Rescue of the cardiac defect in ErbB2 mutant mice reveals essential roles of ErbB2 in peripheral nervous system development.

ErbB2 receptor tyrosine kinase plays a role in neuregulin signaling and is expressed in the developing nervous system. We genetically rescued the cardiac defect of erbB2 null mutant embryos, which otherwise died at E11. These rescued erbB2 mutant mice die at birth and display a severe loss of both motor and sensory neurons. Motor and sensory axons are severely defasciculated and aberrantly projected within their final target tissues. Schwann cells are completely absent in the peripheral nerves. Schwann cell precursors are present within the DRG and proliferate normally, but their ability to migrate is decreased. Acetylcholine receptors cluster within the central band of the mutant diaphragm muscle. However, these clusters are dispersed and morphologically different from those in control muscle. Our results reveal an important role for erbB2 during normal peripheral nervous system development.

Patients with congenital myasthenia associated with end-plate acetylcholinesterase deficiency show normal sequence, mRNA splicing, and assembly of catalytic subunits.

A congenital myasthenic condition has been described in several patients characterized by a deficiency in end-plate acetylcholinesterase (AChE). The characteristic form of AChE in the end-plate basal lamina has the catalytic subunits disulfide linked to a collagen-like tail unit. Southern analysis of the gene encoding the catalytic subunits revealed no differences between patient and control DNA. Genomic DNA clones covering exon 4 and the alternatively spliced exons 5 and 6 were analyzed by nuclease protection and sequencing. Although allelic differences were detected between controls, we found no differences in exonic and intronic areas that might yield distinctive splicing patterns in patients and controls. The ACHE gene was cloned from genomic libraries from a patient and a control. Transfection of the cloned genes revealed identical species of mRNA and expressed AChE. Cotransfection of the genes expressing the catalytic subunits with a cDNA from Torpedo encoding the tail unit yielded asymmetric species that require assembly of catalytic subunits and tail unit. thus the catalytic subunits of AChE expressed in the congenital myasthenic syndrome appear identical in sequence, arise from similar splicing patterns, and assemble normally with a tail unit to form a heteromeric species.

Influence of polyfluorination of the phenylalanine ring of angiotensin II on conformation and biological activity.

[Phe(F5)8]angiotensin II was synthesized by the solid phase method and purified by reverse-phase HPLC. In rat uterus and rabbit aorta bioassays the analogue had 10 and 50%, respectively, of the contractile activity of angiotensin II and demonstrated antagonist properties. These findings illustrate that inversion of the Phe8 ring quadrupole moment in angiotensin II decreases agonist activity and invokes antagonist properties. 1H-NMR studies at 400 MHz in DMSO-d6 demonstrated the presence of cis and trans isomers in the ratio 1:3 due to restricted rotation of the His-Pro bond. Downfield shifts of the His C2 and C4 protons in [Phe(F5)]ANG II compared to ANG II suggest that the Phe(F5) residue may be involved in a parallel-plate ring pairing interaction with the imidazole group. However heteronuclear NOE studies, carried out by measuring the proton difference spectrum before and after saturation of the fluorine resonances, showed the absence of any NOE enhancement illustrating that electrostatic influences of the Phe(F5) ring occur at relatively long range.

Structural determinants of haloenol lactone-mediated suicide inhibition of canine myocardial calcium-independent phospholipase A2.

Haloenol lactones are potent mechanism-based inhibitors of a novel class of calcium-independent phospholipases A2 which have been implicated as the enzymic mediators of membrane dysfunction during myocardial ischemia (Hazen, S. L.; et al. J. Biol. Chem. 1991, 266, 7227-7232). Herein we demonstrate that the ring size, hydrophobic group, and cryptic electrophile in the haloenol lactone moiety are important and modifiable determinants of the inhibitory potency of haloenol lactone-mediated inhibition of calcium-independent phospholipase A2. Direct comparisons between haloenol lactone-mediated inhibition of calcium-independent phospholipase A2 and the absence of inhibition with calcium-dependent phospholipase A2 further underscore the marked differences in the catalytic strategy employed by these two classes of intracellular phospholipases A2.

Conformationally constrained [p-(omega-aminoalkyl)phenacetyl]-L-seryl-L-lysyl dipeptide amides as potent peptidomimetic inhibitors of Candida albicans and human myristoyl-CoA:protein N-myristoyl transferase.

