Resistance of K-RasBV12 proteins to farnesyltransferase inhibitors in Rat1 cells.

Benzodiazepine (BZA)-5B, a CAAX farnesyl-transferase inhibitor, was previously shown to block the farnesylation of H-Ras and to reverse the transformed morphology of Rat1 cells expressing oncogenic H-RasV12. Non-transformed Rat1 cells were not affected by BZA-5B, suggesting that they produce a form of Ras whose prenylation is not blocked by this compound. The likely candidate is K-RasB, which differs from H-Ras primarily in the terminal 24 amino acids. In the current study we examined the effect of BZA-5B on the prenylation of a chimeric oncogenic Ras protein designated H/K-RasBV12, consisting of the first 164 amino acids of H-RasV12 followed by the last 24 amino acids of K-RasB. BZA-5B failed to block the prenylation of this chimera and was thus unable to reverse the transformed morphology of Rat1 cells in which it was expressed. Another potent inhibitor of H-Ras farnesylation, L-739,749, also failed to block prenylation of H/K-RasBV12. Similar results were obtained in transfected cells expressing a widely used version of K-RasBV12 containing a 10-amino acid extension at its NH2 terminus. Neither BZA-5B nor L-739,749 reversed the transformed morphology of cells expressing H/K-RasBV12. The resistance of K-RasB to farnesyltransferase inhibition provides a likely explanation for the resistance of nontransformed cells to the growth inhibitory effects of BZA-5B and L-739,749.

Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B.

The objective of this study was to elucidate the role of the proteasome pathway or multicatalytic proteinase complex in the induction of immunologic nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages activated by lipopolysaccharide. Macrophages were incubated in the presence of lipopolysaccharide plus test agent for up to 24 hr. Culture media were analyzed for accumulation of stable oxidation products of NO (NO2- + N03-, designated as NOX-), cellular RNA was extracted for determination of iNOS mRNA levels by Northern blot analysis, and nuclear extracts were prepared for determination of NF-kappa B by electrophoretic mobility-shift assay. Inhibitors of calpain (alpha-N-acetyl-Leu-Leu-norleucinal; N-benzyloxycarbonyl-Leu-leucinal) and the proteasome (N-benzyloxycarbonyl-Ile-Glu-(O-t-Bu)-Ala-leucinal) markedly inhibited or abolished the induction of iNOS in macrophages. The proteinase inhibitors interfered with lipopolysaccharide-induced NOX- production by macrophages, and this effect was accompanied by comparable interference with the appearance of both iNOS mRNA and NF-kappa B. Calpain inhibitors elicited effects at concentrations of 1-100 microM, whereas the proteasome inhibitor was 1000-fold more potent, producing significant inhibitory effects at 1 nM. The present findings indicate that the proteasome pathway is essential for lipopolysaccharide-induced expression of the iNOS gene in rat alveolar macrophages. Furthermore, the data support the view that the proteasome pathway is directly involved in promoting the activation of NF-kappa B and that the induction of iNOS by lipopolysaccharide involves the transcriptional action of NF-kappaB.

Peptides from conserved regions of paramyxovirus fusion (F) proteins are potent inhibitors of viral fusion.

The synthetic peptides DP-107 and DP-178 (T-20), derived from separate domains within the human immunodeficiency virus type 1 (HIV-1) transmembrane (TM) protein, gp4l, are stable and potent inhibitors of HIV-1 infection and fusion. Using a computer searching strategy (computerized antiviral searching technology, C.A.S.T.) based on the predicted secondary structure of DP-107 and DP-178 (T-20), we have identified conserved heptad repeat domains analogous to the DP-107 and DP-178 regions of HIV-1 gp41 within the glycoproteins of other fusogenic viruses. Here we report on antiviral peptides derived from three representative paramyxoviruses, respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV-3), and measles virus (MV). We screened crude preparations of synthetic 35-residue peptides, scanning the DP-178-like domains, in antiviral assays. Peptide preparations demonstrating antiviral activity were purified and tested for their ability to block syncytium formation. Representative DP-178-like peptides from each paramyxovirus blocked homologous virus-mediated syncytium formation and exhibited EC50 values in the range 0.015-0.250 microM. Moreover, these peptides were highly selective for the virus of origin. Identification of biologically active peptides derived from domains within paramyxovirus F1 proteins analogous to the DP-178 domain of HIV-1 gp4l is compelling evidence for equivalent structural and functional features between retroviral and paramyxoviral fusion proteins. These antiviral peptides provide a novel approach to the development of targeted therapies for paramyxovirus infections.

Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors.

The observed in vitro and in vivo benefit of combination treatment with anti-human immunodeficiency virus (HIV) agents prompted us to examine the potential of resistance development when two protease inhibitors are used concurrently. Recombinant HIV-1 (NL4-3) proteases containing combined resistance mutations associated with BMS-186318 and A-77003 (or saquinavir) were either inactive or had impaired enzyme activity. Subsequent construction of HIV-1 (NL4-3) proviral clones containing the same mutations yielded viruses that were severely impaired in growth or nonviable, confirming that combination therapy may be advantageous. However, passage of BMS-186318-resistant HIV-1 (RF) in the presence of either saquinavir or SC52151, which represented sequential drug treatment, produced viable viruses resistant to both BMS-186318 and the second compound. The predominant breakthrough virus contained the G48V/A71T/V82A protease mutations. The clone-purified RF (G48V/A71T/V82A) virus, unlike the corresponding defective NL4-3 triple mutant, grew well and displayed cross-resistance to four distinct protease inhibitors. Chimeric virus and in vitro mutagenesis studies indicated that the RF-specific protease sequence, specifically the Ile at residue 10, enabled the NL4-3 strain with the triple mutant to grow. Our results clearly indicate that viral genetic background will play a key role in determining whether cross-resistance variants will arise.

Apolipoprotein E4 allele as a predictor of cholinergic deficits and treatment outcome in Alzheimer disease.

Apolipoprotein E (apoE) is critical in the modulation of cholesterol and phospholipid transport between cells of different types. Human apoE is a polymorphic protein with three common alleles, APO epsilon 2, APO epsilon 3, and APO epsilon 4. ApoE4 is associated with sporadic and late-onset familial Alzheimer disease (AD). Gene dose was shown to have an effect on risk of developing AD, age of onset, accumulation of senile plaques in the brain, and reduction of choline acetyltransferase (ChAT) activity in the hippocampus of AD subjects. To characterize the possible impact of the apoE4 allele on cholinergic markers in AD, we examined the effect of apoE4 allele copy number on pre- and postsynaptic markers of cholinergic activity. ApoE4 allele copy number showed an inverse relationship with residual brain ChAT activity and nicotinic receptor binding sites in both the hippocampal formation and the temporal cortex of AD subjects. AD cases lacking the apoE4 allele showed ChAT activities close or within age-matched normal control values. The effect of the apoE4 allele on cholinomimetic drug responsiveness was assessed next in a group (n = 40) of AD patients who completed a double-blind, 30-week clinical trial of the cholinesterase inhibitor tacrine. Results showed that > 80% of apoE4-negative AD patients showed marked improvement after 30 weeks as measured by the AD assessment scale (ADAS), whereas 60% of apoE4 carriers had ADAS scores that were worse compared to baseline. These results strongly support the concept that apoE4 plays a crucial role in the cholinergic dysfunction associated with AD and may be a prognostic indicator of poor response to therapy with acetylcholinesterase inhibitors in AD patients.

Activated Drosophila Ras1 is selectively suppressed by isoprenyl transferase inhibitors.

Ras CAAX (C = cysteine, A = aliphatic amino acid, and X = any amino acid) peptidomimetic inhibitors of farnesyl protein transferase suppress Ras-dependent cell transformation by preventing farnesylation of the Ras oncoprotein. These compounds are potential anticancer agents for tumors associated with Ras mutations. The peptidomimetic FTI-254 was tested for Ras1-inhibiting activity in whole animals by injection of activated Ras1val12 Drosophila larvae. FTI-254 decreased the ability of Ras1val12 to form supernumerary R7 photoreceptor cells in the compound eye of transformed flies. In contrast, it had no effect on the related supernumerary R7 phenotypes of flies transformed with either the activated sevenless receptor tyrosine kinase, Raf kinase, or a chimeric Ras1val12 protein that is membrane associated through myristylation instead of isoprenylation. Therefore, FTI-254 acts as an isoprenylation inhibitor to selectively inhibit Ras1val12 signaling activity in a whole-animal model system.

