Structural and kinetic analyses of the protease from an amprenavir-resistant human immunodeficiency virus type 1 mutant rendered resistant to saquinavir and resensitized to amprenavir.

Recent drug regimens have had much success in the treatment of human immunodeficiency virus (HIV)-infected individuals; however, the incidence of resistance to such drugs has become a problem that is likely to increase in importance with long-term therapy of this chronic illness. An analysis and understanding of the molecular interactions between the drug(s) and the mutated viral target(s) is crucial for further progress in the field of AIDS therapy. The protease inhibitor amprenavir (APV) generates a signature set of HIV type 1 (HIV-1) protease mutations associated with in vitro resistance (M46I/L, I47V, and I50V [triple mutant]). Passage of the triple-mutant APV-resistant HIV-1 strain in MT4 cells, in the presence of increasing concentrations of saquinavir (SQV), gave rise to a new variant containing M46I, G48V, I50V, and I84L mutations in the protease and a resulting phenotype that was resistant to SQV and, unexpectedly, resensitized to APV. This phenotype was consistent with a subsequent kinetic analysis of the mutant protease, together with X-ray crystallographic analysis and computational modeling which elucidated the structural basis of these observations. The switch in protease inhibitor sensitivities resulted from (i) the I50V mutation, which reduced the area of contact with APV and SQV; (ii) the compensating I84L mutation, which improved hydrophobic packing with APV; and (iii) the G-to-V mutation at residue 48, which introduced steric repulsion with the P3 group of SQV. This analysis establishes the fine detail necessary for understanding the loss of protease binding for SQV in the quadruple mutant and gain in binding for APV, demonstrating the powerful combination of virology, molecular biology, enzymology, and protein structural and modeling studies in the elucidation and understanding of viral drug resistance.

Broad-spectrum antiviral activity of the IMP dehydrogenase inhibitor VX-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon.

The enzyme IMP dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotides, namely, the conversion of IMP to XMP. The major event occurring in cells exposed to competitive IMPDH inhibitors such as ribavirin or uncompetitive inhibitors such as mycophenolic acid (MPA) is a depletion of the intracellular GTP and dGTP pools. Ribavirin is approved as an inhaled antiviral agent for treatment of respiratory syncytial virus (RSV) infection and orally, in combination with alpha interferon (IFN-alpha), for the treatment of chronic hepatitis C virus (HCV) infection. VX-497 is a potent, reversible uncompetitive IMPDH inhibitor which is structurally unrelated to other known IMPDH inhibitors. Studies were performed to compare VX-497 and ribavirin in terms of their cytotoxicities and their efficacies against a variety of viruses. They included DNA viruses (hepatitis B virus [HBV], human cytomegalovirus [HCMV], and herpes simplex virus type 1 [HSV-1]) and RNA viruses (respiratory syncytial virus [RSV], parainfluenza-3 virus, bovine viral diarrhea virus, Venezuelan equine encephalomyelitis virus [VEEV], dengue virus, yellow fever virus, coxsackie B3 virus, encephalomyocarditis virus [EMCV], and influenza A virus). VX-497 was 17- to 186-fold more potent than ribavirin against HBV, HCMV, RSV, HSV-1, parainfluenza-3 virus, EMCV, and VEEV infections in cultured cells. The therapeutic index of VX-497 was significantly better than that of ribavirin for HBV and HCMV (14- and 39-fold, respectively). Finally, the antiviral effect of VX-497 in combination with IFN-alpha was compared to that of ribavirin with IFN-alpha in the EMCV replication system. Both VX-497 and ribavirin demonstrated additivity when coapplied with IFN-alpha, with VX-497 again being the more potent in this combination. These data are supportive of the hypothesis that VX-497, like ribavirin, is a broad-spectrum antiviral agent.

Crystal structure of JNK3: a kinase implicated in neuronal apoptosis.