MyristoylCoA:protein N-myristoyltransferase (NMT) covalently attaches the 14-carbon saturated fatty acid myristate, via an amide bond, to the N-terminal glycine residues of a variety of cellular proteins. Genetic studies have shown that NMT is essential for the viability of the principal fungal pathogens which cause systemic infection in immunosuppressed humans and hence is a target for development of fungicidal drugs. We have generated a class of potent peptidomimetic inhibitors of the NMT from one such fungal pathogen, Candida albicans. The N-terminal tetrapeptide from a substrate analog inhibitor, ALYASKL-NH2, was replaced with an omega-aminoalkanoyl moiety having an optimal 11-carbon chain for inhibition (11-aminoundecanoyl-SKL-NH2, 3a, IC50 = 1.2 +/- 0.14 microM). A series of replacements for the C-terminal Leu established that residues containing a lipophilic side chain were most effective, with cyclohexylalanine having the greatest potency (3g, IC50 = 0.36 +/- 0.06 microM). Removal of the carboxamide moiety led to a metabolically stable dipeptide inhibitor containing an N-(cyclohexylethyl)lysinamide (17e, IC50 = 0.11 +/- 0.03 microM). Partial rigidification of the flexible aminoundecanoyl chain produced the dipeptide p-(omega-aminohexyl)phenacetyl-L-seryl-L-lysyl-N-(cyclohexyleth yl)amide (26b, IC50 = 0.11 +/- 0.04 microM). Subsequent incorporation of an alpha-methyl substituent into 26b provided the dipeptide analog [2-[p-(omega-aminohexyl)phenyl]propionyl]-L-seryl-L-lysyl-N-(cyclohex ylethyl)amide, a very potent inhibitor (48, IC50 = 0.043 +/- 0.006 microM), which retained the three essential elements required for recognition by the acyl transferase's peptide binding site.

Design and synthesis of novel imidazole-substituted dipeptide amides as potent and selective inhibitors of Candida albicans myristoylCoA:protein N-myristoyltransferase and identification of related tripeptide inhibitors with mechanism-based antifungal activity.

A new class of antifungal agents has been discovered which exert their activity by blockade of myristoylCoA: protein N-myristoyltransferase (NMT; EC 2.1.3.97). Genetic experiments have established that NMT is needed to maintain the viability of Candida albicans and Cryptococcus neoformans,the two principal causes of systemic fungal infections in immunocompromised humans. Beginning with a weak octapeptide inhibitor ALYASKLS-NH2 (2, Ki = 15.3 +/- 6.4 microM), a series of imidazole-substituted Ser-Lys dipeptide amides have been designed and synthesized as potent and selective inhibitors of Candida albicans NMT. The strategy that led to these inhibitors evolved from the identification of those functional groups in the high-affinity octapeptide substrate GLYASKLS-NH2 1a necessary for tight binding, truncation of the C-terminus, replacement of the four amino acids at the N-terminus by a spacer group, and substitution of the glycine amino group with an N-linked 2-methylimidazole moiety. Initial structure-activity studies led to the identification of 31 as a potent and selective peptidomimetic inhibitor with an IC50 of 56 nM and 250-fold selectivity versus human NMT. 2-Methylimidazole as the N-terminal amine replacement in combination with a 4-substituted phenacetyl moiety imparts remarkable potency and selectivity to this novel class of inhibitors. The (S,S) stereochemistry of serine and lysine residues is critical for the inhibitory activity, since the (R,R) enantiomer 40 is 10(3)-fold less active than the (S,S) isomer 31. The inhibitory profile exhibited by this new class of NMT ligands is a function of the pKa of the imidazole substituent as illustrated by the benzimidazole analog 35 which is about 10-fold less potent than 31. The measured pKa (7.1 +/- 0.5) of 2-methylimidazole in 31 is comparable with the estimated pKa (approximately 8.0) of the glycyl residue in the high-affinity substrate 1a. Groups bulkier than methyl, such as ethyl, isopropyl, or iodo, at the imidazole 2-position have a detrimental effect on potency. Further refinement of 31 by grafting an alpha-methyl group at the benzylic position adjacent to the serine residue led to 61 with an IC50 of 40 nM. Subsequent chiral chromatography of 61 culminated in the discovery of the most potent Candida NMT inhibitor 61a reported to date with an IC50 of 20 nM and 400-fold selectivity versus the human enzyme. Both 31 and 61a are competitive inhibitors of Candida NMT with respect to the octapeptide substrate GNAASARR-NH2 with Ki(app) = 30 and 27 nM, respectively. The potency and selectivity displayed by these inhibitors are dependent upon the size and orientation of the alpha-substituent. An alpha-methyl group with the R configuration corresponding to the (S)-methyl-4-alanine in 2 confers maximum potency and selectivity. Structural modification of 31 and 61 by appending an (S)-carboxyl group beta to the cyclohexyl moiety provided the less potent tripeptide inhibitors 73a and 73b with an IC50 of 1.45 +/- 0.08 and 0.38 +/- 0.03 microM, respectively. However, these tripeptides (73a and 73b) exhibited a pronounced selectivity of 560- and 2200-fold versus the human NMT. More importantly 73a displayed fungistatic activity against C albicans with an EC50 of 51 +/- 17 microM in cell culture. Compound 73b also exhibited a similar antifungal activity. An Arf protein gel mobility shift assay for monitoring intracellular myristoylation revealed that a single dose of 200 microM of 73a or 73b produced < 50% reduction in Arf N-myristoylation, after 24 and 48 h, consistent with their fungistatic rather than fungicidal activity. In contrast, the enantiomer 73d which had an IC50 > 1000 microM against C. albicans NMT did not exhibit antifungal activity and produced no detectable reduction in Arf N-myristoylation in cultures of C. albicans. These studies confirm that the observed antifungal activity of 73a and 73b is due to the attenuation of NMT activity and that NMT represents an attractive tar

