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.

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.

Establishing and Validating Cellular Functional Target Engagement Assay for Selective IRAK4 Inhibitor Discovery.

One of the main reasons for the lack of drug efficacy in late-stage clinical trials is the lack of specific and selective target engagement. To increase the likelihood of success of new therapeutics, one approach is to conduct proximal target engagement testing during the early phases of preclinical drug discovery. To identify and optimize selective IRAK4 inhibitors, a kinase that has been implicated in multiple inflammatory and autoimmune diseases, we established an electrochemiluminescence (ECL)-based cellular endogenous IRAK1 activation assay as the most proximal functional evaluation of IRAK4 engagement to support structure-activity relationship (SAR) studies. Since IRAK1 activation is dependent on both the IRAK4 scaffolding function in Myddosome formation and IRAK4 kinase activity for signal transduction, this assay potentially captures inhibitors with different mechanisms of action. Data from this IRAK1 assay with compounds representing different structural classes showed statistically significant correlations when compared with results from both IRAK4 biochemical kinase activity and functional peripheral blood mononuclear cell (PBMC)-derived tumor necrosis factor α (TNFα) secretion assays, validating the biological relevancy of the IRAK1 target engagement as a biomarker of the IRAK4 activity. Plate uniformity and potency reproducibility evaluations demonstrated that this assay is amenable to high throughput. Using Bland-Altman assay agreement analysis, we demonstrated that incorporating such proximal pharmacological assessment of cellular target engagement to an in vitro screening funnel for SAR studies can prevent compound optimization toward off-target activity.

Development of High-Throughput Assays for Evaluation of Hematopoietic Progenitor Kinase 1 Inhibitors.

Hematopoietic progenitor kinase 1 (HPK1), also referred to as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a serine/threonine kinase that negatively regulates T-cell signaling by phosphorylating Ser376 of Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76), a critical mediator of T-cell receptor activation. HPK1 loss of function mouse models demonstrated enhanced immune cell activation and beneficial antitumor activity. To enable discovery and functional characterization of high-affinity small-molecule HPK1 inhibitors, we have established high-throughput biochemical, cell-based, and novel pharmacodynamic (PD) assays. Kinase activity-based time-resolved fluorescence energy transfer (TR-FRET) assays were established as the primary biochemical approach to screen for potent inhibitors and assess selectivity against members of MAP4K and other closely related kinases. A proximal target engagement (TE) assay quantifying pSLP-76 levels as a readout and a distal assay measuring IL-2 secretion as a functional response were established using human peripheral blood mononuclear cells (PBMCs) from two healthy donors. Significant correlations between biochemical and cellular assays as well as excellent correlation between the two donors for the cellular assays were observed. pSLP-76 levels were further used as a PD marker in the preclinical murine model. This effort required the development of a novel ultrasensitive single-molecule array (SiMoA) assay to monitor pSLP-76 changes in mouse spleen.

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.

From Screening to Targeted Degradation: Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9.

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.

Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia model.

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara-C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara-C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara-C were elucidated by whole-exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara-C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.

Spatial and temporal studies of metabolic activity: contrasting biochemical kinetics in tissues and pathways during fasted and fed states.

The regulation of nutrient homeostasis, i.e., the ability to transition between fasted and fed states, is fundamental in maintaining health. Since food is typically consumed over limited (anabolic) periods, dietary components must be processed and stored to counterbalance the catabolic stress that occurs between meals. Herein, we contrast tissue- and pathway-specific metabolic activity in fasted and fed states. We demonstrate that knowledge of biochemical kinetics that is obtained from opposite ends of the energetic spectrum can allow mechanism-based differentiation of healthy and disease phenotypes. Rat models of type 1 and type 2 diabetes serve as case studies for probing spatial and temporal patterns of metabolic activity via [2H]water labeling. Experimental designs that capture integrative whole body metabolism, including meal-induced substrate partitioning, can support an array of research surrounding metabolic disease; the relative simplicity of the approach that is discussed here should enable routine applications in preclinical models.

Novel, highly potent systemic glucokinase activators for the treatment of Type 2 Diabetes Mellitus.