Peptide inhibitors of peptidyltransferase alter the conformation of domains IV and V of large subunit rRNA: a model for nascent peptide control of translation.

Peptides of 5 and 8 residues encoded by the leaders of attenuation regulated chloramphenicol-resistance genes inhibit the peptidyltransferase of microorganisms from the three kingdoms. Therefore, the ribosomal target for the peptides is likely to be a conserved structure and/or sequence. The inhibitor peptides "footprint" to nucleotides of domain V in large subunit rRNA when peptide-ribosome complexes are probed with dimethyl sulfate. Accordingly, rRNA was examined as a candidate for the site of peptide binding. Inhibitor peptides MVKTD and MSTSKNAD were mixed with rRNA phenol-extracted from Escherichia coli ribosomes. The conformation of the RNA was then probed by limited digestion with nucleases that cleave at single-stranded (T1 endonuclease) and double-stranded (V1 endonuclease) sites. Both peptides selectively altered the susceptibility of domains IV and V of 23S rRNA to digestion by T1 endonuclease. Peptide effects on cleavage by V1 nuclease were observed only in domain V. The T1 nuclease susceptibility of domain V of in vitro-transcribed 23S rRNA was also altered by the peptides, demonstrating that peptide binding to the rRNA is independent of ribosomal protein. We propose the peptides MVKTD and MSTSKNAD perturb peptidyltransferase center catalytic activities by altering the conformation of domains IV and V of 23S rRNA. These findings provide a general mechanism through which nascent peptides may cis-regulate the catalytic activities of translating ribosomes.

Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is the major target for antiretroviral therapy of the acquired immunodeficiency syndrome (AIDS). While some inhibitors exhibit activity against most retroviral RTs, others are specific for the HIV-1 enzyme. To develop an animal model for the therapy of the HIV-1 infection with RT inhibitors, the RT of the simian immunodeficiency virus (SIV) was replaced by the RT of HIV-1. Macaques infected with this SIV/HIV-1 hybrid virus developed AIDS-like symptoms and pathology. The HIV-1-specific RT inhibitor LY300046.HCl, but not zidovudine [3'-azido-3'-deoxythymidine (AZT)] delayed the appearance of plasma antigenemia in macaques infected with a high dose of the chimeric virus. Infection of macaques with the chimeric virus seems to be a valuable model to study the in vivo efficacy of new RT inhibitors, the emergence and reversal of drug resistance, the therapy of infections with drug-resistant viruses, and the efficacy of combination therapy.

Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition.

Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of PI2-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by PI2, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by PI2. PI2-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.

Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors.

A combination of transient kinetic and equilibrium titration methods has been used to show that both primer/template and nucleotide binding to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase are two-step processes. In both cases, after initial formation of relatively weakly bound states, isomerization reactions lead to tightly bound states. In the case of deoxynucleotide binding to the reverse transcriptase-primer/template complex, the second step in the interaction is rate-limiting in the overall reaction during processive polymerization. Discrimination against incorrect nucleotides occurs both in the initial weak binding and in the second step but is purely kinetic in the second step (as opposed to thermodynamic in the first step). Nonnucleoside inhibitors have a relatively small effect on nucleotide-binding steps (overall affinity is reduced by a factor of ca. 10), while the affinity of the primer/template duplex is increased by at least a factor of 10. The major effect of nonnucleoside inhibitors is on the chemical step (nucleotide transfer).

Inhibitors of human heart chymase based on a peptide library.