Promoter elements and transcriptional regulation of the acetylcholinesterase gene.

The 5' region of the acetylcholinesterase gene from the electric ray Torpedo californica has been cloned and its cap site identified. The 5' untranslated region is divided into two exons where a small exon extending between bp -22 to -60 is alternatively spliced. Cap sites are defined at two positions, bp -138 and -143. Twenty-one base pairs 5' of the -143 cap site a repeating TATA sequence is found. Further upstream in the gene consensus sequences for Sp1, AP1, and AP2 factors are evident. The promoter region of the acetylcholinesterase gene enhances transcription of a luciferase reporter gene transfected into C2 myoblasts. However, increased transcription was not evident after C2 myoblasts were induced to form myotubes. Cotransfection of this construct with c-Jun (AP1) and AP2 expression vectors shows marked increases of transcription rates in HepG2 and C2 cells. Protein kinase A elicited regulation of expression is also evident in quail fibroblasts. In gel retardation experiments both recombinant c-Jun (AP1) and AP2 proteins bind to the appropriate Torpedo sequences. Cellular extracts from the Torpedo electric organ exhibit AP2 binding activity. Thus, although all facets of specific regulation expected upon differentiation of mammalian muscle cells were not evident, the 5'-flanking region from the Torpedo AChE gene contains consensus sequences and functional promoter elements typical of mammalian nerve and muscle systems.

Structure and regulation of expression of the acetylcholinesterase gene.

Acetylcholinesterase, an enzyme essential for the termination of the action of acetylcholine, is encoded by a single gene. Alternative mRNA processing gives rise to the expression of enzyme forms with three distinct carboxyl-termini. These structural differences govern the cellular disposition of the expressed enzyme but do not influence catalytic activity. Alternative polyadenylation signals give rise to distinct 3' non-coding regions which are likely to affect mRNA stability. Alternative splicing also occurs at the 5' end of the gene where two promoter regions can be identified. Hence, regulation of expression of the gene occurs at 3 levels, transcriptional through alternative promoters, translational by affecting mRNA stability and processing of distinct mRNAs and post-translationally by giving rise to distinct peptide chains which are processed differently. Recombinant DNA studies have also been extended to modifying protein structure through site-specific mutagenesis and studying the function of the mutant enzymes.

Transcription factor repression and activation of the human acetylcholinesterase gene.

