Novel inhibitors of HIV protease: design, synthesis and biological evaluation of picomolar inhibitors containing cyclic P1/P2 scaffolds.

A novel series of HIV protease inhibitors containing cyclic P1/P2 scaffolds has been synthesized and evaluated for biological activity. The trans 3,5-dibenzyl-2-oxo pyrrolidinone ring system resulted in a 50 pM enzyme inhibitor against HIV protease in vitro when combined with an indanolamine derived P'-backbone. This compound also shows comparable activity to currently marketed drugs in the MT-4 cell-based antiviral assay.

Metabolism of amprenavir in liver microsomes: role of CYP3A4 inhibition for drug interactions.

Amprenavir (141W94, VX-478, KVX-478) is metabolized primarily by CYP3A4 (cytochrome P450 3A4) in recombinant systems and human liver microsomes (HLM). The effects of ketoconazole, terfenadine, astemizole, rifampicin, methadone, and rifabutin upon amprenavir metabolism were examined in vitro using HLM. Ketoconazole, terfenadine, and astemizole were observed to inhibit amprenavir depletion, consistent with their known specificity for CYP3A4. The HIV protease inhibitors, indinavir, saquinavir, ritonavir, and nelfinavir, were included in incubations containing amprenavir to examine the interactions of HIV protease inhibitors in vitro. The order of amprenavir metabolism inhibition in human liver microsomes was observed to be: ritonavir > indinavir > nelfinavir > saquinavir. The Ki value for amprenavir-mediated inhibition of testosterone hydroxylation in human liver microsomes was found to be approximately 0.5 microM. Studies suggest that amprenavir inhibits CYP3A4 to a greater extent than saquinavir, and to a much lesser extent than ritonavir. Amprenavir, nelfinavir, and indinavir appear to inhibit CYP3A4 to a moderate extent, suggesting a selected number of coadministration restrictions.

Design, synthesis, and conformational analysis of a novel series of HIV protease inhibitors.

A combination of structure-based design and both solution, and solid-phase synthesis were utilized to derive a potent (nM) series of HIV-1 protease inhibitors bearing a structurally novel backbone. Detailed structural analysis of several inhibitors prepared in this series has suggested that rigidification of the P1/P2 region of this class of molecules may result in compounds with improved potency.

Design and synthesis of novel conformationally restricted HIV protease inhibitors.

A set of HIV protease inhibitors represented by compound 2 has previously been described. Structural and conformational analysis of this compound suggested that conformational restriction of the P1/P2 portion of the molecule could lead to a novel set of potent protease inhibitors. Thus, probe compounds 3-7 were designed, synthesized, and found to be potent inhibitors of HIV protease.

Weak binding of VX-478 to human plasma proteins and implications for anti-human immunodeficiency virus therapy.

VX-478 is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) protease (Ki, 0.6 nM) and of HIV-1 replication in antiviral assays (IC90, 80 nM). The fractional binding of VX-478 to human plasma and to purified plasma proteins was determined by equilibrium dialysis and difference UV spectrophotometry. Binding to alpha 1-acid glycoprotein (89% at 2 microM total drug concentration, Kd of 4 microM) accounts for its fractional binding in plasma (93%). Stopped-flow spectrophotometry methods showed that binding is a reversible two-step process. The measured dissociation rate constant approaches 100 s-1. The antiviral effect of VX-478 was determined in the presence of 45% human plasma, in which the IC90 increased by 1.5-fold compared with control experiments in the presence of 15% fetal bovine serum. The effects of protein binding on the antiviral activity of VX-478 are minor, as expected for a weak drug-protein interaction.

Design, synthesis and structure of non-macrocyclic inhibitors of FKBP12, the major binding protein for the immunosuppressant FK506.

We have synthesized a series of non-macrocyclic ligands to FKBP12 that are comparable in binding potency and peptidyl prolyl isomerase (PPIase) inhibition to FK506 itself. We have also solved the structure of one of these ligands in complex with FKBP12, and have compared that structure to the FK506-FKBP12 complex. Consistent with the observed inhibitory equipotency of these compounds, we observe a strong similarity in the conformation of the two ligands in the region of the protein that mediates PPIase activity. Our compounds, however, are not immunosuppressive. In the FKBP12-FK506 complex, a significant portion of the FK506 ligand, its 'effector domain', projects beyond the envelope of the binding protein in a manner that is suggestive of a potential interaction with a second protein, the calcium-dependent phosphatase, calcineurin, whose inhibition by the FKBP 12-FK506 complex interrupts the T-cell activation events leading to immunosuppression. In contrast, our compounds bind within the surface envelope of FKBP12, and induce significant changes in the structure of the FKBP12 protein which may also affect calcineurin binding indirectly.

Immunosuppressive activity of [MeBm2t]1-, D-diaminobutyryl-8-, and D-diaminopropyl-8-cyclosporin analogues correlates with inhibition of calcineurin phosphatase activity.

