L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. II. Isolation and structure.

A novel HIV-1 protease inhibitor, L-696,474 (C30H39NO4, 477), was isolated from the fermentations of the fungus Hypoxylon fragiforme (ATCC 20995, MF5511) and purified by silica gel chromatography followed by crystallization. Spectroscopic studies have shown the competitive inhibitor L-696,474 to be a novel cytochalasin. Two related novel cytochalasins were also isolated and had no effect on the enzyme.     

L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. I. The producing organism and its fermentation.

A novel cytochalasin, L-696,474, (18-dehydroxy cytochalasin H) that inhibits HIV-1 protease was discovered in fermentations of a bark-inhabiting Ascomycete, Hypoxylon fragiforme. The product was first identified from extracts of an agar medium. Fermentation studies on a number of media indicated that the product can be made on several solid and liquid media. Optimum production was obtained from growth in a complex medium composed of glycerol, glucose, citrate, Ardamine, soybean meal, tomato paste, and inorganic salts. Other Hypoxylon spp., related species of Xylariales, and other fungi known to produce cytochalasins, were also surveyed for their ability to make L-696,474. Only one other Hypoxylon fragiforme isolate was found to make this novel cytochalasin; none of the other cultures surveyed made L-696,474 or any other compounds which inhibit HIV-1 protease.     

Inhibition of HIV-1 replication by a peptide dimerization inhibitor of HIV-1 protease

Peptides based on the amino (N) and carboxy (C)-terminal regions of human immunodeficiency virus type-1 (HIV-1) protease and on the C-terminus of p6* can inhibit HIV-1 protease activity by preventing dimerization. We developed a peptide dimerization inhibitor, P27, that included these domains and a cell permeable domain derived from HIV-1 Tat. P27 inhibited wild type (WT) and protease inhibitor (PI)-resistant HIV-1 protease (IC50: 0.23-0.32 microM). Kinetic and biochemical assays confirmed that P27 inhibits protease dimerization. Fluorescein-labeled peptide accumulated in MT-2 cells and protected acutely infected MT-2 cells from HIV-1-induced cytotoxicity (IC50: 5.1 microM). P27 also inhibited p24 accumulation from H9 and U937 cells chronically infected with WT or PI-resistant HIV-1. Immunoblot analysis on the supernatants and infected cells revealed a block in virus release by P27 rather than an inhibition of polyprotein processing. However, inhibition of p55 Gag processing by active-site inhibitors was enhanced when combined with P27, suggesting that P27 can affect protease function in maturing virions. Although P27 was rationally designed to block dimerization of the mature HIV-1 protease, the effects of P27 on HIV-1 replication may be related to partial inhibition of Gag-Pol processing leading to a disruption in virus release.     

Inhibition of HIV-1 protease by a boron-modified polypeptide

Six boronated tetrapeptides with the carboxy moiety of phenylalanine replaced by dihydroxyboron were synthesized, and their activities against human immunodeficiency virus 1 (HIV-1) protease subsequently investigated. The sequences of these peptides were derived from HIV-1 protease substrates, which included the C-terminal part of the scissile bond (Phe-Pro) within the gag-pol polyprotein. Enzymatic studies showed that these compounds were competitive inhibitors of HIV-1 protease with K(i) values ranging from 5 to 18 microM when experiments were performed at high enzyme concentrations (above 5 x 10(-8) M); however, at low protease concentrations inhibition was due in part to an increase of the association constants of the protease subunits. Ac-Thr-Leu-Asn-PheB inhibited HIV-1 protease with a K(i) of 5 microM, whereas the non-boronated parental compound was inactive at concentrations up to 400 microM, which indicates the significance of boronation in enzyme inhibition. The boronated tetrapeptides were inhibitory to an HIV-1 protease variant that is resistant to several HIV-1 protease inhibitors. Finally, fluorescence analysis showed that the interactions between the boronated peptide Ac-Thr-Leu-Asn-PheB and HIV-1 protease resulted in a rapid decrease of fluorescence emission at 360 nm, which suggests the formation of a compound/enzyme complex. Boronated peptides may provide useful reagents for studying protease biochemistry and yield valuable information toward the development of protease dimerization inhibitors.     

A novel inositol mono-phosphatase inhibitor from Memnoniella echinata. Producing organism, fermentation, isolation, physicochemical and in vitro biological properties.

A novel inositol mono-phosphatase inhibitor, L-671,776 (1), was discovered from a culture of the hyphomycete, Memnoniella echinata (ATCC 20928). 1 has a molecular weight of 388 and a molecular formula of C23H32O5. The mode of inhibition is non-competitive, with a Ki of 450 microM. It shows no inhibition of myo-inositol 1,4-bisphosphate 1-phosphatase or myo-inositol 1,4,5-triphosphate 5-phosphatase, although it weakly inhibits myo-inositol 1,4,5-triphosphate 3-kinase (IC50 = 3 mM). It elevates inositol monophosphates in rat parotid slices (EC50 approximately 3 mM), but abolishes agonist effects. It also produces short-lived contraction of guinea pig trachea at 300 microM.     

