A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Design and discovery of C2-fluoroalkyl iminothiazine dioxides as BACE inhibitors.

This paper describes the structure-activity-relationships of novel fluoroalkyl substituents at the C2 position of iminothiazine dioxide beta secretase inhibitors. Key discoveries include reduced amidine basicity and its effect on Pgp, cell potency, and efficacy in various preclinical in vivo efficacy animal models. Findings from these structure-activity-relationships are discussed.

Modeling a crowdsourced definition of molecular complexity.

This paper brings together the concepts of molecular complexity and crowdsourcing. An exercise was done at Merck where 386 chemists voted on the molecular complexity (on a scale of 1-5) of 2681 molecules taken from various sources: public, licensed, and in-house. The meanComplexity of a molecule is the average over all votes for that molecule. As long as enough votes are cast per molecule, we find meanComplexity is quite easy to model with QSAR methods using only a handful of physical descriptors (e.g., number of chiral centers, number of unique topological torsions, a Wiener index, etc.). The high level of self-consistency of the model (cross-validated R(2) ∼0.88) is remarkable given that our chemists do not agree with each other strongly about the complexity of any given molecule. Thus, the power of crowdsourcing is clearly demonstrated in this case. The meanComplexity appears to be correlated with at least one metric of synthetic complexity from the literature derived in a different way and is correlated with values of process mass intensity (PMI) from the literature and from in-house studies. Complexity can be used to differentiate between in-house programs and to follow a program over time.

Genomic Discovery and Structure-Activity Exploration of a Novel Family of Enzyme-Activated Covalent Cyclin-Dependent Kinase Inhibitors.

Fungi have historically been the source of numerous important medicinal compounds, but full exploitation of their genetic potential for drug development has been hampered in traditional discovery paradigms. Here we describe a radically different approach, top-down drug discovery (TD3), starting with a massive digital search through a database of over 100,000 fully genomicized fungi to identify loci encoding molecules with a predetermined human target. We exemplify TD3 by the selection of cyclin-dependent kinases (CDKs) as targets and the discovery of two molecules, 1 and 2, which inhibit therapeutically important human CDKs. 1 and 2 exhibit a remarkable mechanism, forming a site-selective covalent bond to the CDK active site Lys. We explored the structure-activity relationship via semi- and total synthesis, generating an analog, 43, with improved kinase selectivity, bioavailability, and efficacy. This work highlights the power of TD3 to identify mechanistically and structurally novel molecules for the development of new medicines.

Structure based design of iminohydantoin BACE1 inhibitors: identification of an orally available, centrally active BACE1 inhibitor.

From an initial lead 1, a structure-based design approach led to identification of a novel, high-affinity iminohydantoin BACE1 inhibitor that lowers CNS-derived Aß following oral administration to rats. Herein we report SAR development in the S3 and F' subsites of BACE1 for this series, the synthetic approaches employed in this effort, and in vivo data for the optimized compound.

STimulator of INterferon Genes Agonism Accelerates Antitumor Activity in Poorly Immunogenic Tumors.

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.

Discovery of MK-1454: A Potent Cyclic Dinucleotide Stimulator of Interferon Genes Agonist for the Treatment of Cancer.

Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.

Structure-Based Discovery of Proline-Derived Arginase Inhibitors with Improved Oral Bioavailability for Immuno-Oncology.

Recent data suggest that the inhibition of arginase (ARG) has therapeutic potential for the treatment of a number of indications ranging from pulmonary and vascular disease to cancer. Thus, high demand exists for selective small molecule ARG inhibitors with favorable druglike properties and good oral bioavailability. In light of the significant challenges associated with the unique physicochemical properties of previously disclosed ARG inhibitors, we use structure-based drug design combined with a focused optimization strategy to discover a class of boronic acids featuring a privileged proline scaffold with superior potency and oral bioavailability. These compounds, exemplified by inhibitors 4a, 18, and 27, demonstrated a favorable overall profile, and 4a was well tolerated following multiple days of dosing at concentrations that exceed those required for serum arginase inhibition and concomitant arginine elevation in a syngeneic mouse carcinoma model.