Acetylcholinesterase in man is encoded by a single gene, ACHE, located on chromosome 7q22. In this study, the transcription start sites and major DNA promoter elements controlling the expression of this gene have been characterized by structural and functional studies. Immediately upstream of the first untranslated exon of the gene are GC-rich sequences containing consensus binding sites for several transcription factors, including Sp1, EGR-1 and AP2. In vitro transcription studies and RNase protection analyses of mRNA isolated from human NT2/D1 teratocarcinoma cells reveal that two closely spaced transcription cap sites are located at a consensus initiator (Inr) element similar to that found in the terminal transferase gene. Transient transfection of mutant genes shows that removal of three bases of this initiator sequence reduces promoter activity by 98% in NT2/D1 cells. In vitro transcription studies and transient transfection of a series of 5' deletion mutants of the ACHE promoter linked to a luciferase reporter show an Sp1 site at -71 to be essential for promoter activity. Purified Sp1 protein protects this site from DNase cleavage during in vitro footprinting experiments. A conserved AP2 consensus binding site, located between the GC box elements and the Inr, is protected by recombinant AP2 protein in DNase footprinting experiments, induces a mobility shift with AP2 protein and AP2-containing cell extracts, and fosters inhibition of transcription by AP2 as measured by transient transfection in mouse and human cell lines and in in vitro transcription reactions. These results indicate that AP2 functions as a repressor of human ACHE and mouse Ache transcription.

The human gene encoding acetylcholinesterase is located on the long arm of chromosome 7.

Acetylcholinesterase (AChE) is a secreted enzyme essential for regulating cholinergic neurotransmission at neuronal and neuromuscular synapses. In view of the altered expression of AChE in some central neurological and neuromuscular disorders with a probable genetic basis, we have identified the chromosomal location of the gene encoding AChE. Chromosomal in situ suppression hybridization analysis revealed a single gene to be at 7q22, a result which was confirmed by PCR analysis of genomic DNA from a human/hamster somatic cell hybrid containing a single human chromosome 7. The AChE gene thus maps to the same region in which frequent nonrandom chromosome 7 deletions occur in leukemias of myeloid cell precursors known to express the enzyme during normal differentiation.

Gene structure of mammalian acetylcholinesterase. Alternative exons dictate tissue-specific expression.

The genes encoding mouse and human acetylcholinesterases have been cloned from genomic and cosmid libraries. Restriction analysis and a comparison of sequence with the cDNAs have defined the exon-intron boundaries. In mammals, three invariant exons encode the signal peptide and the amino-terminal 535 amino acids common to all forms of the enzyme whereas alternative exon usage of the next exon accounts for the structural divergence in the carboxyl termini of the catalytic subunits. mRNA protection studies show that the cDNA encoding the hydrophilic catalytic subunits represents the dominant mRNA species in mammalian brain and muscle whereas divergent mRNA species are evident in cells of hematopoietic origin (bone marrow cells and a erythroleukemia cell line). Analyses of mRNA species in these cells and the genomic sequence have enabled us to define two alternative exons in addition to the one found in the cDNAs; they encode unique carboxyl-terminal sequences. One mRNA consists of a direct extension through the intervening sequence between the common exon and the 3' exon deduced from the cDNA. This sequence encodes a subunit lacking the cysteine critical to oligomer formation. Another mRNA results from a splice that encodes a stretch of hydrophobic amino acids immediately upstream of a stop codon. This exon, when spliced to the upstream invariant exons, should encode glycophospholipid-linked species of the enzyme. Homologous sequence, identity of exon-intron junctions, and identity of position of the stop codon are seen for this region in mouse and human. Polymerase chain reactions carried out across the expected intron region and mRNA protection studies show that this splice occurs in mouse bone marrow and erythroleukemia cells yielding the appropriate cDNA.

Kinetic and structural evidence for a sequential ordered Bi Bi mechanism of catalysis by Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

The mechanism of catalysis of Escherichia coli-derived Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase (NMT) has been characterized. Previous studies indicated that a high affinity reaction intermediate forms between NMT and myristoyl-CoA in the absence of a peptide substrate. This complex has been further characterized using S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog. Binding studies involving this analog, as well as myristoylpeptide and CoA, have indicated that the CoA moiety of the acyl substrate is retained in the acyl-NMT complex prior to peptide addition. These structural data, along with kinetic studies of myristoylpeptide and CoA product inhibition, indicate that the mechanism of catalysis of NMT is ordered Bi Bi, with myristoyl-CoA binding to NMT occurring prior to peptide binding and CoA release taking place before release of acyl peptide. Further analyses of the interactions between NMT, acyl peptide, and CoA demonstrate that NMT is able to deacylate a myristoylpeptide in the presence of CoA.

Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2.

The majority of phospholipase A2 activity in myocardium is calcium-independent and selective for hydrolysis of plasmalogen substrate (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303; Hazen, S. L., Stuppy, R. J., and Gross, R. W. (1990) J. Biol. Chem. 265, 10622-10630). Accordingly, identification of an inhibitor which selectively targets calcium-independent phospholipases A2 would facilitate elucidation of the biologic significance of this class of intracellular phospholipases. We now report that the haloenol lactone, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (Compound 1), is a potent, irreversible, mechanism-based inhibitor of myocardial calcium-independent phospholipase A2 which is greater than 1000-fold specific for inhibition of myocardial calcium-independent phospholipase A2 in comparisons with multiple calcium-dependent phospholipases A2. Mechanism-based inhibition of myocardial cytosolic calcium-independent phospholipase A2 by Compound 1 was established by demonstrating: 1) time-dependent irreversible inactivation; 2) covalent binding of [3H]Compound 1 to the purified phospholipase A2; 3) ablation of covalent binding of [3H]Compound 1 after chemical inactivation of phospholipase A2 enzymic activity; 4) identical inhibition of myocardial phospholipase A2 by Compound 1 in the absence or presence of nucleophilic scavengers; 5) Compound 1 is a substrate for myocardial calcium-independent phospholipase A2 resulting in the generation of the electrophilic alpha-bromomethyl ketone; 6) phospholipase A2 inhibition requires the in situ generation of the reactive electrophile (i.e. neither the alpha-bromomethyl ketone nor the diproteoenol lactone analog are inhibitory); and 7) concomitant attenuation of the inhibitory potency and the extent of covalent adduct formation in the presence of saturating substrate. Collectively, these results demonstrate that the haloenol lactone, Compound 1, is a substrate for, covalently binds to, and irreversibly inhibits canine myocardial cytosolic calcium-independent phospholipase A2.

alpha-Alkyl-alpha-amino-beta-sulphone hydroxamates as potent MMP inhibitors that spare MMP-1.

A series of alpha-alkyl-alpha-amino-beta-sulphone hydroxamates was prepared and evaluated for potency versus MMP-2 and MMP-13, and for selectivity versus MMP-1. Low nanomolar potency was obtained with selectivity versus MMP-1 ranging from >10 to >1000. Selected compounds were orally bioavailable.

SC-52151, a novel inhibitor of the human immunodeficiency virus protease.

SC-52151 is a potent, selective, tight-binding human immunodeficiency virus (HIV) protease inhibitor containing the novel (R)-(hydroxyethyl) urea isostere. The mean 50% effective concentration for lymphotropic, monocytotropic strains and field isolates of HIV type 1 (HIV-1), HIV-2, and simian immunodeficiency virus is 26 ng/ml (43 nM). The combination of SC-52151 and nucleoside reverse transcriptase inhibitors synergistically inhibited HIV-1 replication without additive toxicity. An extended postantiviral effect correlates with inhibition of gag and gag-pol polyprotein processing. SC-52151 is highly protein bound ( >90%) in human plasma, and the level of partitioning into erythrocytes is low. Physiological concentrations of alpha-1-acid glycoprotein, but not albumin, substantially affect the antiviral potency of SC-52151. The oral bioavailability of [14C]SC-52151 is 17% when it is administered as an elixir to the rat, dog, or monkey. Oxidation of the t-butyl moiety is the major route of biotransformation, and elimination is mainly by biliary excretion. No toxicologically significant effects have been observed in animals. Pharmacokinetic and metabolism studies in multiple animal species predict 20 to 30% systemic bioavailability, an elimination half-life of 1 to 2 h, and a volume of distribution of greater than 3 liters/kg in humans.

Scanning alanine mutagenesis and de-peptidization of a Candida albicans myristoyl-CoA:protein N-myristoyltransferase octapeptide substrate reveals three elements critical for molecular recognition.