BACKGROUND: The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein (MAP) kinase family, and regulate signal transduction in response to environmental stress. Activation and nuclear localization of JNK3, a neuronal-specific isoform of JNK, has been associated with hypoxic and ischemic damage of CA1 neurons in the hippocampus. Knockout mice lacking JNK3 showed reduced apoptosis of hippocampal neurons and reduced seizure induced by kainic acid, a glutamate-receptor agonist. Thus, JNK3 may be important in the pathology of neurological disorders and is of significant medical interest. RESULTS: We report here the structure of unphosphorylated JNK3 in complex with adenylyl imidodiphosphate, an ATP analog. JNK3 has a typical kinase fold, with the ATP-binding site situated within a cleft between the N- and C-terminal domains. In contrast to other known MAP kinase structures, the ATP-binding site of JNK3 is well ordered; the glycine-rich nucleotide-binding sequence forms a beta-strand-turn-beta-strand structure over the nucleotide. Unphosphorylated JNK3 assumes an open conformation, in which the N- and C-terminal domains are twisted apart relative to their positions in cAMP-dependent protein kinase. The rotation leads to the misalignment of some of the catalytic residues. The phosphorylation lip of JNK3 partially blocks the substrate-binding site. CONCLUSIONS: This is the first JNK structure to be determined, providing a unique opportunity to compare structures from the three MAP kinase subfamilies. The structure reveals atomic-level details of the shape of JNK3 and the interactions between the kinase and the nucleotide. The misalignment of catalytic residues and occlusion of the active site by the phosphorylation lip may account for the low activity of unphosphorylated JNK3. The structure provides a framework for understanding the substrate specificity of different JNK isoforms, and should aid the design of selective JNK3 inhibitors.

Purification and characterization of the NS3 serine protease domain of hepatitis C virus expressed in Saccharomyces cerevisiae.

cDNA encoding the putative core of the hepatitis C virus NS3 serine protease domain (residues 1-181 of NS3; NS3 (181)) was expressed as an N-terminally (His)6-tagged fusion protein in Saccharomyces cerevisiae. NS3 (181) protease activity was found in soluble cell lysates, and the N-terminal metal-chelating domain facilitated the efficient purification of active enzyme, using immobilized metal affinity chromatography. The purified NS3(181), protease activity was characterized by assaying the trans-cleavage of in vitro transcription-translation generated substrates, and subsequently a previously unobserved cleavage site within the NS5A region was identified. The inhibitory effect of known protease inhibitors was also examined. It is hoped that availability of this method for the expression and purification of the NS3(181) protease will facilitate the development of anti-hepatitis C therapies.

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.

Obtaining a family of high-affinity, high-specificity protein inhibitors of plasmin and plasma kallikrein.

Human lipoprotein-associated coagulation inhibitor (LACI) is a serum protein containing three Kunitz domains. We displayed the first domain (LACI-D1) on the III protein of phage M13 and made libraries of this domain. We iteratively varied 13 residues in the region corresponding to the BPTI-trypsin interface and selected for binding to human plasmin (PLA) and human plasma kallikrein (pKAL). For PLA, our first-round best binder, EPI-P211, had KD = 2 nM. Using information from the first selection, we made a PLA-biased library containing approximately 500,000 proteins and selected from these a protein, EPI-P302, having a KD for PLA of 87 pM. EPI-P302 inhibits pKAL with KD approximately 250 nM (approximately 2800-fold higher than for PLA) and KD values for other proteases are higher yet. From the same initial LACI-D1 library, we selected an inhibitor of pKAL, EPI-K401, with a KD for pKAL of 287 pM. We used information from this selection to construct a pKAL-biased library from which we selected EPI-K502, which has a KD for pKAL of 40 pM. EPI-K502 inhibits PLA with KD approximately 20 nM (500-fold higher than for pKAL); KD values for other proteases are much higher. For both targets and for both selections, there are families of proteins having a few differences and a range of affinities for their targets. These proteins are candidate drugs and imaging agents for indications involving excess PLA or pKAL. Structure-activity relationships of PLA and pKAL binders will allow design of small molecules that are specific for these targets.

Iterative optimization of high-affinity proteases inhibitors using phage display. 1. Plasmin.