We have synthesized two sets of noncleavable peptide-inhibitor libraries to map the S and S' subsites of human heart chymase. Human heart chymase is a chymotrypsin-like enzyme that converts angiotensin I to angiotensin II. The first library consists of peptides with 3-fluorobenzylpyruvamides in the P1 position. (Amino acid residues of substrates numbered P1, P2, etc., are toward the N-terminal direction, and P'1, P'2, etc., are toward the C-terminal direction from the scissile bond.) The P'1 and P'2 positions were varied to contain each one of the 20 naturally occurring amino acids and P'3 was kept constant as an arginine. The second library consists of peptides with phenylalanine keto-amides at P1, glycine in P'1, and benzyloxycarbonyl (Z)-isoleucine in P4. The P2 and P3 positions were varied to contain each of the naturally occurring amino acids, except for cysteine and methionine. The peptides of both libraries are attached to a solid support (pins). The peptides are evaluated by immersing the pins in a solution of the target enzyme and evaluating the amount of enzyme absorbed. The pins with the best inhibitors will absorb most enzyme. The libraries select the best and worst inhibitors within each group of peptides and provide an approximate ranking of the remaining peptides according to Ki. Through this library, we determined that Z-Ile-Glu-Pro-Phe-CO2Me and (F)-Phe-CO-Glu-Asp-ArgOMe should be the best inhibitors of chymase in this collection of peptide inhibitors. We synthesized the peptides and found Ki values were 1 nM and 1 microM, respectively. The corresponding Ki values for chymotrypsin were 10 nM and 100 microM. The use of libraries of inhibitors has advantages over the classical method of synthesis of potential inhibitors in solution: the libraries are reusable, the same libraries can be used with a variety of different serine proteases, and the method allows the screening of hundreds of compounds in short periods of time.

Suppression of the breakthrough of human immunodeficiency virus type 1 (HIV-1) in cell culture by thiocarboxanilide derivatives when used individually or in combination with other HIV-1-specific inhibitors (i.e., TSAO derivatives).

Five structurally related thiophene and furane analogues of the oxathiin carboxanilide derivative NSC 615985 (UC84) (designated UC10, UC68, UC81, UC42, and UC16) were identified as potent inhibitors of HIV-1 replication in cell culture and HIV-1 reverse transcriptase activity. These compounds were markedly active against a series of mutant HIV-1 strains, containing the Leu-100-->Ile, Val-106-->Ala, Glu-138-->Lys, or Tyr-181-->Cys mutations in their reverse transcriptase. However, the thiocarboxanilide derivatives selected for mutations at amino acid positions 100 (Leu-->Ile), 101 (Lys-->Ile/Glu), 103 (Lys-->Thr/Asp) and 141 (Gly-->Glu) in the HIV-1 reverse transcriptase. The compounds completely suppressed HIV-1 replication and prevented the emergence of resistant virus strains when used at 1.3-6.6 microM--that is, 10- to 25-fold lower than the concentration required for nevirapine and bis(heteroaryl)piperazine (BHAP) U90152 to do so. If UC42 was combined with the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)]-beta-D-pentofuranosyl (TSAO) derivative of N3-methylthymine (TSAO-m3T), virus breakthrough could be prevented for a much longer time, and at much lower concentrations, than if the compounds were used individually. Virus breakthrough could be suppressed for even longer, and at lower drug concentrations, if BHAP was added to the combination of UC42 with TSAO-m3T, which points to the feasibility of two- or three-drug combinations in preventing virus breakthrough and resistance development.

DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.

Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased > 300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.

Protein structure-based design of potent orally bioavailable, nonpeptide inhibitors of human immunodeficiency virus protease.

A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species.

Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans.

Attachment of Ras protein to the membrane, which requires farnesylation at its C terminus, is essential for its biological activity. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyltransferase. We use Caenorhabditis elegans vulval differentiation, which is controlled by a Ras-mediated signal transduction pathway, as a model system to test previously identified farnesyltransferase inhibitors. We show here that two farnesyltransferase inhibitors, manumycin and gliotoxin, suppress the Multivulva phenotype resulting from an activated let-60 ras mutation, but not the Multivulva phenotype resulting from mutations in the lin-1 gene or the lin-15 gene, which act downstream and upstream of let-60 ras, respectively, in the signaling pathway. These results are consistent with the idea that the suppression of the Multivulva phenotype of let-60 ras by the two inhibitors is specific for Ras protein and that the mutant Ras protein might be more sensitive than wild-type Ras to the farnesyltransferase inhibitors. This work suggests that C. elegans vulval development could be a simple and effective in vivo system for evaluation of farnesyltransferase inhibitors against Ras-activated tumors.