Design-rules for stapled peptides with in vivo activity and their application to Mdm2/X antagonists.

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these 'design rules' to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif.

A high-throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell-based assays.

Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t 1/2 = 1.1-2.8 min) after microfluidic vortex shedding (µVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DµVS), that integrates a µVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DµVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DµVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.

Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

Bioactivation of GPR40 Agonist MK-8666: Formation of Protein Adducts in Vitro from Reactive Acyl Glucuronide and Acyl CoA Thioester.

MK-8666, a selective GPR40 agonist developed for the treatment of type 2 diabetes mellitus, was discontinued in phase I clinical trials due to liver safety concerns. To address whether chemically reactive metabolites played a causative role in the observed drug induced liver injury (DILI), we characterized the metabolism, covalent binding to proteins, and amino acid targets of MK-8666 in rat and human hepatocytes or cofactor-fortified liver microsomes. MK-8666 was primarily metabolized to an acyl glucuronide in hepatocytes of both species and a taurine conjugate in rat hepatocytes. Similar levels of covalent binding to proteins were observed in rat and human hepatocytes following incubation with [3H]MK-8666. After protease digestion of hepatocyte pellets, amino acid adducts A1, A2, and A3 were identified as transacylated products with lysine, serine, and cysteine residues, respectively. Amino acid adducts A4a-c were identified as glycation adducts resulting from rearrangement of MK-8666-1-O-ß-acyl glucuronide to ring-opened aldehydes which further condensed with lysine residues of proteins into imine adducts. Adducts A1-A3 and A4a-c were detected in rat and human liver microsomes fortified with UDPGA. Adducts A1-A3 were detected in rat and human liver microsomes fortified with CoA and ATP. Additionally, a trace amount of CoA thioester metabolite of MK-8666 and its transacylated GSH adduct were detected in human liver microsomes fortified with CoA, ATP, and GSH. Higher levels of covalent binding to protein were observed when [3H]MK-8666 was incubated in liver microsomes supplemented with CoA and ATP compared to UDPGA. Addition of GSH attenuated levels of CoA thioester-mediated covalent binding by 41-45%. Collectively, these studies indicated that metabolism of the -COOH moiety of MK-8666 can form a reactive acyl glucuronide and an acyl CoA thioester, which covalently modifies proteins and may represent one causative mechanism of the observed DILI.

Design, synthesis and biological evaluation of indane derived GPR40 agoPAMs.

GPR40 (FFAR1 or FFA1) is a G protein-coupled receptor, primarily expressed in pancreatic islet ß-cells and intestinal enteroendocrine cells. When activated by fatty acids, GPR40 elicits increased insulin secretion from islet ß-cells only in the presence of elevated glucose levels. Towards this end, studies were undertaken towards discovering a novel GPR40 Agonist whose mode of action is via Positive Allosteric Modulation of the GPR40 receptor (AgoPAM). Efforts were made to identify a suitable GPR40 AgoPAM tool molecule to investigate mechanism of action and de-risk liver toxicity of GPR40 AgoPAMs due to reactive acyl-glucuronide (AG) metabolites.

Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40.

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40-MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Design and Synthesis of Novel, Selective GPR40 AgoPAMs.

GPR40 is a G-protein-coupled receptor expressed primarily in pancreatic islets and intestinal L-cells that has been a target of significant recent therapeutic interest for type II diabetes. Activation of GPR40 by partial agonists elicits insulin secretion only in the presence of elevated blood glucose levels, minimizing the risk of hypoglycemia. GPR40 agoPAMs have shown superior efficacy to partial agonists as assessed in a glucose tolerability test (GTT). Herein, we report the discovery and optimization of a series of potent, selective GPR40 agoPAMs. Compound 24 demonstrated sustained glucose lowering in a chronic study of Goto Kakizaki rats, showing no signs of tachyphylaxis for this mechanism.

Substituted 4-morpholine N-arylsulfonamides as γ-secretase inhibitors.