Comprehensive Strategies to Bicyclic Prolines: Applications in the Synthesis of Potent Arginase Inhibitors.

Comprehensive synthetic strategies afforded a diverse set of structurally unique bicyclic proline-containing arginase inhibitors with a high degree of three-dimensionality. The analogs that favored the Cγ-exo conformation of the proline improved the arginase potency over the initial lead. The novel synthetic strategies reported here not only enable access to previously unknown stereochemically complex proline derivatives but also provide a foundation for the future synthesis of bicyclic proline analogs, which incorporate inherent three-dimensional character into building blocks, medicine, and catalysts and could have a profound impact on the conformation of proline-containing peptides and macrocycles.

Piperazine sulfonamide BACE1 inhibitors: design, synthesis, and in vivo characterization.

With collaboration between chemistry, X-ray crystallography, and molecular modeling, we designed and synthesized a series of novel piperazine sulfonamide BACE1 inhibitors. Iterative exploration of the non-prime side and S2' sub-pocket of the enzyme culminated in identification of an analog that potently lowers peripheral Abeta(40) in transgenic mice with a single subcutaneous dose.

Discovery and Optimization of Rationally Designed Bicyclic Inhibitors of Human Arginase to Enhance Cancer Immunotherapy.

The action of arginase, a metalloenzyme responsible for the hydrolysis of arginine to urea and ornithine, is hypothesized to suppress immune-cell activity within the tumor microenvironment, and thus its inhibition may constitute a means by which to potentiate the efficacy of immunotherapeutics such as anti-PD-1 checkpoint inhibitors. Taking inspiration from reported enzyme-inhibitor cocrystal structures, we designed and synthesized novel inhibitors of human arginase possessing a fused 5,5-bicyclic ring system. The prototypical member of this class, 3, when dosed orally, successfully demonstrated serum arginase inhibition and concomitant arginine elevation in a syngeneic mouse carcinoma model, despite modest oral bioavailability. Structure-based design strategies to improve the bioavailability of this class, including scaffold modification, fluorination, and installation of active-transport recognition motifs were explored.

An orally available non-nucleotide STING agonist with antitumor activity.

Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Overcoming Time-Dependent Inhibition (TDI) of Cytochrome P450 3A4 (CYP3A4) Resulting from Bioactivation of a Fluoropyrimidine Moiety.

Herein we describe structure-activity relationship (SAR) and metabolite identification (Met-ID) studies that provided insight into the origin of time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4) by compound 1. Collectively, these efforts revealed that bioactivation of the fluoropyrimidine moiety of 1 led to reactive metabolite formation via oxidative defluorination and was responsible for the observed TDI. We discovered that substitution at both the 4- and 6-positions of the 5-fluoropyrimidine of 1 was necessary to ameliorate this TDI as exemplified by compound 19.

The BACE1 inhibitor verubecestat (MK-8931) reduces CNS ß-amyloid in animal models and in Alzheimer's disease patients.

ß-Amyloid (Aß) peptides are thought to be critically involved in the etiology of Alzheimer's disease (AD). The aspartyl protease ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of Aß, and BACE1 inhibition is thus an attractive target for the treatment of AD. We show that verubecestat (MK-8931) is a potent, selective, structurally unique BACE1 inhibitor that reduced plasma, cerebrospinal fluid (CSF), and brain concentrations of Aß40, Aß42, and sAPPß (a direct product of BACE1 enzymatic activity) after acute and chronic administration to rats and monkeys. Chronic treatment of rats and monkeys with verubecestat achieved exposures >40-fold higher than those being tested in clinical trials in AD patients yet did not elicit many of the adverse effects previously attributed to BACE inhibition, such as reduced nerve myelination, neurodegeneration, altered glucose homeostasis, or hepatotoxicity. Fur hypopigmentation was observed in rabbits and mice but not in monkeys. Single and multiple doses were generally well tolerated and produced reductions in Aß40, Aß42, and sAPPß in the CSF of both healthy human subjects and AD patients. The human data were fit to an amyloid pathway model that provided insight into the Aß pools affected by BACE1 inhibition and guided the choice of doses for subsequent clinical trials.

