◽  
Author(s):  
Wai Y. Phong ◽  
Nicole J. Moreland ◽  
Siew P. Lim ◽  
Daying Wen ◽  
Prasad N. Paradkar ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Protease Activity ◽  
Structural Integrity ◽  
Glutathione Transferase ◽  
Human Monoclonal Antibody ◽  
Site Formation ◽  
Single Chain ◽  
Ns3 Protease

Flaviviral NS3 serine proteases require the NS2B cofactor region (cNS2B) to be active. Recent crystal structures of WNV (West Nile virus) protease in complex with inhibitors revealed that cNS2B participates in the formation of the protease active site. No crystal structures of ternary complexes are currently available for DENV (dengue virus) to validate the role of cNS2B in active site formation. In the present study, a GST (glutathione transferase) fusion protein of DENV-2 cNS2B49–95 was used as a bait to pull down DENV-2 protease domain (NS3pro). The affinity of NS3pro for cNS2B was strong (equilibrium-binding constant <200 nM) and the heterodimeric complex displayed a catalytic efficiency similar to that of single-chain DENV-2 cNS2B/NS3pro. Various truncations and mutations in the cNS2B sequence showed that conformational integrity of the entire 47 amino acids is critical for protease activity. Furthermore, DENV-2 NS3 protease can be pulled down and transactivated by cNS2B cofactors from DENV-1, -3, -4 and WNV, suggesting that mechanisms for activation are conserved across the flavivirus genus. To validate NS2B as a potential target in allosteric inhibitor development, a cNS2B-specific human monoclonal antibody (3F10) was utilized. 3F10 disrupted the interaction between cNS2B and NS3 in vitro and reduced DENV viral replication in HEK (human embryonic kidney)-293 cells. This provides proof-of-concept for developing assays to find inhibitors that block the interaction between NS2B and NS3 during viral translation.

scholarly journals Reversible autoinhibitory regulation ofEscherichia colimetallopeptidase BepA for selective β-barrel protein degradation

2020 ◽  
Vol 117 (45) ◽  
pp. 27989-27996
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Ryoji Miyazaki ◽  
Yohei Hizukuri ◽  
Hiroyuki Mori ◽  
...  
Keyword(s):  
Active Site ◽  
Protease Activity ◽  
Underlying Mechanism ◽  
Zinc Ion ◽  
Wild Type ◽  
Protease Domain ◽  
Loop Region ◽  
Assembly Pathway

Escherichia coliperiplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246–mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

scholarly journals Crystal structures of hFPPS in complex with novel anticancer drug leads

2014 ◽  
Vol 70 (a1) ◽  
pp. C712-C712
Author(s):  
Jaeok Park ◽  
Chun Leung ◽  
Yih-Shyan Lin ◽  
Joris De Schutter ◽  
Youla Tsantrizos ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Crystal Structures ◽  
Anticancer Agents ◽  
Active Site ◽  
Farnesyl Pyrophosphate ◽  
Allosteric Site ◽  
Cellular Processes ◽  
Inhibitory Site ◽  
Active Site Cavity

Human farnesyl pyrophosphate synthase (hFPPS) produces farnesyl pyrophosphate, an isoprenoid required for a variety of essential cellular processes. Inhibition of hFPPS has been well established as the mechanism of action of the nitrogen-containing bisphosphonate (N-BP) drugs, currently best known for their anti-bone resorptive effects. Recent investigations indicate that hFPPS inhibition also produces potent anticancer effects both in vitro and vivo: N-BPs inhibit proliferation, motility, and viability of tumor cells, and act in synergy with other anticancer agents [1,2]. However, the physicochemical properties of the current N-BP drugs seriously compromise their full anticancer potential in non-skeletal tissues. They show poor membrane permeability and extreme affinity to bone, due mainly to their highly charged bisphosphonate moiety, which mimics the pyrophosphate of the substrates of hFPPS. Both the substrates and N-BPs bind to hFPPS via Mg ion-mediated interactions between their pyrophosphate/bisphosphonate moiety and two aspartate-rich surfaces of the enzyme's active site cavity. Recently, we took a structure-guided approach to develop bisphosphonates with higher lipophilicity for enhanced uptake into non-skeletal tissues. Surprisingly, some of the new compounds were found to bind to hFPPS even in the absence of Mg ions. Crystal structures of hFPPS in complex with a representative compound revealed that this bisphosphonate binds to the enzyme's active site in the presence of Mg ions, but also to a nearby allosteric inhibitory site in their absence. Furthermore, removal of a phosphonate group from the bisphosphonate moiety of this compound resulted in an inhibitor that binds exclusively to the allosteric site. Based on the crystal structures with these lead compounds, we generated of a novel class of non-bisphosphonate, allosteric inhibitors of hFPPS with superior physicochemical properties than those of the current N-BP drugs for broader tissue distribution.

