U. Helena Danielson

4‐Hydroxyalk‐2‐enals are substrates for glutathione transferase

The 4‐hydroxyalk‐2‐enals are established products of lipid peroxidation that are conjugated with intracellular glutathione. Cytosolic glutathione transferases from rat liver were shown to give high specific activities with 4‐hydroxynonenal and 4‐hydroxydecenal. The isoenzyme giving the highest specific activity was glutathione transferase 4‐4. The rate of the spontaneous conjugation reaction is negligible in comparison with the rate calculated for the cellular concentration of the glutathione transferases. It is proposed that a major biological function of the glutathione transferases is to protect the cell against products of oxidative metabolism, such as epoxides, organic hydroperoxides, and 4‐hydroxyalkenals.

Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing

Seven major isoenzymes of glutathione transferase with isoelectric points ranging from pH 6.9 to 10 were isolated from rat liver cytosol. The purification procedure included affinity chromatography on immobilized S-hexylglutathione followed by high-performance liquid chromatofocusing. Characteristics, such as physical properties, reactions with antibodies, specific activities with various substrates, kinetic constants, and sensitivities to a set of inhibitors, are given for discrimination and identification of the different isoenzymes. The multiple forms of the enzyme correspond to glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 in the recently introduced nomenclature [W.B. Jakoby et al. (1984) Biochem. Pharmacol. 33, 2539-2540]. A seventh form appears to be a heterodimeric protein composed of subunit 3 and an as yet unidentified subunit.

Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-length NS3 (protease-helicase/NTPase): A comparative study of different C-terminals

Synthesis and inhibitory potencies of three types of protease inhibitors of the hepatitis C virus (HCV) full-length NS3 (protease-helicase/NTPase) are reported: (i) inhibitors comprising electrophilic serine traps (pentafluoroethyl ketones, alpha-keto acids, and alpha-ketotetrazoles), (ii) product-based inhibitors comprising a C-terminal carboxylate group, and (iii) previously unexplored inhibitors comprising C-terminal carboxylic acid bioisosteres (tetrazoles and acyl sulfonamides). Bioisosteric replacement with the tetrazole group provided inhibitors equally potent to the corresponding carboxylates, and substitution with the phenyl acyl sulfonamide group yielded more potent inhibitors. The hexapeptide inhibitors Suc-Asp-D-Glu-Leu-Ile-Cha-Nva-NHSO(2)Ph and Suc-Asp-D-Glu-Leu-Ile-Cha-ACPC-NHSO(2)Ph with K(i) values of 13.6 and 3.8 nM, respectively, were approximately 20 times more potent than the corresponding inhibitors with a C-terminal carboxylate and were comparable to the carboxylate-based inhibitor containing the native cysteine, Suc-Asp-D-Glu-Leu-Ile-Cha-Cys-OH (K(i)=28 nM). The acyl sulfonamide group constitutes a very promising C-terminal functionality that allows for prime site optimization.

Biophysics in drug discovery: impact, challenges and opportunities

Over the past 25 years, biophysical technologies such as X-ray crystallography, nuclear magnetic resonance spectroscopy, surface plasmon resonance spectroscopy and isothermal titration calorimetry have become key components of drug discovery platforms in many pharmaceutical companies and academic laboratories. There have been great improvements in the speed, sensitivity and range of possible measurements, providing high-resolution mechanistic, kinetic, thermodynamic and structural information on compound–target interactions. This Review provides a framework to understand this evolution by describing the key biophysical methods, the information they can provide and the ways in which they can be applied at different stages of the drug discovery process. We also discuss the challenges for current technologies and future opportunities to use biophysical methods to solve drug discovery problems.

Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase

ABSTRACT The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5- to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains “thumb” and “fingers.” These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.

Expression and purification of recombinant full-length NS3 protease-helicase from a new variant of Hepatitis C virus.