Glucokinase (GK, hexokinase IV) is a unique hexokinase that plays a central role in mammalian glucose homeostasis. Glucose phosphorylation by GK in the pancreatic ß-cell is the rate-limiting step that controls glucose-stimulated insulin secretion. Similarly, GK-mediated glucose phosphorylation in hepatocytes plays a major role in increasing hepatic glucose uptake and metabolism and possibly lowering hepatic glucose output. Small molecule GK activators (GKAs) have been identified that increase enzyme activity by binding to an allosteric site. GKAs offer a novel approach for the treatment of Type 2 Diabetes Mellitus (T2DM) and as such have garnered much attention. We now report the design, synthesis, and biological evaluation of a novel series of 2,5,6-trisubstituted indole derivatives that act as highly potent GKAs. Among them, Compound 1 was found to possess high in vitro potency, excellent physicochemical properties, and good pharmacokinetic profile in rodents. Oral administration of Compound 1 at doses as low as 0.03mg/kg led to robust blood glucose lowering efficacy in 3week high fat diet-fed mice.

Discovery of orally active hepatoselective glucokinase activators for treatment of Type II Diabetes Mellitus.

Systemically acting glucokinase activators (GKA) have been demonstrated in clinical trials to effectively lower blood glucose in patients with type II diabetes. However, mechanism-based hypoglycemia is a major adverse effect that limits the therapeutic potential of these agents. We hypothesized that the predominant mechanism leading to hypoglycemia is GKA-induced excessive insulin secretion from pancreatic ß-cells at (sub-)euglycemic levels. We further hypothesized that restricting GK activation to hepatocytes would maintain glucose-lowering efficacy while significantly reducing hypoglycemic risk. Here we report the discovery of a novel series of carboxylic acid substituted GKAs based on pyridine-2-carboxamide. These GKAs exhibit preferential distribution to the liver versus the pancreas in mice. SAR studies led to the identification of a potent and orally active hepatoselective GKA, compound 6. GKA 6 demonstrated robust glucose lowering efficacy in high fat diet-fed mice at doses ⩾10mpk, with ⩾70-fold liver:pancreas distribution, minimal effects on plasma insulin levels, and significantly reduced risk of hypoglycemia.

Identification of DGAT2 Inhibitors Using Mass Spectrometry.

Mass spectrometry offers significant advantages over other detection technologies in the areas of hit finding, hit validation, and medicinal chemistry compound optimization. The foremost obvious advantage is the ability to directly measure enzymatic product formation. In addition, the inherent sensitivity of the liquid chromatography/mass spectrometry (LC/MS) approach allows the execution of enzymatic assays at substrate concentrations typically at or below substrate Km. Another advantage of the LC/MS approach is the ability to assay impure enzyme systems that would otherwise be difficult to prosecute with traditional labeled methods. This approach was used to identify inhibitors of diacylglycerol O-acyltransferase-2 (DGAT2), a transmembrane enzyme involved in the triglyceride (TG) production pathway. The LC/MS approach was employed because of its increased assay window (compared with control membranes) of more than sevenfold compared with less than twofold with a conventional fluorogenic assay. The ability to generate thousands of dose-dependent IC50 data was made possible by the use of a staggered parallel LC/MS system with fast elution gradients. From the hit-deconvolution efforts, several structural scaffold series were identified that inhibit DGAT2 activity. Additional profiling of one chemotype in particular identified two promising reversible and selective compounds (compound 15 and compound 16) that effectively inhibit TG production in mouse primary hepatocytes.

Discovery and Pharmacology of a Novel Class of Diacylglycerol Acyltransferase 2 Inhibitors.

DGAT2 plays a critical role in hepatic triglyceride production, and data suggests that inhibition of DGAT2 could prove to be beneficial in treating a number of disease states. This article documents the discovery and optimization of a selective small molecule inhibitor of DGAT2 as well as pharmacological proof of biology in a mouse model of triglyceride production.

Development of a fluorescence polarization binding assay for folate receptor.

Folate receptor (FR) has been actively investigated for targeted delivery of therapeutics into cancer cells because this receptor is selectively and highly expressed in carcinomas. Because FR rapidly cycles between the cell surface and cytoplasm, folic acid conjugated to a therapeutic agent can drive targeted therapeutic delivery to cancer cells. We prepared a novel fluorescent ligand Cy5-folate and used it to develop a fluorescence polarization (FP) FR binding assay to determine the binding affinities of FR-targeted molecules. The assay was performed in 96-well microplates using membrane preparations from human KB cells as a source of FR and Cy5 fluorophore-labeled folic acid as a tracer. This high-throughput homogeneous assay demonstrates advantages over existing multistep methods in that it minimizes both time and resources spent determining binding affinities. At the optimized conditions, a Z' of 0.64 was achieved in a 96-well format.

The location and nature of general anesthetic binding sites on the active conformation of firefly luciferase; a time resolved photolabeling study.

Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP-driven production of light. We have used time-resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [(3)H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC-MSMS revealed a similar conformation-dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule) whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.

