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.

Multiple Synthetic Routes to the Mini-Protein Omomyc and Coiled-Coil Domain Truncations.

The Myc transcription factor represents an "undruggable" target of high biological interest due to its central role in various cancers. An abbreviated form of the c-Myc protein, called Omomyc, consists of the Myc DNA-binding domain and a coiled-coil region to facilitate dimerization of the 90 amino acid polypeptide. Here we present our results to evaluate the synthesis of Omomyc using three complementary strategies: linear Fmoc solid-phase peptide synthesis (SPPS) using several advancements for difficult sequences, native chemical ligation from smaller peptide fragments, and a high-throughput bacterial expression and assay platform for rapid mutagenesis. This multifaceted approach allowed access to up to gram quantities of the mini-protein and permitted in vitro and in vivo SAR exploration of this modality. DNA-binding results and cellular activity confirm that Omomyc and analogues presented here, are potent binders of the E-box DNA engaged by Myc for transcriptional activation and that this 90-amino acid mini-protein is cell permeable and can inhibit proliferation of Myc-dependent cell lines. We also present additional results on covalent homodimerization through disulfide formation of the full-length mini-protein and show the coiled-coil region can be truncated while preserving both DNA binding and cellular activity. Altogether, our results highlight the ability of advanced peptide synthesis to achieve SAR tractability in a challenging synthetic modality.

Discovery and optimization of 2-pyridinone aminal integrase strand transfer inhibitors for the treatment of HIV.

HIV integrase strand transfer inhibitors (InSTIs) represent an important class of antiviral therapeutics with proven efficacy and excellent tolerability for the treatment of HIV infections. In 2007, Raltegravir became the first marketed strand transfer inhibitor pioneering the way to a first-line therapy for treatment-naïve patients. Challenges with this class of therapeutics remain, including frequency of the dosing regimen and the genetic barrier to resistance. To address these issues, research towards next-generation integrase inhibitors has focused on imparting potency against RAL-resistent mutants and improving pharmacokinetic profiles. Herein, we detail medicinal chemistry efforts on a novel class of 2-pyridinone aminal InSTIs, inpsired by MK-0536, which led to the discovery of important lead molecules for our program. Systematic optimization carried out at the amide and aminal positions on the periphery of the core provided the necessary balance of antiviral activity and physiochemical properties. These efforts led to a novel aminal lead compound with the desired virological profile and preclinical pharmacokinetic profile to support a once-daily human dose prediction.

cGMP production of the radiopharmaceutical [18 F]MK-6240 for PET imaging of human neurofibrillary tangles.

Fluorine-18-labelled 6-(fluoro)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine ([18 F]MK-6240) is a novel potent and selective positron emission tomography (PET) radiopharmaceutical for detecting human neurofibrillary tangles, which are made up of aggregated tau protein. Herein, we report the fully automated 2-step radiosynthesis of [18 F]MK-6240 using a commercially available radiosynthesis module, GE Healthcare TRACERlab FXFN . Nucleophilic fluorination of the 5-diBoc-6-nitro precursor with potassium cryptand [18 F]fluoride (K[18 F]/K222 ) was performed by conventional heating, followed by acid deprotection and semipreparative high-performance liquid chromatography under isocratic conditions. The isolated product was diluted with formulation solution and sterile filtered under Current Good Manufacturing Practices, and quality control procedures were established to validate this radiopharmaceutical for human use. At the end of synthesis, 6.3 to 9.3 GBq (170-250 mCi) of [18 F]MK-6240 was formulated and ready for injection, in an uncorrected radiochemical yield of 7.5% ± 1.9% (relative to starting [18 F]fluoride) with a specific activity of 222 ± 67 GBq/µmol (6.0 ± 1.8 Ci/µmol) at the end of synthesis (90 minutes; n = 3). [18 F]MK-6240 was successfully validated for human PET studies meeting all Food and Drug Administration and United States Pharmacopeia requirements for a PET radiopharmaceutical. The present method can be easily adopted for use with other radiofluorination modules for widespread clinical research use.

Direct and enantioselective {alpha}-allylation of ketones via singly occupied molecular orbital (SOMO) catalysis.

The first enantioselective organocatalytic α-allylation of cyclic ketones has been accomplished via singly occupied molecular orbital catalysis. Geometrically constrained radical cations, forged from the one-electron oxidation of transiently generated enamines, readily undergo allylic alkylation with a variety of commercially available allyl silanes. A reasonable latitude in both the ketone and allyl silane components is readily accommodated in this new transformation. Moreover, three new oxidatively stable imidazolidinone catalysts have been developed that allow cyclic ketones to successfully participate in this transformation. The new catalyst platform has also been exploited in the first catalytic enantioselective α-enolation and α-carbooxidation of ketones.

Discovery and characterization of novel peptide inhibitors of the NRF2/MAFG/DNA ternary complex for the treatment of cancer.