Calcineurin, a Ca2+/calmodulin-dependent phosphatase, has recently been identified as a common target for cyclophilin A-cyclosporin A and FK506 binding protein 12-FK506 complexes. This study has examined the structure activity relationships of cyclosporin A (CsA) and three functionally distinct analogues, [MeBm2t]1-CsA, D-diaminobutyryl-8-CsA (Dab8-CsA), and D-diaminopropyl-8-CsA (Dap8-CsA). Immunosuppressive potency in T cell activation models, NF kappa B activation, and IL-2 mRNA transcription has been compared with analogue affinity for cyclophilin A and inhibition of calcineurin phosphatase activity. CsA, Dap8-CsA, and Dab8-CsA bind to cyclophilin A with a similar affinity (Ki 4 to 5 nM as measured by inhibition of prolyl cis-trans isomerase activity), however, Dap8-CsA and Dab8-CsA inhibit T cell activation less than CsA. Although [MeBm2t]-CsA has weak affinity for cyclophilin A (Ki 540 nM), its immunosuppressive potency is similar to that of CsA. Both cyclophilin A-CsA and cyclophilin A-[MeBm2t]1-CsA complexes inhibit calcineurin phosphatase activity in vitro (Ki 114 and 67 nM, respectively). In Jurkat cells exposed to CsA or the analogues for 2 h, endogenous calcineurin phosphatase activity in cell lysates was inhibited by CsA and [MeBm2t]1-CsA (drug concentrations causing 50% reduction in 32PO4 release of 8 and 55 nM, respectively) in proportion to inhibition of T cell activation, IL-2 mRNA transcription, and NF kappa B activation. Dap8-CsA and Dab8-CsA had a minimal effect on endogenous calcineurin phosphatase activity in Jurkat cell lysates. These findings correlate the functional activity of CsA and structural analogues with calcineurin phosphatase activity and support calcineurin as a target for drug action. The Dap8 and Dab8 modifications of CsA, occurring in residue 8, which is exposed to solvent in the cyclophilin A-CsA complex, appears to significantly alter complex affinity for calcineurin.

Development of a novel class of cyclic hexapeptide oxytocin antagonists based on a natural product.

A new structural class of cyclic hexapeptide oxytocin antagonists derived from Streptomyces silvensis and typified by L-365,209 (cyclo-[L-prolyl1-D-phenylalanyl2-L- isoleucyl3-D-dehydropiperazyl4-L-dehydroperazyl5-D-(N- methyl)phenylalanyl6]) was recently reported. In this paper we further delineate the structure-activity profile for this new class by systematic study of L-365,209 analogs obtained by total synthesis. The optimal combination of cyclic amino acid ring sizes at positions 1, 4, and 5 and the role of the N-alkyl substituent at position 6 was elucidated. The lipophilic amino acids at positions 2 and 3 and the unusual amino acid D-dehydropiperazic acid at position 4 were found to be the most critical residues for obtaining good oxytocin receptor affinity. Analogs containing a basic side chain at the less critical 5- and 6-positions maintained good receptor affinity and also had useful levels of water solubility for intravenous formulation. By combining potency- and solubility-enhancing substitutions, several analogs were identified that have the desired combination of properties in vitro (22, cyclo-[L-prolyl-D-tryptophanyl-L-isoleucyl-D-pipecolyl-L-pipeco lyl-D- histidyl]; 25, cyclo-[L-prolyl-D-2-naphthylalanyl-L-isoleucyl-D-pipecolyl-L -pipecolyl-D- histidyl]; 26, cyclo-[L-prolyl-D-tryptophanyl-L-isoleucyl-D-dehydropiperazyl-L-++ pipecolyl-D-histidyl]; 33, cyclo-[L-prolyl-D-tryptophanyl-L-isoleucyl-D-pipecolyl-L- piperazinylcarboxy-D-(N-methyl)phenylalanyl]; 34, cyclo-[L-prolyl-D-phenylalanyl-L-isoleucyl-D-dehydropiperazyl-L-or nithyl- D-(N-methyl)phenylalanyl]). In general, this class exhibited good selectivity for binding to the oxytocin receptor versus the arginine vasopressin V1a and V2 receptor subtypes, although increased V2 receptor affinity was observed in one case (32, cyclo[L-prolyl-D-2-naphthylalanyl-L-isoleucyl-D-pipecolyl-L- lysyl-D-(N- methyl)phenylalanyl]). Unexpectedly, compound 33 was found to stimulate contractions of the isolated rat uterus via activation of the uterine bradykinin receptor. Compounds 22, 25, 26, 33, and 34 were found to be potent antagonists of oxytocin-stimulated contraction of the rat uterus in vitro and in vivo. Compounds 22 and 25 were additionally characterized as potent antagonists of oxytocin-stimulated uterine contractions in the near-term pregnant rhesus monkey. These studies thus demonstrate the selectivity and efficacy of certain members of this novel class of antagonists and suggest their use as pharmacological tools in further defining the role of oxytocin in both term and preterm labor.

In vitro pharmacological profile of a novel structural class of oxytocin antagonists.

A number of structurally novel cyclic hexapeptides have been characterized as potent and selective oxytocin (OT) antagonists in vitro. As a representative of this class of compounds, L-366,948 [[cyclo(L-prolyl-D-2-naphthylalanyl-L-isoleucyl-D-pipecolyl- L-pipecolyl-D- histidyl)]] exhibited a high binding affinity (Ki, low nanomolar) for OT receptors in rat (uterus and mammary) and primate (pregnant rhesus and human myometrium) tissue with a several hundred-fold binding selectivity vs. rat arginine vasopressin (AVP)-V1 (liver) and AVP-V2 (kidney medulla) receptors. In functional assays, L-366,948 was a pure OT antagonist, blocking both OT-stimulated contraction of the isolated rat uterus (pA2, 8.5) and phosphatidylinositol turnover in uterine slices (IC50, 40 vs. 3 nM OT), with no evidence of partial agonist activity. L-366,948 was comparatively weak as an antagonist of AVP-induced contraction of the isolated rat tail artery (AVP-V1 receptor) and AVP-stimulated adenylate cyclase (AVP-V2 receptor) activity in rat kidney medulla and did not influence prostaglandin F2 alpha- or bradykinin-induced contractions of the isolated rat uterus. L-366,948 and related compounds described in this report represent new experimental tools for the study of the pharmacology and physiology of OT.