HTS in the new millennium: the role of pharmacology and flexibility.

Over the past decade, high throughput screening (HTS) has become the focal point for discovery programs within the pharmaceutical industry. The role of this discipline has been and remains the rapid and efficient identification of lead chemical matter within chemical libraries for therapeutics development. Recent advances in molecular and computational biology, i.e., genomic sequencing and bioinformatics, have resulted in the announcement of publication of the first draft of the human genome. While much work remains before a complete and accurate genomic map will be available, there can be no doubt that the number of potential therapeutic intervention points will increase dramatically, thereby increasing the workload of early discovery groups. One current drug discovery paradigm integrates genomics, protein biosciences and HTS in establishing what the authors refer to as the "gene-to-screen" process. Adoption of the "gene-to-screen" paradigm results in a dramatic increase in the efficiency of the process of converting a novel gene coding for a putative enzymatic or receptor function into a robust and pharmacologically relevant high throughput screen. This article details aspects of the identification of lead chemical matter from HTS. Topics discussed include portfolio composition (molecular targets amenable to small molecule drug discovery), screening file content, assay formats and plating densities, and the impact of instrumentation on the ability of HTS to identify lead chemical matter.

Mechanistic role of an NS4A peptide cofactor with the truncated NS3 protease of hepatitis C virus: elucidation of the NS4A stimulatory effect via kinetic analysis and inhibitor mapping.

Infection by hepatitis C viruses (HCVs) is a serious medical problem with no broadly effective treatment available for the progression of chronic hepatitis. The catalytic activity of a viral serine protease located in the N-terminal one-third of nonstructural protein 3 (NS3) is required for polyprotein processing at four site-specific junctions. The three-dimensional crystal structure of the NS3-NS4A co-complex [Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O'Malley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A., Caron, P. R., & Thomson, J. A. (1996) Cell 87, 343-355] delineates a small hydrophobic region within the 54-residue NS4A protein that intercalates with and makes extensive contacts to the core of the protease. The current investigation addresses the mechanism of NS3 protease catalytic activation by NS4A utilizing a small synthetic NS4A peptide (residues 1678-1691 of the virus polyprotein sequence) and the recombinantly expressed protease domain of NS3. The addition of NS4A dramatically increased NS3 kcat and kcat/Km catalytic parameters when measured against small peptide substrates representing the different site-specific junctions of the polyprotein. The catalytic effect of natural and non-natural amino acid substitutions at the P1 position in a 5A/5B peptide substrate was investigated. NS3-NS4A demonstrated a marked catalytic preference for the cysteine residue commonly found in authentic substrates. The pH dependence of the NS3 hydrolysis reaction is not affected by the presence of NS4A. This result suggests that NS4A does not change the pKa values of the active site residues of NS3 protease. A steady state kinetic analysis was performed and indicated that the binding of NS4A and the peptide substrate occurs in an ordered fashion during the catalytic cycle, with NS4A binding first. Two distinct kinetic classes of peptidyl inhibitors based upon the 5A/5B cleavage site were identified. An NS4A-independent class is devoid of prime residues. A second class of inhibitors is NS4A-dependent and contains a natural or non-natural cyclic amino acid substituted for the commonly found P1' residue serine. These inhibitors display an up to 80-fold increase in affinity for NS3 protease in the presence of NS4A. Sequential truncation of prime and P residues from this inhibitor class demonstrated the fact that the P4' and P1' residues are crucial for potent inhibition. The selectivity of this NS4A effect is interpreted using a model of the 5A/5B decapeptide substrate bound to the active site of the NS3-NS4A structure.

Polynucleotide modulation of the protease, nucleoside triphosphatase, and helicase activities of a hepatitis C virus NS3-NS4A complex isolated from transfected COS cells.