We generated a series of libraries having variants of the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1, also known as tissue-factor pathway inhibitor-I) displayed on bacteriophage M13 as pIII-fusions. We varied LACI-DI iteratively in two regions: the P1 region (positions 10-21) and the "second loop", (positions 31-39), which together form one end of the domain. Display-phage library Lib#1 allows 31 200 amino-acid sequences in P1 region (residues 13, 16-19). Preliminary, we screened Lib#1 against human plasmin (PLA, EC 3.4.21.7) immobolized on agarose to enrich for phage displaying variants with PLA affinity. We introduced a 1600-fold increase in second-loop diversity (residues 31, 32, 34, 39) into the population of selectants from Lib#1, yielding Lib#2. Lib#2 (allowing approximately 50 million amino-acid sequences) was screened against PLA-agarose to isolate highest affinity binders. Protein EPI-P211, derived from the best isolate of Lib#2, inhibits PLA with Ki = 2 nM (at least 500-fold better than LACI-D1) and with high specificity. We used amino-acid sequences of PLA-binding selectants to design a PLA-biased library (Lib#3) which we screened against PLA. The protein EPI-P302 (derived from the best binder obtained from Lib#3) has Ki for PLA inhibition of 87 pM, which is 25-fold better than the first-round best binder and > or = 12 500-fold better than LACI-D1. EPI-P302 also shows very high specificity for PLA vs other human proteases and is resistant to inactivation by oxidants and extremes of temperature or pH. Thus, one can use selectants from one library to design target-tailored combinatorial libraries and obtain quite stable, highly specific, very high-affinity binding molecules while maintaining an essentially human framework.

Iterative optimization of high-affinity protease inhibitors using phage display. 2. Plasma kallikrein and thrombin.

As discussed in the accompanying paper [Markland, W., Ley, A. C., & Ladner, R. C. (1996) Biochemistry 35, 8045-8057], we generated libraries from the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1) using multivalent M13 III display and derived potent inhibitors of human plasmin (PLA) by iterative variegation and selection. Here, we show that high-affinity, high-specificity binders to human plasma kallikrein (pKAL) and human thrombin (THBN) can be obtained starting from the identical library and employing the same iterative variegation procedures used to obtain PLA inhibitors. Lib#1 (allowing 31 200 variants involving five positions near the P1 residue of LACI-D1) and its pKAL-biased derivative, Lib#4 (allowing an additional 1600 variants at residues 31, 32, 34, and 39), were screened against pKAL, yielding potent inhibitors. One of these, EPI-K401, has Ki = 284 pM, very high specificity, and excellent stability. We used information from Lib#4 selectants to design Lib#5 (allowing 1.5 x 10(6) amino-acid sequences involving nine varied positions) from which we obtained an inhibitor (EPI-K503) having high affinity for pKAL (Ki = 40 pM) and retaining the high specificity of EPI-K401. When we screened Lib#1 and its THBN-tailored derivative, Lib#6, against THBN, we obtained a different and very homogeneous population of selected molecules. The purified proteins derived from Lib#6 selectants bound to THBN-agarose beads but did not inhibit proteolytic activity of THBN, suggesting that these selectants bind to a site on THBN other than the catalytic site. Thus, a single large combinatorial library can serve as a source to obtain highly specific, high-affinity binding molecules for each of several targets. Furthermore, the results with THBN show that the binding of Kunitz domains to other proteins is not limited to the catalytic sites of trypsin-homologous proteases.

Affinity maturation of proteins displayed on surface of M13 bacteriophage as major coat protein fusions.

This chapter described the preparation and fractionation of libraries of M13 phage displaying proteins as fusions to the major coat protein. High titer (10(13) pfu/ml) phage libraries can readily be generated using a single vector and the level of display surpasses that of gene III fusion phage. Since the synthetic VIII fusion gene can be customized, this system should provide the flexibility required to construct phage libraries displaying a variety of different peptides and proteins and to select variants possessing the highest affinity for target molecules of a diverse chemical nature.

M13 bacteriophage displaying disulfide-constrained microproteins.

