Cell-Free Expression of G Protein-Coupled Receptors.

The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes.

Identification of small-molecule inhibitors against SecA by structure-based virtual ligand screening.

The rapid rise of antibiotic-resistant bacteria is one of the major concerns in modern medicine. Therefore, to treat bacterial infections, there is an urgent need for new antibacterials-preferably directed against alternative bacterial targets. One such potential target is the preprotein translocation motor SecA. SecA is a peripheral membrane ATPase and a key component of the Sec secretion pathway, the major route for bacterial protein export across or into the cytoplasmic membrane. As SecA is essential for bacterial viability, ubiquitous and highly conserved in bacteria, but not present in eukaryotic cells, it represents an attractive antibacterial target. Using an in silico approach, we have defined several potentially druggable and conserved pockets on the surface of SecA. We show that three of these potentially druggable sites are important for SecA function. A starting collection of ~500 000 commercially available small-molecules was virtually screened against a predicted druggable pocket in the preprotein-binding domain of Escherichia coli SecA using a multi-step virtual ligand screening protocol. The 1040 top-scoring molecules were tested in vitro for inhibition of the translocation ATPase activity of E. coli SecA. Five inhibitors of the translocation ATPase, and not of basal or membrane ATPase, were identified with IC50 values <65 µm. The most potent inhibitor showed an IC50 of 24 µm. The antimicrobial activity was determined for the five most potent SecA inhibitors. Two compounds were found to possess weak antibacterial activity (IC50 ~198 µm) against E. coli, whereas some compounds showed moderate antibacterial activity (IC50 ~100 µm) against Staphylococcus aureus.

Traffic jam at the bacterial sec translocase: targeting the SecA nanomotor by small-molecule inhibitors.

The rapid rise of drug-resistant bacteria is one of the most serious unmet medical needs facing the world. Despite this increasing problem of antibiotic resistance, the number of different antibiotics available for the treatment of serious infections is dwindling. Therefore, there is an urgent need for new antibacterial drugs, preferably with novel modes of action to potentially avoid cross-resistance with existing antibacterial agents. In recent years, increasing attention has been paid to bacterial protein secretion as a potential antibacterial target. Among the different protein secretion pathways that are present in bacterial pathogens, the general protein secretory (Sec) pathway is widely considered as an attractive target for antibacterial therapy. One of the key components of the Sec pathway is the peripheral membrane ATPase SecA, which provides the energy for the translocation of preproteins across the bacterial cytoplasmic membrane. In this review, we will provide an overview of research efforts on the discovery and development of small-molecule SecA inhibitors. Furthermore, recent advances on the structure and function of SecA and their potential impact on antibacterial drug discovery will be discussed.

Synthesis and antibacterial evaluation of a novel series of 2-(1,2-dihydro-3-oxo-3H-pyrazol-2-yl)benzothiazoles.

The 2-(1,2-dihydro-3-oxo-3H-pyrazol-2-yl)benzothiazole scaffold was selected as a central core structure for the discovery of novel antibacterial compounds. A systematic variation of the substituents on the oxo-pyrazole moiety, as well as on the benzo moiety, led to the creation of a small and focused library of benzothiazoles that was subjected to antibacterial screening. In a first round of screening, activity of the compounds against six representative microorganisms was established. For the most potent congeners, MIC values against S. aureus and P. aeruginosa were determined. The structure-activity relationship study clearly revealed that subtle structural variations influence the antibacterial activity to a large extent. The most potent congeners displayed MIC values of 3.30 µM.

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors.

A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli (E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ~27,000 compounds and proved to be highly reliable (average Z' factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors.

Synthesis of novel 5-amino-thiazolo[4,5-d]pyrimidines as E. coli and S. aureus SecA inhibitors.

An efficient synthesis of a library of 5-amino-thiazolo[4,5-d]pyrimidines is reported. Regioselective displacements of chlorines, as well as regioselective diazotation reactions are described, which allow the introduction of structural diversity on the scaffold by consecutive reactions. Screening of this focused library led to the discovery of SecA inhibitors from Escherichia coli and Staphylococcus aureus.