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) is a 70-kDa multifunctional enzyme with three known catalytic activities segregated in two somewhat independent domains. The essential machinery of a serine protease is localized in the N-terminal one-third of the protein, and nucleoside triphosphatase (NTPase) and helicase activities reside in the remaining C-terminal region. NS4A is a 54-residue protein expressed immediately downstream of NS3 in the viral polyprotein, and a central stretch of hydrophobic residues in NS4A form an integral structural component of the NS3 serine protease domain. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic processing in vivo. This may reflect economical packaging of essential viral replicative components, but it could also mean that there is functional interdependence between the two domains. In this study, a full-length NS3-NS4A complex was isolated after expression and autoprocessing in transiently transfected COS cells. The protein was used to examine the effects of polynucleotides on the NTPase, helicase, and protease activities. Unlike the previously reported behavior of a separately expressed NS3 helicase domain, the full NS3-NS4A complex demonstrated optimal NTPase activity between pH 7.5 and 8.5. All three NS3-NS4A activities were modulated by polynucleotides, with poly(U) having the most remarkable effect. These findings suggest that the domains within NS3 may influence the activity of one another and that the interplay of HCV genomic elements may regulate the enzyme activities of this complex HCV replicase component.

Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide.

An estimated 1% of the global human population is infected by hepatitis C viruses (HCVs), and there are no broadly effective treatments for the debilitating progression of chronic hepatitis C. A serine protease located within the HCV NS3 protein processes the viral polyprotein at four specific sites and is considered essential for replication. Thus, it emerges as an attractive target for drug design. We report here the 2.5 angstrom resolution X-ray crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator peptide. The protease has a chymotrypsin-like fold and features a tetrahedrally coordinated metal ion distal to the active site. The NS4A peptide intercalates within a beta sheet of the enzyme core.

C-terminal zinc-containing peptide required for RNA recognition by a class I tRNA synthetase.

Escherichia coli isoleucyl-tRNA synthetase is one of five closely related class I tRNA synthetases. The active site of the 939 amino acid polypeptide is in an N-terminal domain which contains an insertion believed essential for interactions with the tRNA acceptor helix. The enzyme was shown previously to contain an essential (for function in vivo) zinc bound to a Cys4 cluster at the C-terminal end of the polypeptide. The specific function of this zinc has been unknown. We show here that aminoacylation activity can be reconstituted in vitro by combining a 53 amino acid zinc-containing C-terminal peptide with a protein consisting of the remaining 886 amino acids. Reconstitution of aminoacylation is zinc-dependent. In contrast, the zinc-containing peptide is dispensable for synthesis of isoleucyl adenylate. Affinity coelectrophoresis showed that the 53 amino acid C-terminal peptide is required specifically for tRNA binding. We propose that the zinc-containing peptide curls back to the active site to make contact with the acceptor helix of bound tRNA, but not with isoleucine or ATP. It is the first example of a zinc-containing peptide in a class I tRNA synthetase that is essential for tRNA binding interactions. The design of this enzyme may be part of a more general scheme for class I tRNA synthetases to acquire acceptor helix binding elements during the development of the genetic code.

Thiol ligation of two zinc atoms to a class I tRNA synthetase: evidence for unshared thiols and role in amino acid binding and utilization.

Class I tRNA synthetases generally contain a characteristic N-terminal catalytic core joined to a C-terminal domain that is idiosyncratic to the enzyme. The closely related class I Escherichia coli methionyl- and isoleucyl-tRNA synthetases each have a single zinc atom coordinated to ligands contained in the catalytic domain. Isoleucyl-tRNA synthetase has a second, functionally essential, zinc bound to ligands at the C-terminal end of the 939 amino acid polypeptide. Recent evidence suggested that this structure curls back and interacts directly or indirectly with the active site. We show here by X-ray absorption spectroscopy that the average Zn environment contains predominantly sulfur ligands with a Zn-S distance of 2.33 A. A model with eight coordinated thiolates divided between two Zn(Cys)4 structures best fit the data which are not consistent with a thiolate-bridged Zn2(Cys)6 structure joining the C-terminal end with the N-terminal active site domain. We also show that zinc bound to the N-terminal catalytic core is important specifically for amino acid binding and utilization, although a direct interaction with zinc is unlikely. We suggest that, in addition to idiosyncratic sequences for tRNA acceptor helix interactions incorporated into the class-defining catalytic domain common to class I enzymes, the architecture of at least some parts of the amino acid binding sites may differ from enzyme to enzyme and include motifs that bind zinc.