A Novel Anti CD23 Fully Human Monoclonal Antibody Potentially Useful for B-CLL Therapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5047-5047
Author(s):  
Marc Delcommenne ◽  
Hans-Georg Klingemann ◽  
Stephanie A. Gregory
Keyword(s):  
Monoclonal Antibody ◽  
B Lymphocytes ◽  
Human Monoclonal Antibody ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Normal Peripheral Blood ◽  
Human Igg1

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common hematological malignancies and is, in most cases, characterized by an increased expression of CD23 on the cell surface. Since cross-linking CD23 induces B-CLL apoptosis, it is an attractive target for B-CLL antibody-based immunotherapy. In this study we show that an anti-CD23 human IgG1 monoclonal antibody, C6F5, may be useful in treating B-CLL. This antibody is derived from the human single chain antibody (scFv) C6F5 that was originally raised against the RPMI-8226 multiple myeloma cell line using the antibody phage display technique. While the C6F5 scFv did not bind to other myeloma cell lines, it was able to bind weakly to normal peripheral blood B lymphocytes and strongly to EBV transformed B cells and B-CLL cells. The antigen recognized by C6F5 was also upregulated on B lymphocytes that had been stimulated by CD40 ligand. Immunoprecipitations by the scFv C6F5 identified a protein of 45 kDa which co-migrated with CD23. Furthermore, this protein was recognized by an anti-CD23 mouse mAb in Western blot analyses. Immunofluorescence staining with the C6F5 scFv was inhibited if cells were preincubated with an anti-CD23 polyclonal antiserum. Taken together, these results verify that C6F5 recognizes CD23. The VH and VL regions of C6F5 antibody were then cloned into a baculovirus transfer vector encoding the human IgG1 heavy and light chains so that fully human C6F5 IgG1 antibody could be produced in baculovirus infected SF9 cells. Since C6F5 binding specificity was preserved in the IgG1 format, this antibody is ready to be tested in in vitro cytotoxic assays against B-CLL cells.

scholarly journals Protease Activity, Secretion, Cell Entry, Cytotoxicity, and Cellular Targets of Secreted Autotransporter Toxin of Uropathogenic Escherichia coli

2006 ◽  
Vol 74 (11) ◽  
pp. 6124-6134 ◽  
Author(s):  
Nathalie M. Maroncle ◽  
Kelsey E. Sivick ◽  
Rebecca Brady ◽  
Faye-Ellen Stokes ◽  
Harry L. T. Mobley
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Active Site ◽  
Protease Activity ◽  
Host Cells ◽  
Single Mutation ◽  
Wild Type ◽  
Passenger Domain ◽  
Hek 293

ABSTRACT The secreted autotransporter toxin (Sat), found predominantly in uropathogenic Escherichia coli, is a member of the SPATE (serine protease autotransporters of Enterobacteriaceae) family and, as such, has serine protease activity and causes cytopathic effects on various cell types. To assess the contribution of the serine protease active site to the mechanism of action of Sat, mutations were made in the first (S256I), in the second (S258A), or in both (S256I/S258A) serine residues within the active site motif. Mutations in the first or both serines reduced protease activity to background levels (P < 0.001); a single mutation in the second serine reduced activity by 60% compared to wild type (P < 0.001). After reversion of the S256I mutation to wild type (I256S), we confirmed S256 as the catalytically active serine. None of these mutations affected secretion of the mature passenger domain or release into the supernatant. The S256I mutation, however, abrogated the cytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK 293) epithelial cells, characterized by rounding and elongation, respectively, and a high level of cell detachment. Moreover, S256 is essential for Sat to mediate cytoskeletal contraction and actin loss in host cells as well as to degrade specific membrane/cytoskeletal (fodrin and leukocyte function-associated molecule 1) and nuclear [microtubule-associated proteins, LIM domain-only protein 7, Rap GTPase-activating protein, poly(ADP-ribose) polymerase] proteins in vitro. Lastly, Sat was internalized by host cells and localized to the cytoskeletal fraction where membrane/cytoskeletal target proteins reside.

