Synthesis and evaluation of inhibitors of cytochrome P450 3A (CYP3A) for pharmacokinetic enhancement of drugs.

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2-P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.

Synthesis, antiviral activity, and conformational studies of a P3 aza-peptide analog of a potent macrocyclic tripeptide HCV protease inhibitor.

BILN 2061 is a macrocyclic tripeptide inhibitor of hepatitis C virus NS3-4A protease that has shown efficacy in the clinic for treating patients infected with HCV. We have synthesized a P3 aza-peptide analog of a potent macrocyclic tripeptide inhibitor closely related to BILN 2061. This aza-derivative was found to be >2 orders of magnitude less active than the parent macrocycle in both isolated enzyme (HCV NS3-4A) and HCV subgenomic replicon assays. NMR studies of P3 aza-peptides revealed these compounds adopt a beta-turn conformation stabilized by an intramolecular H-bonding interaction. Molecular models of these structures indicate a D-like configuration of the P3 aza-residue. Thus, the configurationally undefined nature at P3 in the aza-peptide allows the compound to adopt an H-bond stabilized conformation that is substantially different from that necessary for tight binding to the active site of HCV NS3 protease.

Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1.

Little is known about how a neuron undergoes site-specific changes in intrinsic excitability during neuronal activity. We provide evidence for a novel mechanism for mTORC1 kinase-dependent translational regulation of the voltage-gated potassium channel Kv1.1 messenger RNA (mRNA). We identified a microRNA, miR-129, that repressed Kv1.1 mRNA translation when mTORC1 was active. When mTORC1 was inactive, we found that the RNA-binding protein, HuD, bound to Kv1.1 mRNA and promoted its translation. Unexpectedly, inhibition of mTORC1 activity did not alter levels of miR-129 and HuD to favor binding to Kv1.1 mRNA. However, reduced mTORC1 signaling caused the degradation of high affinity HuD target mRNAs, freeing HuD to bind Kv1.1 mRNA. Hence, mTORC1 activity regulation of mRNA stability and high affinity HuD-target mRNA degradation mediates the bidirectional expression of dendritic Kv1.1 ion channels.

Synthesis and biological characterization of B-ring amino analogues of potent benzothiadiazine hepatitis C virus polymerase inhibitors.

Benzothiadiazine inhibitors of the HCV NS5B RNA-dependent RNA polymerase are an important class of non-nucleoside inhibitors that have received considerable attention in the search for novel HCV therapeutics. Research in our laboratories has identified a novel series of tetracyclic benzothiadiazine inhibitors of HCV polymerase bearing a benzylamino substituent on the B-ring. Compounds in this series exhibit low-nanomolar activities in both genotypes 1a and 1b polymerase inhibition assays and subgenomic replicon assays. Optimization of pharmacokinetic properties in rat led to compound 30, which has good oral bioavailability (F = 56%) and a favorable tissue distribution drug profile, with high liver to plasma ratios. Compound 30 is a potent inhibitor in replicon assays, with EC(50) values of 10 and 6 nM against genotypes 1a and 1b, respectively.

Synthesis, antiviral activity, and pharmacokinetic evaluation of P3 pyridylmethyl analogs of oximinoarylsulfonyl HIV-1 protease inhibitors.

As a continuation of the recently communicated discovery of oximinoarylsulfonamides as potent inhibitors of HIV-1 aspartyl protease, compounds bearing pyridylmethyl substituents at P3 were designed and synthesized. Potent analogs in this series provided low single-digit nanomolar EC50 values against both wild-type HIV and resistant mutant virus (A17), attenuated some 3- to 12-fold in the presence of 50% human serum. Pharmacokinetic results for compounds in this series showed good to excellent exposure when co-administered orally with an equal amount of ritonavir (5mg/kg each) in the rat, with average AUC >8 microg h/mL. Similar dosing in dog resulted in significantly lower plasma levels (average AUC <2 microg h/mL). The 3-pyridylmethyl analog 30 gave the best overall exposure (rat AUC=7.1 microg h/mL and dog AUC=4.9 microg h/mL), however, this compound was found to be a potent inhibitor of cytochrome P450 3A (Ki=2.4 nM).

Synthesis and antiviral activity of P1' arylsulfonamide azacyclic urea HIV protease inhibitors.

A series of novel azacyclic urea HIV protease inhibitors bearing a benzenesulfonamide group at P1' were synthesized utilizing a parallel synthesis method. Structural studies of early analogs bound in the enzyme active site were used to design more potent inhibitors. The effects of substituting the P1' benzenesulfonyl group on antiviral activity and protein binding are described.

Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro.

BILN 2061 is a novel, specific hepatitis C virus (HCV) NS3 serine protease inhibitor discovered by Boehringer Ingelheim that has shown potent activity against HCV replicons in tissue culture and is currently under clinical investigation for the treatment of HCV infection. The poor fidelity of the HCV RNA-dependent RNA polymerase will likely lead to the development of drug-resistant viruses in treated patients. The development of resistance to BILN 2061 was studied by the in vitro passage of HCV genotype 1b replicon cells in the presence of a fixed concentration of the drug. Three weeks posttreatment, four colonies were expanded for genotypic and phenotypic characterization. The 50% inhibitory concentrations of BILN 2061 for these colonies were 72- to 1,228-fold higher than that for the wild-type replicon. Sequencing of the individual colonies identified several mutations in the NS3 serine protease gene. Molecular clones containing the single amino acid substitution A156T, R155Q, or D168V resulted in 357-fold, 24-fold, and 144-fold reductions in susceptibility to BILN 2061, respectively, compared to the level of susceptibility shown by the wild-type replicon. Modeling studies indicate that all three of these residues are located in close proximity to the inhibitor binding site. These findings, in addition to the three-dimensional structure analysis of the NS3/NS4A serine protease inhibitor complex, provide a strategic guide for the development of next-generation inhibitors of HCV NS3/NS4A serine protease.