Zinc-dependent cell growth conferred by mutant tRNA synthetase.

We present evidence that zinc bound near the C terminus of a long tRNA synthetase polypeptide, and at a location far in the sequence from the catalytic domain, is needed to sustain cell growth and is, therefore, essential for enzyme function. Several class I and class II tRNA synthetases contain bound zinc, including the 939-amino acid class I Escherichia coli isoleucyl-tRNA synthetase, which has two zinc atoms coordinated to cysteine sulfhydryls. The functional significance of these bound zinc atoms has been unclear. Like other class I tRNA synthetases, the isoleucine enzyme has a class-defining conserved N-terminal domain that contains the catalytic site. The C-terminal domain is variable in sequence and structure and not conserved among all of the class I enzymes. Using split proteins, we localized a zinc binding site to the C-terminal end of isoleucyl-tRNA synthetase. Serine substitutions of single cysteines at a thiol-containing putative zinc binding site that is less than 40 amino acids from the C terminus confer a zinc-dependent growth phenotype on cells harboring the mutant enzymes. We propose that zinc bound near the C terminus is part of a structure that interacts directly or indirectly with the active site. A structure at the C terminus that provides a functional link between the conserved N-terminal catalytic and non-conserved C-terminal domain may be common to several class I enzymes.

The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: mechanistic and crystallographic evidence for stereospecific alkylation by (R)-alpha-phenylglycidate.

The mechanism of irreversible inactivation of mandelate racemase (MR) from Pseudomonas putida by alpha-phenylglycidate (alpha PGA) has been investigated stereochemically and crystallographically. The (R) and (S) enantiomers of alpha PGA were synthesized in high enantiomeric excess (81% ee and 83% ee, respectively) using Sharpless epoxidation chemistry. (R)-alpha PGA was determined to be a stereospecific and stoichiometric irreversible inactivator of MR. (S)-alpha PGA does not inactivate MR and appears to bind noncovalently to the active site of MR with less affinity than that of (R)-alpha PGA. The X-ray crystal structure (2.0-A resolution) of MR inactivated by (R)-alpha PGA revealed the presence of a covalent adduct formed by nucleophilic attack of the epsilon-amino group of Lys 166 on the distal carbon on the epoxide ring of (R)-alpha PGA. The proximity of the alpha-proton of (S)-mandelate to Lys 166 [configurationally equivalent to (R)-alpha PGA] was corroborated by the crystal structure (2.1-A resolution) of MR complexed with the substrate analog/competitive inhibitor, (S)-atrolactate [(S)-alpha-methylmandelate]. These results support the proposal that Lys 166 is the polyvalent acid/base responsible for proton transfers on the (S) face of mandelate. In addition, the high-resolution structures also provide insight into the probable interactions of mandelate with the essential Mg2+ and functional groups in the active site.

C-terminal peptide appendix in a class I tRNA synthetase needed for acceptor-helix contacts and microhelix aminoacylation.