Pathway activating mutations of the transcription factor NRF2 and its negative regulator KEAP1 are strongly correlative with poor clinical outcome with pemetrexed/carbo(cis)platin/pembrolizumab (PCP) chemo-immunotherapy in lung cancer. Despite the strong genetic support and therapeutic potential for a NRF2 transcriptional inhibitor, currently there are no known direct inhibitors of the NRF2 protein or its complexes with MAF and/or DNA. Herein we describe the design of a novel and high-confidence homology model to guide a medicinal chemistry effort that resulted in the discovery of a series of peptides that demonstrate high affinity, selective binding to the Antioxidant Response Element (ARE) DNA and thereby displace NRF2-MAFG from its promoter, which is an inhibitory mechanism that to our knowledge has not been previously described. In addition to their activity in electrophoretic mobility shift (EMSA) and TR-FRET-based assays, we show significant dose-dependent ternary complex disruption of NRF2-MAFG binding to DNA by SPR, as well as cellular target engagement by thermal destabilization of HiBiT-tagged NRF2 in the NCI-H1944 NSCLC cell line upon digitonin permeabilization, and SAR studies leading to improved cellular stability. We report the characterization and unique profile of lead peptide 18, which we believe to be a useful in vitro tool to probe NRF2 biology in cancer cell lines and models, while also serving as an excellent starting point for additional in vivo optimization toward inhibition of NRF2-driven transcription to address a significant unmet medical need in non-small cell lung cancer (NSCLC).

A Series of Novel, Highly Potent, and Orally Bioavailable Next-Generation Tricyclic Peptide PCSK9 Inhibitors.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. Starting from second-generation lead structures such as 2, we were able to refine these structures to obtain extremely potent bi- and tricyclic PCSK9 inhibitor peptides. Optimized molecules such as 44 demonstrated sufficient oral bioavailability to maintain therapeutic levels in rats and cynomolgus monkeys after dosing with an enabled formulation. We demonstrated target engagement and LDL lowering in cynomolgus monkeys essentially identical to those observed with the clinically approved, parenterally dosed antibodies. These molecules represent the first report of highly potent and orally bioavailable macrocyclic peptide PCSK9 inhibitors with overall profiles favorable for potential development as once-daily oral lipid-lowering agents. In this manuscript, we detail the design criteria and multiparameter optimization of this novel series of PCSK9 inhibitors.

Inhibition of Myc transcriptional activity by a mini-protein based upon Mxd1.

Myc, a transcription factor with oncogenic activity, is upregulated by amplification, translocation, and mutation of the cellular pathways that regulate its stability. Inhibition of the Myc oncogene by various modalities has had limited success. One Myc inhibitor, Omomyc, has limited cellular and in vivo activity. Here, we report a mini-protein, referred to as Mad, which is derived from the cellular Myc antagonist Mxd1. Mad localizes to the nucleus in cells and is 10-fold more potent than Omomyc in inhibiting Myc-driven cell proliferation. Similar to Mxd1, Mad also interacts with Max, the binding partner of Myc, and with the nucleolar upstream binding factor. Mad binds to E-Box DNA in the promoters of Myc target genes and represses Myc-mediated transcription to a greater extent than Omomyc. Overall, Mad appears to be more potent than Omomyc both in vitro and in cells.

Preclinical Safety Evaluation and Human Dosimetry of [18F]MK-6240, a Novel PET Tracer for Imaging Neurofibrillary Tangles.

PURPOSE: [18F]MK-6240 is a selective, high-affinity positron emission tomography tracer for imaging neurofibrillary tangles, a key pathological signature that correlates with cognitive decline in Alzheimer disease. This report provides safety information from preclinical toxicology studies and first-in-human whole-body biodistribution and dosimetry studies of [18F]MK-6240 for its potential application in human brain imaging studies. PROCEDURES: MK-6240 was administered intravenously (IV) in a 7-day rat toxicity study at × 50, × 100, and × 1000 dose margins relative to projected highest clinical dose of 0.333 µg/kg. The IV formulation of MK-6240 for clinical use and the formulation used in the 7-day rat toxicity study was tested for hemolysis potential in human and Wistar rat whole blood. Sequential whole-body positron emission tomography scans were performed in three healthy young subjects after IV bolus injection of 180 ± 0.3 MBq [18F]MK-6240 to characterize organ biodistribution and estimate whole-body radiation exposure (effective dose). RESULTS: MK-6240 administered IV in a 7-day rat toxicity study did not show any test article-related changes. The no-observed-adverse-effect level in rats was ≥ 333 µg/kg/day which provides a margin 1000-fold over an anticipated maximum clinical dose of 0.333 µg/kg. Additionally, the MK-6240 formulation was not hemolytic in human or Wistar rat blood. [18F]MK-6240 activity was widely distributed to the brain and the rest of the body, with organ absorbed doses largest for the gall bladder (202 µGy/MBq). The average (±SD) effective dose was 29.4 ± 0.6 µSv/MBq, which is in the typical range for F-18 radiolabeled ligands. CONCLUSIONS: Microdoses of [18F]MK-6240 are safe for clinical positron emission tomography imaging studies. Single IV administration of 185 MBq (5 mCi) [18F]MK-6240 is anticipated to result in a total human effective dose of 5.4 mSv and thus allows multiple positron emission tomography scans of the same subject per year.

Cycle-specific organocascade catalysis: application to olefin hydroamination, hydro-oxidation, and amino-oxidation, and to natural product synthesis.

United in effort: The combined application of iminium (Im) and enamine (En) catalysts can effect a range of valuable asymmetric transformations including 1,2-hydroamination, -hydro-oxidation, and -amino-oxidation of olefins (see picture). An enantioselective organocascade catalysis was also applied in the synthesis of a complex natural product.