Structure-Based Design of an Iminoheterocyclic ß-Site Amyloid Precursor Protein Cleaving Enzyme (BACE) Inhibitor that Lowers Central Aß in Nonhuman Primates.

We describe successful efforts to optimize the in vivo profile and address off-target liabilities of a series of BACE1 inhibitors represented by 6 that embodies the recently validated fused pyrrolidine iminopyrimidinone scaffold. Employing structure-based design, truncation of the cyanophenyl group of 6 that binds in the S3 pocket of BACE1 followed by modification of the thienyl group in S1 was pursued. Optimization of the pyrimidine substituent that binds in the S2'-S2″ pocket of BACE1 remediated time-dependent CYP3A4 inhibition of earlier analogues in this series and imparted high BACE1 affinity. These efforts resulted in the discovery of difluorophenyl analogue 9 (MBi-4), which robustly lowered CSF and cortex Aß40 in both rats and cynomolgus monkeys following a single oral dose. Compound 9 represents a unique molecular shape among BACE inhibitors reported to potently lower central Aß in nonrodent preclinical species.

Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer's Disease.

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aß levels in rats and nonhuman primates and CSF Aß levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

Design and development of BACE-1 inhibitors.

In early 1999, beta-amyloid cleaving enzyme-1 (BACE-1) was identified as the protease responsible for the critical first step in the processing of beta-amyloid precursor protein that ultimately leads to the production of Abeta peptides in the brain. Accumulation of these peptides has been implicated in the pathology of Alzheimer's disease (AD). An inhibitor of BACE-1 would therefore have therapeutic potential to slow or halt the progression of this debilitating and ultimately fatal disease. This review provides a perspective on the recent developments in the design of BACE-1 inhibitors. An overview of early research is also included, with particular emphasis on a comprehensive survey of the patent literature.

Discovery of an Orally Available, Brain Penetrant BACE1 Inhibitor that Affords Robust CNS Aß Reduction.

Inhibition of BACE1 to prevent brain Aß peptide formation is a potential disease-modifying approach to the treatment of Alzheimer's disease. Despite over a decade of drug discovery efforts, the identification of brain-penetrant BACE1 inhibitors that substantially lower CNS Aß levels following systemic administration remains challenging. In this report we describe structure-based optimization of a series of brain-penetrant BACE1 inhibitors derived from an iminopyrimidinone scaffold. Application of structure-based design in tandem with control of physicochemical properties culminated in the discovery of compound 16, which potently reduced cortex and CSF Aß40 levels when administered orally to rats.

Design and validation of bicyclic iminopyrimidinones as beta amyloid cleaving enzyme-1 (BACE1) inhibitors: conformational constraint to favor a bioactive conformation.

On the basis of our observation that the biaryl substituent of iminopyrimidinone 7 must be in a pseudoaxial conformation to occupy the contiguous S1-S3 subsites of BACE1, we have designed a novel fused bicyclic iminopyrimidinone scaffold intended to favor this bioactive conformation. Strategic incorporation of a nitrogen atom in the new constrained ring allowed us to develop SAR around the S2' binding pocket and ultimately resulted in analogues with low nanomolar potency for BACE1. In particular, optimization of the prime side substituent led to major improvements in potency by displacement of two conserved water molecules from a region near S2'. Further optimization of the pharmacokinetic properties of this fused pyrrolidine series, in conjunction with facile access to a rat pharmacodynamic model, led to identification of compound 43, which is an orally active, brain penetrant inhibitor that reduces Aß(40) in the plasma, CSF, and cortex of rats in a dose-dependent manner.

Discovery of cyclic acylguanidines as highly potent and selective beta-site amyloid cleaving enzyme (BACE) inhibitors: Part I--inhibitor design and validation.

A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit (3), with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores (10 and 23) were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180 degrees in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates, as revealed by 3. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.