The design, synthesis, SAR, and biological profile of a substituted 4-morpholine sulfonamide series of γ-secretase inhibitors (GSIs) were described. In several cases, the resulting series of GSIs reduced CYP liabilities and improved γ-secretase inhibition activity compared to our previous research series. Selected compounds demonstrated significant reduction of amyloid-ß (Aß) after acute oral dosing in a transgenic animal model of Alzheimer's disease (AD).

Discovery of MK-8831, A Novel Spiro-Proline Macrocycle as a Pan-Genotypic HCV-NS3/4a Protease Inhibitor.

We have been focused on identifying a structurally different next generation inhibitor to MK-5172 (our Ns3/4a protease inhibitor currently under regulatory review), which would achieve superior pangenotypic activity with acceptable safety and pharmacokinetic profile. These efforts have led to the discovery of a novel class of HCV NS3/4a protease inhibitors containing a unique spirocyclic-proline structural motif. The design strategy involved a molecular-modeling based approach, and the optimization efforts on the series to obtain pan-genotypic coverage with good exposures on oral dosing. One of the key elements in this effort was the spirocyclization of the P2 quinoline group, which rigidified and constrained the binding conformation to provide a novel core. A second focus of the team was also to improve the activity against genotype 3a and the key mutant variants of genotype 1b. The rational application of structural chemistry with molecular modeling guided the design and optimization of the structure-activity relationships have resulted in the identification of the clinical candidate MK-8831 with excellent pan-genotypic activity and safety profile.

Discovery of a Novel, Potent Spirocyclic Series of γ-Secretase Inhibitors.

In the present paper, we described the design, synthesis, SAR, and biological profile of a novel spirocyclic sulfone series of γ-secretase inhibitors (GSIs) related to MRK-560. We utilized an additional spirocyclic ring system to stabilize the active chair conformation of the parent γ-secretase inhibitors. The resulting series is devoid of the CYP2C9 inhibition liability of MRK-560. A few representative analogs were assessed in a nontransgenic animal model of Alzheimer's disease (AD), demonstrating reduction of amyloid-ß (Aß) in the CNS after acute oral dosing. A spirocyclic phosphonate was identified as the optimal ring system for both potency and pharmacokinetics. Compared to GSIs studied in the clinic, representative spirocyclic phosphonate 18a(-) features improved selectivity for the inhibition of the PS-1 isoform of γ-secretase (33-fold vs PS-2), which may alleviate the adverse effect profile of the clinical GSIs.

Iminopyrimidinones: a novel pharmacophore for the development of orally active renin inhibitors.

The development of renin inhibitors with favorable oral pharmacokinetic profiles has been a longstanding challenge for the pharmaceutical industry. As part of our work to identify inhibitors of BACE1, we have previously developed iminopyrimidinones as a novel pharmacophore for aspartyl protease inhibition. In this letter we describe how we modified substitution around this pharmacophore to develop a potent, selective and orally active renin inhibitor.

Structure activity relationship studies of tricyclic bispyran sulfone γ-secretase inhibitors.

An investigation is detailed of the structure activity relationships (SAR) of two sulfone side chains of compound (-)-1a (SCH 900229), a potent, PS1-selective γ-secretase inhibitor and clinical candidate for the treatment of Alzheimer's disease. Specifically, 4-CF(3) and 4-Br substituted arylsulfone analogs, (-)-1b and (-)-1c, are equipotent to compound (-)-1a. On the right hand side chain, linker size and terminal substituents of the pendant sulfone group are also investigated.

The discovery of fused oxadiazepines as gamma secretase modulators for treatment of Alzheimer's disease.

In an attempt to further improve overall profiles of the oxadiazine series of GSMs, in particular the hERG activity, conformational modifications of the core structure resulted in the identification of fused oxadiazepines such as 7i which had an improved hERG inhibition profile and was a highly efficacious GSM in vitro and in vivo in rats. These SAR explorations offer opportunities to identify potential drugs to treat Alzheimer's disease.

Cyclic hydroxyamidines as amide isosteres: discovery of oxadiazolines and oxadiazines as potent and highly efficacious γ-secretase modulators in vivo.

Cyclic hydroxyamidines were designed and validated as isosteric replacements of the amide functionality. Compounds with these structural motifs were found to be metabolically stable and to possess highly desirable pharmacokinetic profiles. These designs were applied in the identification of γ-secretase modulators leading to highly efficacious agents for reduction of central nervous system Aß(42) in various animal models.