A display-phage library (TN2), displaying an 18-residue peptide fused to coat protein III, represents a collection of up to 8.55 x 10(6) peptides encoded by only 1.68 x 10(7) DNA sequences. Each displayed peptide has two fixed cysteine residues (allowing disulfide formation) and six variegated residues, four between the cysteines and one either side of the cysteines. Screening this library against streptavidin (Sv) and the anti-beta-endorphin monoclonal antibody, 3-E7, yielded phage displaying disulfide-constrained microproteins with sequences similar to those published for the linear-peptide display phage. Analysis of selected clones indicated that a disulfide bond is required for high-affinity binding to each of the target proteins. The microproteins selected for binding to Sv and 3-E7 show more stringent sequence specificity than do linear peptides selected for binding to the same targets.

Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors.

We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product. Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin. A point mutation of BPTI [Lys15-->Leu(K15L)] alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated. Phage were eluted from an immobilized protease with step gradients of decreasing pH. Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix. Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.

Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage.

Inhibitors of human neutrophil elastase were engineered by designing and producing a library of phage-displayed protease inhibitory domains derived from wild-type bovine pancreatic trypsin inhibitor and fractionating the library for binding to the target protease. The affinity of one of the engineered variants for human neutrophil elastase (Kd = 1.0 pM) is 3.6 x 10(6)-fold higher than that of the parental protein and exceeds the highest affinity reported for any reversible human neutrophil elastase inhibitor by 50-fold. Thus the display phage method has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target. The technology can be applied to design high-affinity proteins for a wide variety of target molecules.

Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13.

Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.

Association of p62c-yes with polyomavirus middle T-antigen mutants correlates with transforming ability.

A number of mutants of polyomavirus middle T antigen (MTag) were constructed into replication-competent avian retroviruses. To assess the ability of these MTag variants to transform and to associate with the avian p60c-src and p62c-yes proto-oncogene products, we used these viruses to infect chicken embryo fibroblasts. We found that the ability of individual mutant MTags to associate with p62c-yes correlated well with the ability of these mutants to transform, as has been previously shown for the association of MTag with p60c-src. All transformation-competent mutant MTags retained the ability to complex with p62c-yes. Two transformation-defective mutants, RX67 and RX68, which could weakly associate with p60c-src, were unable to associate with p62c-yes.dl1015, a transformation-defective mutant which could associate with p60c-src and with a phosphatidylinositol kinase activity, was also able to associate with p62c-yes. Therefore, some as yet unmeasured biochemical property is defective in this mutant.

Structure-function analysis of tissue-type plasminogen activator by linker-insertion, point and deletion mutagenesis.

We undertook a structure--function analysis of human tissue plasminogen activator (tPA) using linker-scanning and deletion mutagenesis. Synthetic oligonucleotide linkers were introduced into the tPA cDNA at pre-existing restriction enzyme sites. This generated a series of tPA variants which contained small primary sequence alterations consisting of point mutations, deletions or insertions. The majority of the linker-insertion variants demonstrate a significant reduction in amidolytic and fibrinolytic activity in comparison to wild-type tPA. The exceptions are the variants with linker-inserts placed at the BglII(115) and StyI(277) sites of the tPA cDNA (4SLEG5 and 57LEA58 respectively), which encode insertions at the boundaries of the finger domain. The variants with linker-inserts in the light chain (protease domain) of tPA are the lowest in enzymatic activity. Particularly sensitive to mutation are highly conserved amino acids. Heavy chain deletion variants were constructed from point mutants at the domain boundaries of tPA. Deletion of the kringle domains lowers the fibrinolytic activity to a greater extent than deletion of the finger or growth factor domains. We conclude that alterations in any domain of the tPA molecule, and particularly in the highly conserved residues within these domains, can affect fibrinolytic activity.

Tissue-type plasminogen activator variants with domain duplications and rearrangements.

