Craig A. Coburn

In Vivo β-Secretase 1 Inhibition Leads to Brain Aβ Lowering and Increased α-Secretase Processing of Amyloid Precursor Protein without Effect on Neuregulin-1

β-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid β peptide Aβ42, the putative neurotoxic species in Alzheimers disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC50 ∼ 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose- and time-dependent lowering of brain Aβ40 and Aβ42. BACE1 inhibition also led to a robust brain secreted (s)APPβ lowering that was accompanied by an increase in brain sAPPα levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (–/–) mice, in agreement with previous studies, this effect was also observed in 15-day-old heterozygous (+/–) mice, but it was not evident in 30-day-old and 2-year-old BACE1 KO (–/–) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant Aβ lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major β-site cleavage enzyme for APP and that its inhibition can lower brain Aβ and redirect APP processing via the potentially nonamyloidogenic α-secretase pathway, without significantly altering NRG-1 processing.

Discovery of a Pharmacologically Active Antagonist of the Two-Pore-Domain Potassium Channel K2P9.1 (TASK-3)

TWIK‐related acid‐sensitive K+ (K2P9.1, TASK‐3) ion channels have the capacity to regulate the activity of neuronal pathways by influencing the resting membrane potential of neurons on which they are expressed. The central nervous system (CNS) expression of these channels suggests potential roles in neurologic disorders, and it is believed that the development of TASK‐3 antagonists could lead to the therapeutic treatment of a number of neurological conditions. While a therapeutic potential for TASK‐3 channel modulation exists, there are only a few documented examples of potent and selective small‐molecule channel blockers. Herein, we describe the discovery and lead optimization efforts for a novel series of TASK‐3 channel antagonists based on a 5,6,7,8‐tetrahydropyrido[4,3‐d]pyrimidine high‐throughput screening lead from which a subseries of potent and selective inhibitors were identified. One compound was profiled in detail with respect to its physical properties and demonstrated pharmacological target engagement as indicated by its ability to modulate sleep architecture in rodent electroencephalogram (EEG) telemetry models.

Bicyclic pyridones as potent, efficacious and orally bioavailable thrombin inhibitors.

A new class of conformationally constrained thrombin inhibitors is described. These compounds contain a unique bicyclic pyridone scaffold which serves as a P3P2 dipeptide surrogate. The synthesis and antithrombotic activity of these inhibitors is reported.

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.

Assessment of the susceptibility of mutant HIV-1 to antiviral agents

Traditional phenotypic assays used to assess the susceptibility of mutant human immunodeficiency virus type-1 (HIV-1) obtained from infected patients or from resistance selection to antiviral agents in cell culture are rather tedious and time consuming. To improve the efficiency of this process, a novel method was developed in which mutant viruses are captured with magnetic nano-beads and used to infect gag-GFP reporter cells to evaluate the extent of resistance conferred by the mutant viruses against antiviral agents. The optimal timing for measuring the inhibitory potencies of antiviral agents was found to be day 3 post-infection for integrase strand transfer inhibitors and protease inhibitors and day 4 for non-nucleoside reverse transcriptase inhibitors. Comparable EC(50) values were obtained when bead-captured breakthrough virus from in vitro resistance selection experiments and its matched site-directed mutagenesis virus were tested side by side in this assay. This assay protocol was also employed to evaluate the inhibitor susceptibility of breakthrough viruses collected from resistance selections that were conducted in the presence of increasing concentrations of an HIV-1 protease inhibitor. Taken together, these findings suggest that a rapid, sensitive, non-invasive, and homogeneous phenotypic assay has been developed for assessing the antiviral agent susceptibility of mutant viruses that emerge from in vitro resistance selection studies.

Aryl or heteroaryl substituted aminal derivatives of HCV NS5A inhibitor MK-8742.

Herein we describe our research efforts around the aryl and heteroaryl substitutions at the aminal carbon of the tetracyclic indole-based HCV NS5A inhibitor MK-8742. A series of potent NS5A inhibitors are described, such as compounds 45-47, 54, 56, and 65, which showed improved potency against clinically relevant and resistance associated HCV variants. The improved potency profiles of these compounds demonstrated an SAR that can improve the potency against GT2b, GT1a Y93H, and GT1a L31V altogether, which was unprecedented in our previous efforts in NS5A inhibition.

Substituted tetracyclic indole core derivatives of HCV NS5A inhibitor MK-8742

As part of an ongoing effort in NS5A inhibition at Merck we now describe our efforts for introducing substitution around the tetracyclic indole core of MK-8742. Fluoro substitution on the core combined with the fluoro substitutions on the proline ring improved the potency against GT1a Y93H significantly. However, no improvement on GT2b potency was achieved. Limiting the fluoro substitution to C-1 of the tetracyclic indole core had a positive impact on the potency against the resistance associated variants, such as GT1a Y93H and GT2b, and the PK profile as well. Compounds, such as 62, with reduced potency shifts between wild type GT1a to GT2b, GT1a Y93H, and GT1a L31V were identified.

Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors.

A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-L-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.

Discovery of potent macrocyclic HCV NS5A inhibitors

HCV NS5A inhibitors have demonstrated impressive in vitro virologic profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed-dose combination (FDC) regimen for the treatment of HCV infection. Mercks effort in this area identified MK-4882 and MK-8325 as early development leads. Herein, we describe the discovery of potent macrocyclic NS5A inhibitors bearing the MK-8325 or MK-4882 core structure.

Discovery of fused tricyclic core containing HCV NS5A inhibitors with pan-genotype activity.

HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein, we describe research efforts that led to the discovery of a series of fused tricyclic core containing HCV NS5A inhibitors such as 24, 39, 40, 43, and 44 which have pan-genotype activity and are orally bioavailable in the rat.