Structure-activity relationships of cytochalasins in the differentiation of cytolytic T lymphocytes

Four naturally occurring cytochalasins and three synthetic congeners have been studied for their effects on in vitro sensitization of murine lymphocytes to P815 mastocytoma. The relative order of effectiveness of these secondary fungal metabolites in inhibiting cytotoxic T cell development is as follows: cytochalasin D greater than cytochalasin E greater than cytochalasin A greater than cytochalasin B, 21,22- dihydrocytochalasin A greater than 7- acetylcytochalasin D. The 7,20 diacetylcytochalasin B derivative was inactive at the highest level tested (4 X 10(-6) M). Cytochalasin D is the most effective compound, producing at 5 X 10(-8) M a 50% inhibition of 51Cr release in a 4-hr cytolysis assay. This response pattern is in keeping with other test systems that implicate actin involvement, and underscores the contribution of an unsubstituted 7-hydroxyl drug function in receptor recognition. Inhibition produced by the cytochalasins is reversible if the compounds are removed from the tissue culture medium within the first 24 hr of a 4-day culture period. Delayed addition of cytochalasin D inhibits T cell development only within this first 24 hr of culture. These data suggest that the effects of cytochalasins are at an early step in the sensitization process, possibly antigen recognition.     

Synthesis and biological evaluation of the first N-alkyl cage dimeric 4-aryl-1,4-dihydropyridines as novel nonpeptidic HIV-1 protease inhibitors

A first series of novel NH and N-alkyl-substituted cage dimeric 4-aryl-1,4-dihydropyridines 3a-f has been synthesized and evaluated as HIV-1 protease inhibitors in in vitro assays. While the NH and N-methyl derivatives 3a,b,e,f were almost inactive with IC(50) values of about 200 microM, the N-Benzyl compounds exhibited stronger activity with an IC(50) value of 16.2 microM for the presently best compound 3c. The type of HIV-1 protease inhibition of these novel inhibitors was characterized as competitive. With the increase of observed activity from NH and N-methyl derivatives to N-benzyl compounds, respectively, the binding mode may correspond to that of cyclic and azacyclic ureas showing hydrophobic interactions of the four aromatic residues to the S1/S1' and S2/S2' regions of HIV-1 protease.     

Anti-AIDS agents 69. Moronic acid and other triterpene derivatives as novel potent anti-HIV agents

In a continuing structure-activity relationship study of potent anti-HIV agents, seven new triterpene derivatives were designed, synthesized, and evaluated for in vitro antiviral activity. Among them, moronic acid derivatives 19, 20, and 21 showed significant activity in HIV-1 infected H9 lymphocytes. Compounds 19 and 20 were also evaluated against HIV-1 NL4-3 and drug resistant strains in the MT-4 cell line. Compounds 19 and 20 showed better antiviral profiles than the betulinic acid analogue 8 (PA-457), which has successfully completed a Phase IIa clinical trial. Compound 20 showed potent anti-HIV activity with EC50 values of 0.0085 microM against NL4-3, 0.021 microM against PI-R (a multiple protease inhibitor resistant strain), and 0.13 microM against FHR-2 (an HIV strain resistant to 8). Promising compound 20 has become a new lead for modification, and further development of 20-related compounds as clinical trial candidates is warranted.     

Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-1, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 microM. Their activities against HIV-1 protease (K(i) 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC(50) 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC(50) 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 A (1) and 1.85 A (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.     

A novel aspochalasin with HIV-1 integrase inhibitory activity from Aspergillus flavipes

A novel aspochalasin, aspochalasin L (1), was isolated from the fermentation broth of a soil-derived fungal culture identified as Aspergillus flavipes (Deuteromycota). Structure elucidation of 1 was accomplished by detailed spectroscopic data analyses and by comparison with related cytochalasins. Aspochalasin L demonstrated activity against HIV integrase with an IC50 of 71.7microM.     

Inhibition of viral proteases by Zingiberaceae extracts and flavones isolated from Kaempferia parviflora

In order to identify novel lead compounds with antiviral effect, methanol and aqueous extracts of eight medicinal plants in the Zingiberaceae family were screened for inhibition of proteases from human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV) and human cytomegalovirus (HCMV). In general, the methanol extracts inhibited the enzymes more effectively than the aqueous extracts. HIV-1 protease was strongly inhibited by the methanol extract of Alpinia galanga. This extract also inhibited HCV and HCMV proteases, but to a lower degree. HCV protease was most efficiently inhibited by the extracts from Zingiber officinale, with little difference between the aqueous and the methanol extracts. Many of the methanol extracts inhibited HCMV protease, but the aqueous extracts showed weak inhibition. In a first endeavor to identify the active constituents, eight flavones were isolated from the black rhizomes of Kaempferia parviflora. The most effective inhibitors, 5-hydroxy-7-methoxyflavone and 5,7-dimethoxyflavone, inhibited HIV-1 protease with IC50 values of 19 microM. Moreover, 5-hydroxy-3,7-dimethoxyflavone inhibited HCV protease and HCMV protease with IC50 values of 190 and 250 microM, respectively.     