Inhibition of thrombin formation by active site mutated (S360A) activated protein C.

Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg(506) and Arg(306) and of factor VIIIa (FVIIIa) by cleavage at Arg(336) and Arg(562). To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K(D) of 0.11 +/- 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg(506) and not Arg(306) and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg(506) cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg(506) interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.

Screening Outside the Catalytic Site: Inhibition of Macromolecular Inter-actions Through Structure-Based Virtual Ligand Screening Experiments.

During these last 15 years, drug discovery strategies have essentially focused on identifying small molecules able to inhibit catalytic sites. However, other mechanisms could be targeted. Protein-protein interactions play crucial roles in a number of biological processes, and, as such, their disruption or stabilization is becoming an area of intense activity. Along the same line, inhibition of protein-membrane could be of major importance in several disease indications. Despite the many challenges associated with the development of such classes of interaction modulators, there has been considerable success in the recent years. Importantly, through the existence of protein hot-spots and the presence of druggable pockets at the macromolecular interfaces or in their vicinities, it has been possible to find small molecule effectors using a variety of screening techniques, including combined virtual ligand-in vitro screening strategy. Indeed such in silico-in vitro protocols emerge as the method of choice to facilitate our quest of novel drug-like compounds or of mechanistic probes aiming at facilitating the understanding of molecular reactions involved in the Health and Disease process. In this review, we comment recent successes of combined in silico-in vitro screening methods applied to modulating macromolecular interactions with a special emphasis on protein-membrane interactions.

Identification of surface epitopes of human coagulation factor Va that are important for interaction with activated protein C and heparin.

Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.

Multimerin 1 binds factor V and activated factor V with high affinity and inhibits thrombin generation.

Multimerin 1 (MMRN1) is a polymeric, factor V (FV) binding protein that is stored in platelet and endothelial cell secretion granules but is undetectable in normal plasma. In human platelet alpha-granules, FV is stored complexed to MMRN1, predominantly by noncovalent binding interactions. The FV binding site for MMRN1 is located in the light chain, where it overlaps the C1 and C2 domain membrane binding sites essential for activated FV (FVa) procoagulant function. Surface plasmon resonance (SPR), circular dichroism (CD) and thrombin generation assays were used to study the binding of FV and FVa to MMRN1, and the functional consequences. FV and FVa bound MMRN1 with high affinities (K(D): 2 and 7 nM, respectively). FV dissociated more slowly from MMRN1 than FVa in SPR experiments, and CD analyses suggested greater conformational changes in mixtures of FV and MMRN1 than in mixtures of FVa and MMRN1. SPR analyses indicated that soluble phosphatidylserine (1,2-Dicaproylsn-glycero-3-phospho-L-serine) competitively inhibited both FV-MMRN1 and FVa-MMRN1 binding. Furthermore, exogenous MMRN1 delayed and reduced thrombin generation by plasma and platelets, and it reduced thrombin generation by preformed FVa. Exogenous MMRN1 also delayed FV activation, triggered by adding tissue factor to plasma, or by adding purified thrombin or factor Xa to purified FV. The high affinity binding of FV to MMRN1 may facilitate the costorage of the two proteins in platelet alpha-granules. As a consequence, MMRN1 release during platelet activation may limit platelet dependent thrombin generation in vivo.

The role of thrombin exosites I and II in the activation of human coagulation factor V.