Viral mRNA extracted from the serum of a patient infected with HCV strain 1a was used for cloning, expression, and purification of full-length Hepatitis C NS3 protein. Sequencing of the protease gene identified the virus to be a new variant closely related to strain H77, differing in 15 out of 631 amino acids in the NS3 protein, none of which were predicted to be directly involved in catalysis, binding of substrate, or cofactor. A pBAD expression system was used to express the enzyme with an N-terminal tag in Escherichia coli. Purification from the soluble cellular fraction was achieved by Ni(2+)-IMAC and PolyU Sepharose affinity chromatography. The dependence of the proteolytic activity of the full-length NS3 protein on ionic strength, glycerol concentration, and a peptide corresponding to the activating region of NS4A was analyzed and used to design an activity assay that is suitable for inhibition studies. The kinetic constants (k(cat) and K(M)) for catalysis and the inhibitory potencies (IC(50) and K(i)) of five product-based hexapeptide inhibitors were comparable to those reported for the truncated NS3 protein. Detailed kinetic and inhibition studies using this variant of full-length NS3 can increase the understanding of the enzymatic characteristics of NS3, reveal the importance of the substituted amino acids and the significance of the genetic variability for design of effective inhibitors of the virus, and is thus of relevance for drug discovery.

Fragment Library Screening and Lead Characterization Using SPR Biosensors

The transition from high throughput screening of collections of drug-like compounds to screening of fragment libraries via lower throughput methods with high sensitivity has revolutionized early drug discovery. It is highlighting the need for sensitive biophysical techniques for interaction analysis rather than high throughput methods. Biosensors with SPR detection are well suited for this novel scenario. In less than 20 years the technique has been launched, established and become a highly informative method for a variety of applications in drug discovery. It is no longer limited to the detection of proteins or other high molecular weight analytes, but the detection of weakly interacting fragments is now feasible. This paper discusses the theoretical and experimental limitations for such applications and reviews a number of successful studies in the area of fragment-based lead discovery that have recently been published. It can be anticipated that the evolution of this young technique will be significantly influenced by the requirements for efficient fragment-based lead discovery.

Characterization of Ca2+ and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

The interactions between Ca2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca2+ or phosphocholine were injected. Binding of Ca2+ induced a 10-fold higher signal than expected from the molecular weight of Ca2+. It was interpreted to result from the conformational change that occurs on binding of Ca2+. Two sites with different characteristics were distinguished: a high-affinity site with K(D)=0.03 mM and a low-affinity site with K(D)=5.45 mM. The pH dependencies of the two Ca2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K(D)=5 microM at 10 mM Ca2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca2+ is an efficient regulator under physiological conditions.

Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

Significance In this study we take advantage of a recently described chimera of the α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding protein (AChBP), termed α7-AChBP. To date, more than 70 crystal structures have been determined for AChBP in complex with ligands that occupy the orthosteric binding site. Here, we use an innovative screening strategy to discover molecular fragments that occupy allosteric binding sites. In combination with X-ray crystallography we determine a molecular blueprint of three different allosteric sites in α7-AChBP. Using electrophysiological recordings on the human α7 nAChR we demonstrate that each of the three sites is involved in allosteric modulation of the receptor. Our study contributes to understanding the sites of allosteric binding in ion channels. The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential.

Identification of MMP-12 Inhibitors by Using Biosensor-Based Screening of a Fragment Library

Small inhibitors of matrix metalloproteinase 12 (MMP-12) have been identified with a biosensor-based screening strategy and a specifically designed fragment library. The interaction between fragments and three variants of the target and a reference protein with an active-site zinc ion was measured continuously by surface plasmon resonance. The developed experimental design overcame the inherent instability of MMP-12 and allowed the identification of fragments that interacted specifically with the active-site of MMP-12 but not with the reference protein. The interaction with MMP-12 for selected compounds were analyzed for concentration dependence and saturability. Compounds interacting distinctly with the target were further evaluated by an activity-based assay, verifying MMP-12 inhibition. Two effective inhibitors were identified, and the compound with highest affinity was confirmed to be a competitive inhibitor with an IC50 of 290 nM and a ligand efficiency of 0.7 kcal/mol heavy atom. This procedure integrates selectivity and binding site identification into the screening procedure and does not require structure determination.