Fungal phenalenones inhibit HIV-1 integrase

A phenalenone compound, atrovenetinone methyl acetal, was isolated from a culture broth of Penicillium sp. FKI-1463 as an HIV-1 integrase inhibitor, and it showed anti-HIV activity in vitro. HIV-1 integrase inhibition and anti-HIV activity of two other natural phenalenones were also studied. Among the tested compounds, funalenone inhibited HIV-1 integrase with an IC50 value of 10 microM and showed the best selectivity (anti-HIV, IC50=1.7 microM; cytotoxicity, IC50=87 microM).     

Bioanalysis of syn dimeric HIV-1 protease inhibitor N-benzyl 4-aryl-1,4-dihydropyridine H19: metabolic and cytotoxic properties in Hep G2 cells

Syn dimeric N-benzyl 4-aryl-1,4-dihydropyridine H19 is a nonpeptidic HIV-1 protease inhibitor of the dihydroxyethylene type representing novel C2-symmetric inhibitors. Great interest was focussed on the extent of metabolism of these novel inhibitory structures as their functional groups are similar to certain peptidic and non-peptidic HIV-1 protease inhibitors with poor bioavailability due to extensive metabolism. Thus, early characterization of metabolic and toxic properties decisively determines the future prospects of those novel HIV-1 protease inhibitors. Both metabolism and toxicity were evaluated in Hep G2 monolayers. While no phase-I metabolites were found the extent of conjugation in phase-II of biotransformation was poor. Moreover, cytotoxic evaluation of protein and DNA decrease and, furthermore, of membrane toxicity characterized the novel inhibitors as non-toxic. Consequently, the favourable poor metabolism and non-toxic properties encourage further development of these novel HIV-1 protease inhibitors.     

Pharmacokinetic interactions between HIV-1 protease inhibitors in rats: study on combinations of two kinds of HIV-1 protease inhibitors

The drug interactions between four human immune deficiency virus (HIV-1) protease inhibitors have been characterized by in-vitro metabolic studies using rat liver microsomal fractions and in-vivo oral administration. In this study, a new HPLC analytical method developed by us was used for the simultaneous determination of saquinavir and nelfinavir in rat plasma and microsomes. The metabolic clearance rates (Vmax/Km) of saquinavir, nelfinavir, and indinavir were 170.9 +/- 10.9, 126.1 +/- 4-4, and 73.0 +/- 2.0 microL min(-1) (mg protein)(-1), respectively. Ritonavir was the strongest inhibitor with inhibition constants (Ki) of 1.64 microM for saquinavir, 0.95 microM for indinavir, and 1.01 microM for nelfinavir. Nelfinavir was the second strongest inhibitor with Ki's of 2.35 microM for saquinavir and 2.14 microM for indinavir. Indinavir was the third strongest inhibitor with Ki's of 2.76 microM for nelfinavir and 3.55 microM for saquinavir. Saquinavir was the weakest inhibitor for the other three HIV- 1 protease inhibitors. After oral co-administration in combination with another HIV-1 protease inhibitor, the AUCs of saquinavir, indinavir, and nelfinavir were significantly increased compared with mono-treatment. The AUCs of saquinavir were increased about 10.1-, 3.1- and 45.9-fold in the presence of indinavir, nelfinavir and ritonavir, respectively. The AUCs of indinavir were increased about 6.8-, 5.9- and 9.4-fold in the presence of nelfinavir, saquinavir and ritonavir, respectively. The AUCs of nelfinavir were increased about 2.2-, 6.6- and 8.5-fold in the presence of indinavir, saquinavir and ritonavir, respectively. The in-vivo effects observed after co-administration of two kinds of HIV-1 protease inhibitor were not always expected from in-vitro data, suggesting the presence of other interaction processes besides metabolism in the liver. These results provide useful information for the treatment of AIDS patients receiving combination therapy with two HIV-1 protease inhibitors.     

Antiviral activity of UIC-PI, a novel inhibitor of the human immunodeficiency virus type 1 protease

The human immunodeficiency virus type 1 (HIV-1) protease inhibitor UIC-PI (1) was developed via structure-based design and incorporated a novel bis-tetrahydrofuran (bis-THF) ligand in the (R)-(hydroxyethyl)sulfonamide based isostere. The EC(50) and EC(90) of the compound in acutely-infected H9 cells were <1 and approximately 1 nM, respectively. In chronically infected H9/HIV-1(IIIB) cells, the EC(50) and EC(90) were 20 and 50 nM, respectively. In parallel studies comparing UIC-PI and saquinavir in H9/HIV-1(IIIB) cells, viral p24 levels in culture supernatants were an order of magnitude lower with UIC-PI than with saquinavir.