Production by Thrombin of a Proteolytically Modified form of Antithrombin and Release of the Same form from the Antithrombin-Thrombin Complex

1979 ◽  
Author(s):  
W. Fish ◽  
I. Björk
Keyword(s):  
Protease Inhibitor ◽  
Active Site ◽  
Enzyme Inhibitor ◽  
Serine Protease Inhibitor ◽  
Prolonged Treatment ◽  
Stable Complex ◽  
Single Chain ◽  
Inhibitor Complex ◽  
Active Site Region

In addition to the in vitro formation of an inactive, stable complex (ATT) between thrombin (T) and the plasma serine protease inhibitor, antithrombin (AT), a sizeable proportion of AT is converted by T to a form which has lost the ability to inhibit T and which exhibits a reduced affinity for heparin. Under non-denaturing conditions, the isolated modified AT (ATm) could not be differentiated from native AT by hydrodynamic, electrophoretic or immunological analyses. Under reducing/denaturing conditions, ATm yielded two polypeptides, one of about 50000 and one of about 5000 daltons. Preliminary studies indicate that these are the result of a proteolytic cleavage by T of an arg-ser bond in the C-terminal end of the single-chain AT molecule. Dissociation of ATT by prolonged treatment in hot SDS or by treatment with hydoxylamine or ammonia in SDS produced only T and ATm. Under nondenaturing conditions, dissociation by the two latter agents released ATm and up to 40% of the potential T activity from ATT. However, under no conditions tested could intact AT be shown to be produced from dissociation of ATT. These results suggest that scission by T of a specific arg-ser bond in the active site region of AT, or, alternatively, conversion of this bond to a tetrahedral intermediate state, occurs during the formation of the inactive enzyme-inhibitor complex.

scholarly journals Structure-Based Virtual Screening: Identification of a Novel NS2B-NS3 Protease Inhibitor with Potent Antiviral Activity against Zika and Dengue Viruses

2021 ◽  
Vol 9 (3) ◽  
pp. 545
Author(s):  
Hye Jin Shin ◽  
Mi-Hwa Kim ◽  
Joo-Youn Lee ◽  
Insu Hwang ◽  
Gun Young Yoon ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Protease Activity ◽  
Therapeutic Option ◽  
Denv Infection ◽  
Antiviral Drugs ◽  
Protease Inhibition ◽  
Docking Analysis ◽  
Ns3 Protease ◽  
Protease Inhibition Assay

Zika virus (ZIKV), which is associated with severe diseases in humans, has spread rapidly and globally since its emergence. ZIKV and dengue virus (DENV) are closely related, and antibody-dependent enhancement (ADE) of infection between cocirculating ZIKV and DENV may exacerbate disease. Despite these serious threats, there are currently no approved antiviral drugs against ZIKV and DENV. The NS2B-NS3 viral protease is an attractive antiviral target because it plays a pivotal role in polyprotein cleavage, which is required for viral replication. Thus, we sought to identify novel inhibitors of the NS2B-NS3 protease. To that aim, we performed structure-based virtual screening using 467,000 structurally diverse chemical compounds. Then, a fluorescence-based protease inhibition assay was used to test whether the selected candidates inhibited ZIKV protease activity. Among the 123 candidate inhibitors selected from virtual screening, compound 1 significantly inhibited ZIKV NS2B-NS3 protease activity in vitro. In addition, compound 1 effectively inhibited ZIKV and DENV infection of human cells. Molecular docking analysis suggested that compound 1 binds to the NS2B-NS3 protease of ZIKV and DENV. Thus, compound 1 could be used as a new therapeutic option for the development of more potent antiviral drugs against both ZIKV and DENV, reducing the risks of ADE.

scholarly journals Reversible auto-inhibitory regulation of Escherichia coli metallopeptidase BepA for selective β-barrel protein degradation