The 10 class I tRNA synthetases have an N-terminal nucleotide-binding fold which contains the catalytic center. Insertions into the nucleotide-binding fold provide contacts for acceptor-helix interactions, which stabilize the amino acid acceptor end of the tRNA substrate in the active site. A separate and largely nonconserved C-terminal domain provides contacts with distal parts of the tRNA, including the anticodon. For Escherichia coli methionyl tRNA synthetase, whose structure is known, the C-terminal domain is predominantly alpha-helical and forms a loop which interacts with the anticodon trinucleotide located about 76 A from the amino acid attachment site. Fused to the end of this helical domain is a peptide which curls back into the N-terminal nucleotide-binding fold and region of the active site. We show here that mutations in this peptide appendix disrupt aminoacylation and binding of a 7 base pair microhelix substrate based on the acceptor stem of tRNA(fMet), without affecting interactions with ATP or methionine or with the tRNA(fMet) anticodon. The impairment of acceptor-helix interactions by mutation of the C-terminal peptide can offset favorable anticodon interactions and severely reduce aminoacylation of tRNA(fMet). Thus, in addition to, or as an alternative to, acceptor-helix-binding insertions into the N-terminal nucleotide-binding fold, C-terminal peptide epitopes in some class I enzymes may provide a mechanism for facilitating RNA microhelix interactions with the catalytic site.

Evidence for distinct locations for metal binding sites in two closely related class I tRNA synthetases.

Of the ten class I tRNA synthetases, those for methionine and isoleucine are among the most closely related. In recent work we showed that the 676 amino acid E. coli methionine tRNA synthetase has one zinc bound per polypeptide. Zinc may be replaced by spectroscopically observable cobalt with retention of full activity. Bound zinc has been localized to a cysteine cluster within an insertion into the nucleotide binding fold that characterizes all class I enzymes. Mutations which interfere with metal ligation to these cysteines yield proteins that are defective in activity. Additional data presented here show that change of the cobalt oxidation state and coordination geometry of the Co(II)-substituted enzyme results in a complete loss in activity, and that mutations which replace any one of the zinc-binding cysteine sulfhydryls have a small but measurable effect on protein stability. These results further support the importance of the metal for the active site. We also show that, in contrast to methionine tRNA synthetase, the closely related but larger 939 amino acid E. coli isoleucine tRNA synthetase contains 1.5 to 2 molecules of zinc bound per polypeptide. The cobalt-substituted enzyme is active and shows the expected spectrum for tetrahedral coordination to sulfur ligands. Although the site(s) for metal coordination in isoleucine tRNA synthetase has not been rigorously established, one likely sequence element is in a region of the primary structure different from the known metal binding site in methionine tRNA synthetase. Thus, these two closely related proteins have incorporated metal binding sites into distinct parts of their related sequences.

Metal-binding site in a class I tRNA synthetase localized to a cysteine cluster inserted into nucleotide-binding fold.

The 10 class I aminoacyl-tRNA synthetases share a common N-terminal nucleotide-binding fold. Idiosyncratic polypeptide insertions into this fold introduce residues important for activity, including those that interact with the tRNA acceptor helix. The class I Escherichia coli methionyl-tRNA synthetase (L-methionine:tRNA(Met) ligase, EC 6.1.1.10), a 676-amino acid homodimer, was shown previously by others to contain zinc and to have an activity dependent on its presence. We show here by atomic absorption spectroscopy and zinc titrations the presence of 1 mol of zinc per polypeptide. Replacement of zinc with cobalt yields an active enzyme with a visible absorption spectrum characteristic of tetrahedral coordination to sulfur ligands and an intense metal-to-sulfur charge-transfer band at 340 nm. Mapping of the metal-binding site by zinc blotting of recombinant and proteolytic fragments localized the site to a polypeptide insertion between two strands and a beta-sheet in the N-terminal nucleotide-binding fold that contains the catalytic site. Beginning at Cys-145, this insertion contains a Cys-Xaa2-Cys-Xaa9-Cys-Xaa2-Cys motif. Site-directed substitution of these cysteines with serines yielded proteins that were stable but generally devoid of activity. With this result there is now at least one example of a class I and of a class II E. coli tRNA synthetase with a metal-binding domain important for activity inserted into the catalytic domain.

