M. Katharine Holloway

Discovery of Vaniprevir (MK-7009), a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor

A new class of HCV NS3/4a protease inhibitors which contain a P2 to P4 macrocyclic constraint was designed using a molecular-modeling derived strategy. Exploration of the P2 heterocyclic region, the P2 to P4 linker, and the P1 side chain of this class of compounds via a modular synthetic strategy allowed for the optimization of enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 35b (vaniprevir, MK-7009), which is active against both the genotype 1 and genotype 2 NS3/4a protease enzymes and has good plasma exposure and excellent liver exposure in multiple species.

Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor.

A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.

Molecular Modeling Based Approach to Potent P2−P4 Macrocyclic Inhibitors of Hepatitis C NS3/4A Protease

Molecular modeling of inhibitor bound full length HCV NS3/4A protease structures proved to be a valuable tool in the design of a new series of potent NS3 protease inhibitors. Optimization of initial compounds provided 25a. The in vitro activity and selectivity as well as the rat pharmacokinetic profile of 25a compare favorably with the data for other NS3/4A protease inhibitors currently in clinical development for the treatment of HCV.

Thermodynamics of Ligand Binding and Efficiency

Analysis of the experimental binding thermodynamics for approximately 100 protein-ligand complexes provides important insights into the factors governing ligand affinity and efficiency. The commonly accepted correlation between enthalpy and -TΔS is clearly observed for this relatively diverse data set. It is also clear that affinity (i.e., ΔG) is not generally correlated to either enthalpy or -TΔS. This is a worrisome trend since the vast majority of computational structure-based design is carried out using interaction energies for one, or at most a few, ligand poses. As such, these energies are most closely comparable to enthalpies not free energies. Closer inspection of the data shows that in a few cases the enthalpy (or -TΔS) is correlated with free energy. It is tempting to speculate that this could be an important consideration as to why some targets are readily amenable to modeling and others are not. Additionally, analysis of the enthalpy and -TΔS efficiencies shows that the trends observed for ligand efficiencies with respect to molecular size are primarily a consequence of enthalpic, not entropic, effects.

3'-Tetrahydrofuranylglycine as a novel, unnatural amino acid surrogate for asparagine in the design of inhibitors of the HIV protease

The blockade of the HIV protease has become a major target in the search for an effective therapy for AIDS.1 While many reports of potent HIV-1 inhibitors have appeared recently, the compound Ro 31-8959 remains the least selective for the HIV-1 and HIV-2 proteases.2 This property may result in reduced susceptibility to resistance since these represent the genetically most divergent strains of HIV presently known to exist.

Transcription control and neuronal differentiation by agents that activate the LXR nuclear receptor family

LXR and PPAR receptors belong to the nuclear receptor superfamily of transcriptional activating factors. Using ligand-dependent transcription assays, we found that 5-tetradecyloxy-2-furancarboxylic acid (TOFA) transactivates chimeric receptors composed of the glucocorticoid receptor DNA binding domain and the ligand binding regions of PPARalpha, PPARbeta (NUC-1) and LXRbeta (NER) receptors. In the same assays, ligands for PPARs (oleic acid, WY-14643 and L-631,033) and LXRs (hydroxycholesterols) maintain their respective receptor selectivity. TOFA and hydroxycholesterols also stimulate transcription from a minimal fibrinogen promoter that is under the control of AP-1 or NF-kappaB transcription factor binding sites. In addition to their effects on transcription, these LXRbeta activators induce neuronal differentiation in rat pheochromocytoma cells. TOFA and the natural LXR agonist, 22 (R)-hydroxycholesterol, stimulate neurite outgrowth in 55 and 28% of cells, respectively. No neurite outgrowth was induced by the related 22(S)-hydroxycholesterol, which does not activate the LXR family. These results suggest that the hydroxycholesterol signaling pathway has a complex effect on transcription that mediates the activity of TOFA and hydroxycholesterol on neuronal differentiation in pheochromocytoma cells.

Discovery of MK-1220: A Macrocyclic Inhibitor of Hepatitis C Virus NS3/4A Protease with Improved Preclinical Plasma Exposure

The discovery of MK-1220 is reported along with the development of a series of HCV NS3/4A protease inhibitors containing a P2 to P4 macrocyclic constraint with improved preclinical pharmacokinetics. Optimization of the P2 heterocycle substitution pattern as well as the P3 amino acid led to compounds with greatly improved plasma exposure following oral dosing in both rats and dogs while maintaining excellent enzyme potency and cellular activity. These studies led to the identification of MK-1220.

Synthesis and evaluation of pyridyl analogs of L-735,524: Potent HIV-1 protease inhibitors

Abstract Two series of HIV protease inhibitors possessing a hydroxyaminopentanamide transition state isostere were prepared and evaluated in peptide cleavage and whole cell assays. These were found to be effective in low concentrations at halting the spread of the AIDS virus, and a number of these inhibitors were also found to provide reasonable plasma levels after oral dosing in animal models. The most promising, L-748,496 is potent (IC50 = 0.12 nM and IC95 = 6-12 nM) and comparable to L-735,524, which is currently in phase II human clinical trials.

PL-100, a novel HIV-1 protease inhibitor displaying a high genetic barrier to resistance: An in vitro selection study†

The development of new HIV inhibitors with distinct resistance profiles is essential in order to combat the development of multi‐resistant viral strains. A drug discovery program based on the identification of compounds that are active against drug‐resistant viruses has produced PL‐100, a novel potent protease inhibitor (PI) that incorporates a lysine‐based scaffold. A selection for resistance against PL‐100 in cord blood mononuclear cells was performed, using the laboratory‐adapted IIIb strain of HIV‐1, and it was shown that resistance appears to develop slower against this compound than against amprenavir, which was studied as a control. Four mutations in protease (PR) were selected after 25 weeks: two flap mutations (K45R and M46I) and two novel active site mutations (T80I and P81S). Site‐directed mutagenesis revealed that all four mutations were required to develop low‐level resistance to PL‐100, which is indicative of the high genetic barrier of the compound. Importantly, these mutations did not cause cross‐resistance to currently marketed PIs. In contrast, the P81S mutation alone caused hypersensitivity to two other PIs, saquinavir (SQV) and nelfinavir (NFV). Analysis of p55Gag processing showed that a marked defect in protease activity caused by mutation P81S could only be compensated when K45R and M46I were present. These data correlated well with the replication capacity (RC) of the mutant viruses as measured by a standard viral growth assay, since only viruses containing all four mutations approached the RC of wild type virus. X‐ray crystallography provided insight on the structural basis of the resistance conferred by the identified mutations. J. Med. Virol. 80:2053–2063, 2008.

P2‐Quinazolinones and Bis‐Macrocycles as New Templates for Next‐Generation Hepatitis C Virus NS3/4a Protease Inhibitors: Discovery of MK‐2748 and MK‐6325

With the goal of identifying inhibitors of hepatitis C virus (HCV) NS3/4a protease that are potent against a wide range of genotypes and clinically relevant mutant viruses, several subseries of macrocycles were investigated based on observations made during the discovery of MK‐5172. Quinazolinone‐containing macrocycles were identified as promising leads, and optimization for superior cross‐genotype and mutant enzyme potency as well as rat liver and plasma concentrations following oral dosing, led to the development of MK‐2748. Additional investigation of a series of bis‐macrocycles containing a fused 18‐ and 15‐membered ring system were also optimized for the same properties, leading to the discovery of MK‐6325. Both compounds display the broad genotype and mutant potency necessary for clinical development as next‐generation HCV NS3/4a protease inhibitors.