2020 ◽  
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Ryoji Miyazaki ◽  
Yohei Hizukuri ◽  
Hiroyuki Mori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Protease Activity ◽  
Underlying Mechanism ◽  
Zinc Ion ◽  
Loop Region ◽  
Assembly Pathway ◽  
Inhibitory Regulation

AbstractEscherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD, but the underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features, in particular the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has a potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of the BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of the protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

scholarly journals Glutamate-64, a newly identified residue of the functionally conserved electron-sharing network contributes to catalysis and structural integrity of glutathione transferases

2007 ◽  
Vol 402 (2) ◽  
pp. 339-348 ◽  
Author(s):  
Pakorn Winayanuwattikun ◽  
Albert J. Ketterman
Keyword(s):  
Structural Integrity ◽  
Glutathione Transferase ◽  
Product Formation ◽  
Glutathione Transferases ◽  
Nucleophilic Attack ◽  
Anopheles Dirus ◽  
Catalytic Function ◽  
Equivalent Residue ◽  
Vitro Analysis

In Anopheles dirus glutathione transferase D3-3, position 64 is occupied by a functionally conserved glutamate residue, which interacts directly with the γ-glutamate moiety of GSH (glutathione) as part of an electron-sharing network present in all soluble GSTs (glutathione transferases). Primary sequence alignment of all GST classes suggests that Glu64 is one of a few residues that is functionally conserved in the GST superfamily. Available crystal structures as well as consideration of the property of the equivalent residue at position 64, acidic or polar, suggest that the GST electron-sharing motif can be divided into two types. Electrostatic interaction between the GSH glutamyl and carboxylic Glu64, as well as with Arg66 and Asp100, was observed to extend the electron-sharing motif identified previously. Glu64 contributes to the catalytic function of this motif and the ‘base-assisted deprotonation’ that are essential for GSH ionization during catalysis. Moreover, this residue also appears to affect multiple steps in the enzyme catalytic strategy, including binding of GSH, nucleophilic attack by thiolate at the electrophilic centre and product formation, probably through active-site packing effects. Replacement with non-functionally-conserved amino acids alters initial packing or folding by favouring aggregation during heterologous expression. Thermodynamic and reactivation in vitro analysis indicated that Glu64 also contributes to the initial folding pathway and overall structural stability. Therefore Glu64 also appears to impact upon catalysis through roles in both initial folding and structural maintenance.

scholarly journals An approach to optimizing the active site in a glutathione transferase by evolution in vitro

1999 ◽  
Vol 344 (1) ◽  
pp. 93-100 ◽  
Author(s):  
Lars O. HANSSON ◽  
Mikael WIDERSTEN ◽  
Bengt MANNERVIK
Keyword(s):  
Active Site ◽  
Glutathione Transferase ◽  
Single Point ◽  
Active Form ◽  
Catalytic Efficiency ◽  
Order Rate Constant ◽  
Single Point Mutation ◽  
Transition State Analogue ◽  
Active Site Mutants

A glutathione transferase (GST) mutant with four active-site substitutions (Phe10 → Pro/Ala12 → Trp/Leu107 → Phe/Leu108 → Arg) (C36) was isolated from a library of active-site mutants of human GST A1-1 by the combination of phage display and mechanism-based affinity adsorption [Hansson, Widersten and Mannervik (1997) Biochemistry 36, 11252-11260]. C36 was selected on the basis of its affinity for the transition-state analogue 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate. C36 affords a 105-fold rate enhancement over the uncatalysed reaction between reduced glutathione and 1-chloro-2,4-dinitrobenzene (CDNB), as evidenced by the ratio between kcat/Km and the second-order rate constant k2. The present study shows that C36 can evolve to an even higher catalytic efficiency by an additional site-specific mutation. Random mutations of the fifth active-site residue 208 allowed the identification of 18 variants, of which the mutant C36 Met208 → Cys proved to be the most active form. The altered activity was substrate selective such that the catalytic efficiency with CDNB and with 1-chloro-6-trifluoromethyl-2,4-dinitrobenzene were increased 2-3-fold, whereas the activity with ethacrynic acid was decreased by a factor of 8. The results show that a single-point mutation in the active site of an enzyme may modulate the catalytic activity without being directly involved as a functional group in the enzymic mechanism. Such limited modifications are relevant both to the natural evolution and the in vitro redesign of proteins for novel functions.

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