3-Carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida is homologous to the class II fumarase family: a new reaction in the evolution of a mechanistic motif.

The gene (pcaB) for 3-carboxymuconate lactonizing enzyme (CMLE; 3-carboxymuconate cycloisomerase; EC 5.5.1.2) from Pseudomonas putida has been cloned into pMG27NS, a temperature-sensitive expression vector, and expressed in Escherichia coli N4830. The specific activity and kinetic parameters of the recombinant CMLE were comparable to those previously reported. A comparison of the deduced amino acid sequence of CMLE with sequences available in the PIR and Genbank databases revealed that CMLE has highly significant sequence homology to the class II fumarase family, particularly to adenylosuccinate lyase from Bacillus subtilis. CMLE has no significant homology to muconate lactonizing enzyme (MLE) from P. putida, its sister enzyme in the beta-ketoadipate pathway. These findings fully corroborate a prediction made by us on the basis of mechanistic and stereochemical analyses of CMLE and MLE [Chari, R. V. J., Whitman, C. P., Kozarich, J. W., Ngai, K.-L., & Ornston, L. N. (1987) J. Am. Chem. Soc. 109, 5514-5519] and suggest that CMLE and MLE were recruited into this specialized pathway from two different enzyme families.

Isomerization of (R)- and (S)-glutathiolactaldehydes by glyoxalase I: the case for dichotomous stereochemical behavior in a single active site.

The ability of glyoxalase I to isomerize both diastereomeric thiohemiacetals formed between glutathione and alpha-ketoaldehydes has been probed with stereochemically "locked" substrate analogues. Both (R)- and (S)-glutathiolactaldehyde (5 and 5') were unambiguously synthesized by employing the Sharpless epoxidation procedure as a key step. In the presence of human erythrocyte glyoxalase I, high-field 1H NMR analysis reveals that the R and S isomers (approximately 20 mM) are both converted to glutathiohydroxyacetone at rates of 0.8 and 0.4 s-1, respectively. This reaction is characterized by a nonstereospecific proton abstraction followed by a partially shielded proton transfer to the si face of the cis-enediol intermediate. Glyoxalase I catalyzes the exchange of the pro-S proton of glutathiohydroxyacetone with solvent deuterium. Glutathiohydroxyacetone was found to be a good competitive inhibitor of the normal glyoxalase I reaction (KI = 1.46 mM), suggesting that the slow processing rate of these compounds with respect to the normal thiohemiacetals is not due to poor binding. The results are consistent with a nonstereospecific proton abstraction and a stereospecific reprotonation at contiguous substrate carbons.

Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant.

The two preceding papers [Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue); Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R., Kenyon, G. L., & Gerlt, J. A. (1991) Biochemistry (second paper of three in this issue)] suggest that the active site of mandelate racemase (MR) contains two distinct general acid/base catalysts: Lys 166, which abstracts the alpha-proton from (S)-mandelate, and His 297, which abstracts the alpha-proton from (R)-mandelate. In this paper we report on the properties of the mutant of MR in which His 297 has been converted to asparagine by site-directed mutagenesis (H297N). The structure of H297N, solved by molecular replacement at 2.2-A resolution, reveals that no conformational alterations accompany the substitution. As expected, H297N has no detectable MR activity. However, H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)-benzoylformate in 45% yield at a rate equal to that measured for wild-type enzyme; the unreacted p-(bromomethyl)mandelate is recovered as (R)-p-(hydroxymethyl)mandelate. At pD 7.5, H297N catalyzes the stereospecific exchange of the alpha-proton of (S)- but not (R)-mandelate with D2O solvent at a rate 3.3-fold less than that observed for incorporation of solvent deuterium into (S)-mandelate catalyzed by wild-type enzyme. The pD dependence of the rate of the exchange reaction catalyzed by H297N reveals a pKa of 6.4 in D2O, which is assigned to Lys 166.(ABSTRACT TRUNCATED AT 250 WORDS)