Discovery of SCH 900229, a Potent Presenilin 1 Selective γ-Secretase Inhibitor for the Treatment of Alzheimer's Disease.

An exploration of the SAR of the side chain of a novel tricyclic series of γ-secretase inhibitors led to the identification of compound (-)-16 (SCH 900229), which is a potent and PS1 selective inhibitor of γ-secretase (Aß40 IC50 = 1.3 nM). Compound (-)-16 demonstrated excellent lowering of Aß after oral administration in preclinical animal models and was advanced to human clinical trials for further development as a therapeutic agent for the treatment of Alzheimer's disease.

Identification of presenilin 1-selective γ-secretase inhibitors with reconstituted γ-secretase complexes.

Accumulation of the ß-amyloid (Aß) peptides is one of the major pathologic hallmarks in the brains of Alzheimer's disease (AD) patients. Aß is generated by sequential proteolytic cleavage of the amyloid precursor protein (APP) catalyzed by ß- and γ-secretases. Inhibition of Aß production by γ-secretase inhibitors (GSIs) is thus being pursued as a target for treatment of AD. In addition to processing APP, γ-secretase also catalyzes proteolytic cleavage of other transmembrane substrates, with the best characterized one being the cell surface receptor Notch. GSIs reduce Aß production in animals and humans but also cause significant side effects because of the inhibition of Notch processing. The development of GSIs that reduce Aß production and have less Notch-mediated side effect liability is therefore an important goal. γ-Secretase is a large membrane protein complex with four components, two of which have multiple isoforms: presenilin (PS1 or PS2), aph-1 (aph-1a or aph-1b), nicastrin, and pen-2. Here we describe the reconstitution of four γ-secretase complexes in Sf9 cells containing PS1--aph-1a, PS1--aph-1b, PS2--aph-1a, and PS2--aph-1b complexes. While PS1--aph-1a, PS1--aph-1b, and PS2--aph-1a complexes displayed robust γ-secretase activity, the reconstituted PS2--aph-1b complex was devoid of detectable γ-secretase activity. γ-Secretase complexes containing PS1 produced a higher proportion of the toxic species Aß42 than γ-secretase complexes containing PS2. Using the reconstitution system, we identified MRK-560 and SCH 1500022 as highly selective inhibitors of PS1 γ-secretase activity. These findings may provide important insights into developing a new generation of γ-secretase inhibitors with improved side effect profiles.

Design and synthesis of tricyclic sulfones as gamma-secretase inhibitors with greatly reduced Notch toxicity.

A novel series of tricyclic gamma-secretase inhibitors was designed and synthesized via a conformational analysis of literature compounds. The preliminary results have shown that compounds in this new series have much improved in vitro potency and in vivo profiles. More importantly, they have greatly reduced Notch related toxicity that was associated with previous gamma-secretase inhibitors.

Novel orally active morpholine N-arylsulfonamides gamma-secretase inhibitors with low CYP 3A4 liability.

A new class of 2,6-disubstituted morpholine N-arylsulfonamide gamma-secretase inhibitors was designed based on the introduction of a morpholine core in lieu or piperidine in our lead series. This resulted in compounds with improved CYP 3A4 profiles. Several analogs that were active at lowering Abeta levels in Tg CRND8 mice upon oral administration were identified.

Small conformationally restricted piperidine N-arylsulfonamides as orally active gamma-secretase inhibitors.

The design and development of a new class of small 2,6-disubstituted piperidine N-arylsulfonamide gamma-secretase inhibitors is reported. Lowering molecular weight including the use of conformational constraint led to compounds with less CYP 3A4 liability compared to early leads. Compounds active orally in lowering Abeta levels in Tg CRND8 mice were identified as potential treatments for Alzheimer's disease.

Discovery of 2,4,6-trisubstituted N-arylsulfonyl piperidines as gamma-secretase inhibitors.

Development of cis-2,4,6-trisubstituted piperidine N-arylsulfonamides as gamma-secretase inhibitors for the potential treatment of Alzheimer's disease (AD) is reported.