Candida albicans produces a single myristoyl-CoA:protein N-myristoyltransferase (Nmt) that is essential for its viability. An ADP-ribosylation factor (Arf) is included among the few cellular protein substrates of this enzyme. An octapeptide (GLYASKLS-NH2) derived from a N-terminal Arf sequence was used as the starting point to identify elements critical for recognition by the acyltransferases's peptide-binding site. In vitro kinetic studies, employing purified Nmt and a panel of peptides with single Ala substitutions at each position of GLYASKLS-NH2, established that its Gly1, Ser5, and Lys6 residues play predominant roles in binding. ALYASKLS-NH2 was found to be an inhibitor competitive for peptide (Ki = 15.3 +/- 6.4 microM) and noncompetitive for myristoyl-CoA (Ki = 31.2 +/- 0.7 microM). A survey of 26 derivatives of this inhibitor, representing (i) a complete alanine scan, (ii) progressive C-terminal truncations, and (iii) manipulation of the physical-chemical properties of its residues 1, 5, and 6, confirmed the important stereochemical requirements for the N-terminal amine, the beta-hydroxyl of Ser5, and the epsilon-amino group of Lys6. Remarkably, replacement of the the N-terminal tetrapeptide of ALYASKLS-NH2 with an 11-aminoundecanoyl group produced a competitive inhibitor, 11-aminoundecanoyl-SKLS-NH2, that was 38-fold more potent (Ki = 0.40 +/- 0.03 microM) than the starting octapeptide. Removing the primary amine (undecanoyl-SKLS-NH2), or replacing it with a methyl group (dodecanoyl-SKLS-NH2), resulted in 26- and 34-fold increases in IC50, confirming the important contribution of the amine to recognition. Removal of LeuSer from the C terminus (11-aminoundecanoyl-SK-NH2) yielded a competitive dipeptide inhibitor with a Ki (11.7 +/- 0.4 microM) equivalent to that of the starting octapeptide, ALYASKLS-NH2. Substitution of Ser with homoserine, cis-4-hydroxyproline, or tyrosine reduces potency by 3-70-fold, emphasizing the requirement for proper presentation of the hydroxyl group in the dipeptide inhibitor. Substituting D- for L-Lys decreases its inhibitory activity >100-fold, while deletion of the epsilon-amino group (Nle) or masking its charge (epsilon-N-acetyl-lysine) produces 4-7-fold attenuations. L-His, but not its D-isomer, can fully substitute for L-Lys, producing a competitive dipeptide inhibitor with similar potency (Ki = 11.9 +/- 1.0 microM). 11-Aminoundecanoyl-SK-NH2 and 11-aminoundecanoyl-SH-NH2 establish that a simple alkyl backbone can maintain an appropriate distance between three elements critical for recognition by the fungal enzyme's peptide-binding site: a simple omega-terminal amino group, a beta-hydroxyl, and an epsilon-amino group or an imidazole. These compounds contain one peptide bond and two chiral centers, suggesting that it may be feasible to incorporate these elements of recognition, or functionally equivalent mimics, into a fully de-peptidized Nmt inhibitor.

DPC 681 and DPC 684: potent, selective inhibitors of human immunodeficiency virus protease active against clinically relevant mutant variants.

Human immunodeficiency virus (HIV) protease inhibitors (PIs) are important components of many highly active antiretroviral therapy regimens. However, development of phenotypic and/or genotypic resistance can occur, including cross-resistance to other PIs. Development of resistance takes place because trough levels of free drug are inadequate to suppress preexisting resistant mutant variants and/or to inhibit de novo-generated resistant mutant variants. There is thus a need for new PIs, which are more potent against mutant variants of HIV and show higher levels of free drug at the trough. We have optimized a series of substituted sulfonamides and evaluated the inhibitors against laboratory strains and clinical isolates of HIV type 1 (HIV-1), including viruses with mutations in the protease gene. In addition, serum protein binding was determined to estimate total drug requirements for 90% suppression of virus replication (plasma IC(90)). Two compounds resulting from our studies, designated DPC 681 and DPC 684, are potent and selective inhibitors of HIV protease with IC(90)s for wild-type HIV-1 of 4 to 40 nM. DPC 681 and DPC 684 showed no loss in potency toward recombinant mutant HIVs with the D30N mutation and a fivefold or smaller loss in potency toward mutant variants with three to five amino acid substitutions. A panel of chimeric viruses constructed from clinical samples from patients who failed PI-containing regimens and containing 5 to 11 mutations, including positions 10, 32, 46, 47, 50, 54, 63, 71, 82, 84, and 90 had mean IC(50) values of <20 nM for DPC 681 and DPC 681, respectively. In contrast, marketed PIs had mean IC(50) values ranging from 200 nM (amprenavir) to >900 nM (nelfinavir).