Human blood coagulation Factor V (FV) is a plasma protein with little procoagulant activity. Limited proteolysis at Arg(709), Arg(1018), and Arg(1545) by thrombin or Factor Xa (FXa) results in the generation of activated FV, which serves as a cofactor of FXa in prothrombin activation. Both thrombin exosites I and II have been reported to be involved in FV activation, but the relative importance of these regions in the individual cleavages remains unclear. To investigate the role of each exosite in FV activation, we have used recombinant FV molecules with only one of the three activation cleavage sites available, in combination with exosite I- or II-specific aptamers. In addition, structural requirements for exosite interactions located in the B-domain of FV were probed using FV B-domain deletion mutants and comparison with FV activating enzymes from the venom of Russell's viper (RVV-V) and of Levant's viper (LVV-V) known to activate FV by specific cleavage at Arg(1545). Our results indicate that thrombin exosite II is not involved in cleavage at Arg(709) and that both thrombin exosites are important for recognition and cleavage at Arg(1545). Efficient thrombin-catalyzed FV activation requires both the N- and C-terminal regions of the B-domain, whereas only the latter is required by RVV-V and LVV-V. This indicates that proteolysis of FV by thrombin at Arg(709), Arg(1018), and Arg(1545) show different cleavage requirements with respect to interactions mediated by thrombin exosites and areas that surround the respective cleavage sites. In addition, interactions between exosite I of thrombin and FV are primarily responsible for the different cleavage site specificity as compared with activation by RVV-V or LVV-V.

Coagulation factor V and thrombophilia: background and mechanisms.

Human coagulation factor V (FV) is an essential coagulation protein with functions in both the pro- and anticoagulant pathways. Failure to express and control FV functions can either lead to bleeding, or to thromboembolic disease. Both events may develop into a life-threatening condition. Since the first description of APC resistance, and in particular the description of the so-called factor V(Leiden) mutation, in which a prominent activated protein C cleavage site in FV has been abolished through a mutation in the FV gene, FV has been in the center of attention of thrombosis research. In this review we describe how the functions of FV are expressed and regulated and provide an extensive description of the role that FV plays in the etiology of thromboembolic disease.

Design of protein membrane interaction inhibitors by virtual ligand screening, proof of concept with the C2 domain of factor V.

Most orally bioavailable drugs on the market are competitive inhibitors of catalytic sites, but a significant number of targets remain undrugged, because their molecular functions are believed to be inaccessible to drug-like molecules. This observation specifically applies to the development of small-molecule inhibitors of macromolecular interactions such as protein-membrane interactions that have been essentially neglected thus far. Nonetheless, many proteins containing a membrane-targeting domain play a crucial role in health and disease, and the inhibition of such interactions therefore represents a very promising therapeutic strategy. In this study, we demonstrate the use of combined in silico structure-based virtual ligand screening and surface plasmon resonance experiments to identify compounds that specifically disrupt protein-membrane interactions. Computational analysis of several membrane-binding domains revealed they all contain a druggable pocket within their membrane-binding region. We applied our screening protocol to the second discoidin domain of coagulation factor V and screened >300,000 drug-like compounds in silico against two known crystal structure forms. For each C2 domain structure, the top 500 molecules predicted as likely factor V-membrane inhibitors were evaluated in vitro. Seven drug-like hits were identified, indicating that therapeutic targets that bind transiently to the membrane surface can be investigated cost-effectively, and that inhibitors of protein-membrane interactions can be designed.

Structural models of the snake venom factor V activators from Daboia russelli and Daboia lebetina.

Blood coagulation factor V (FV) is a multifunctional protein that circulates in human plasma as a precursor molecule which can be activated by thrombin or activated factor X (FXa) in order to express its cofactor activity in prothrombin activation. FV activation is achieved by limited proteolysis after Arg709, Arg1018, and Arg1545 in the FV molecule. The venoms of Daboia russelli and Daboia lebetina contain a serine protease that specifically activates FV by a single cleavage at Arg1545. We have predicted the three-dimensional structure of these enzymes using comparative protein modeling techniques. The plasminogen activator from Agkistrodon acutus, which shows a high degree of homology with the venom FV activators and for which a high-quality crystallographic structure is available, was used as the molecular template. The RVV-V and LVV-V models provide for the first time a detailed and accurate structure of a snake venom FV activator and explain the observed sensitivity or resistance toward a number of serine protease inhibitors. Finally, electrostatic potential calculations show that two positively charged surface patches are present on opposite sides of the active site. We propose that both FV activators achieve their exquisite substrate specificity for the Arg1545 site via interactions between these exosites and FV.