The interactions between tPA domains that are important for catalysis are poorly understood. We have probed the function of interdomain interactions by generating tPA variants in which domains are duplicated or rearranged. The proteins were expressed in a transient mammalian expression system and tested in vitro for their ability to activate plasminogen, induce fibrinolysis and bind to a forming fibrin clot. Duplication of the heavy chain domains of tPA produced enzymatically active tPA variants, many of which demonstrated similar in vitro amidolytic and fibrinolytic activity and similar fibrin affinity to the parent molecule. Zymographic analysis of the domain duplication tPA variants showed one major active species for each variant. Selection of the residues duplicated and the interdomain spacing were found to be critical considerations in the design of tPA variants with duplicated domains. We also rearranged the domains of tPA such that kringle 1 replaced the second kringle domain and vice versa. An analysis of these variants indicates that the first kringle domain can confer fibrin affinity to a tPA variant and function in place of kringle 2. Therefore, in wild-type tPA, the functions of kringle 1 and kringle 2 must be dependent partially on their orientation within the heavy chain of the protein. The functional autonomy of the heavy and light chains of tPA is demonstrated by the activity of a tPA variant in which the order of the heavy and light chains was reversed.

Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

An in vitro nuclear translocation system is described in which isolated rat liver nuclei were incubated in a defined buffered medium containing radiolabeled or fluorescently labeled exogenous proteins. The nuclei were rapidly recovered, extracted, and analyzed for the presence of associated radiolabeled or fluorescently labeled proteins. The isolated nuclei exhibited the same specificity for protein uptake as seen previously in vivo, accumulating simian virus 40 wild-type large-T antigen and p53 while excluding a cytoplasmic variant of large-T antigen (d10) and bovine serum albumin. The rapid nuclear accumulation of wild-type large-T antigen was shown to be selective and dependent upon the recognition of a wild-type nuclear location signal, ATP and temperature dependent, and unidirectional. Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process.

Mutants of polyomavirus middle-T antigen.

Polyomavirus middle-T antigen induces the transformation of established cell lines in culture and is known to interact with and/or modulate the activity of several enzymes (pp60c.src, protein kinase C and phosphatidylinositol kinase) in vitro. This review is a compilation of the reported mutants of middle-T antigen and their biochemical and biological properties as they relate to the transformation event. The mutants of polyomavirus middle-T antigen have been previously classified phenotypically. Given the now large number of mutants, the classification presented here is based upon the position within the molecule. A model of middle-T is presented in which the protein is considered as consisting of three domains: a hydrophobic domain (the putative membrane-binding domain), the amino-terminal half of the molecule (the putative pp60c.src-binding domain) and the intervening amino acids (the putative modulatory domain). A current model for the induction of transformation by polyomavirus middle-T is presented.

Mapping of the amino-terminal half of polyomavirus middle-T antigen indicates that this region is the binding domain for pp60c-src.

The majority of the carboxy-terminal half of polyomavirus middle-T antigen has been variously mutated and, with the exception of the putative membrane-binding domain (amino acids 394 to 415), was found to be largely dispensible for the transforming activity of the protein. A comparison of the small-T antigen amino acid sequences (equivalent to the region of middle-T encoded by exon 1) of simian virus 40, BK virus, polyomavirus, and a recently described hamster papovavirus highlighted regions of potential interest in mapping functions to the amino-terminal half of polyomavirus middle-T antigen. The regions of interest include amino acids 168 to 191 (previously investigated by this group [S. H. Cheng, W. Markland, A. F. Markham, and A. E. Smith, EMBO J. 5:325-334, 1986]), two cysteine-rich clusters (amino acids 120 to 125 and 148 to 153), and amino acids 92 to 117 (within the limits of the previously described hr-t mutant, SD15). Point mutations, multiple point mutations, and deletions were made by site-specific and site-directed mutagenesis within the cysteine-rich clusters and residues 92 to 117. Studies of the transforming ability of the altered middle-T species demonstrated that this activity is highly sensitive to amino acid changes. All four regions (as defined above) within the amino-terminal half of middle-T have now been studied in detail. The phenotype of the mutants is predominantly transformation defective, and the corresponding variant middle-T species are characterized by being either totally or severely handicapped in the ability to associate actively with pp60c-src. Whether the mutations affect the regions of interaction between middle-T and pp60c-src or simply interfere with the overall conformation of this domain is not known. However, there would appear to be a conformational constraint on this portion of the molecule with regard to its interaction with pp60c-src and by extension to the ability of the middle-T species to transform.