PCSK9 is not involved in the degradation of LDL receptors and BACE1 in the adult mouse brain.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates hepatic low-density lipoprotein receptor (LDLR) levels in humans. PCSK9 has also been shown to regulate the levels of additional membrane-bound proteins in vitro, including the very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (ApoER2) and the beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), which are all highly expressed in the CNS and have been implicated in Alzheimer's disease. To better understand the role of PCSK9 in regulating these additional target proteins in vivo, their steady-state levels were measured in the brain of wild-type, PCSK9-deficient, and human PCSK9 overexpressing transgenic mice. We found that while PCSK9 directly bound to recombinant LDLR, VLDLR, and apoER2 protein in vitro, changes in PCSK9 expression did not alter the level of these receptors in the mouse brain. In addition, we found no evidence that PCSK9 regulates BACE1 levels or APP processing in the mouse brain. In conclusion, our results suggest that while PCSK9 plays an important role in regulating circulating LDL cholesterol levels by reducing the number of hepatic LDLRs, it does not appear to modulate the levels of LDLR and other membrane-bound proteins in the adult mouse brain.

Biochemical characterization of cholesteryl ester transfer protein inhibitors.

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.

Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cdelta.

Protein kinase C (PKC) is an important signal transduction protein that has been proposed to interact with general anesthetics at its cysteine-rich diacylglycerol/phorbol ester-binding domain C1, a tandem repeat of C1A and C1B subdomains. To test this hypothesis, we expressed, purified, and characterized the high affinity phorbol-binding subdomain, C1B, of mouse protein kinase Cdelta, and studied its interaction with general anesthetic alcohols. When the fluorescent phorbol ester, sapintoxin-D, bound to PKCdelta C1B in buffer at a molar ratio of 1:2, its fluorescence emission maximum, lambda(max), shifted from 437 to 425 nm. The general anesthetic alcohols, butanol and octanol, further shifted lambda(max) of the PKCdelta C1B-bound sapintoxin-D in a concentration-dependent, saturable manner to approximately 415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKCdelta C1B was photolabeled with three photo-activable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKCdelta C1B at a stoichiometry of 1:1 in the labeled fraction. The photolabeled PKCdelta C1B was subjected to tryptic digest, the fragments were separated by online chromatography and sequenced by mass spectrometry. Each azialcohol photoincorporated at Tyr-236. Inspection of the known structure of PKCdelta C1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within approximately 10 A of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.

Low chemical specificity of the nicotinic acetylcholine receptor sterol activation site.

The nicotinic acetylcholine receptor (nAcChoR) has an absolute requirement for cholesterol if agonist-stimulated channel opening is to occur [Biochemistry 25 (1986) 830]. Certain non-polar analogs could replace cholesterol in vectorial vesicle permeability assays. Using a stopped-flow fluorescence assay to avoid the limitations of permeability assays imposed by vesicle morphology, it was shown that polar conjugates of cholesterol could also satisfy the sterol requirement [Biochim. Biophys. Acta 1370 (1998) 299]. Here this assay is used to explore the chemical specificity of sterols. Affinity-purified nAcChoRs from Torpedo were reconstituted into bilayers at mole ratios of 58:12:30 [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)/steroid]. When the enantiomer of cholesterol was used, or when the stereochemistry at the 3-hydroxy group was changed from beta to alpha by substituting epicholesterol for cholesterol, activation was still supported. The importance of cholesterol's planar ring structure was tested by comparing planar cholestanol (5alpha-cholestan-3beta-ol) with nonplanar coprostanol (5beta-cholestan-3beta-ol). Both supported activation. Thus, these steroids support activation independent of structural features known to be important for modulation of lipid bilayer properties. This provides indirect support for a steroid binding site possessing very lax structural requirements.

Geometric isomers of a photoactivable general anesthetic delineate a binding site on adenylate kinase.

General anesthetics are a class of drugs whose mode of action is poorly understood. Here, two photoactivable general anesthetics, n-octan-1-ol geometric isomers bearing a diazirine group on either the third or seventh carbon (3- and 7-azioctanol, respectively), were used to locate and delineate an anesthetic site on adenylate kinase. Each photoincorporated at a mole ratio of 1:1 as determined by mass spectrometry. The photolabeled kinase was subjected to tryptic digest, and the fragments were separated by chromatography and sequenced by mass spectrometry. 3-Azioctanol photolabeled His-36, whereas its isomer, 7-azioctanol, photolabeled Asp-41. Inspection of the known structure of adenylate kinase shows that the side chains of these residues are within approximately 5 A of each other. This distance matches the separation of the 3- and 7-positions of an extended aliphatic chain. The alkanol site so-defined spans two domains of adenylate kinase. His-36 is part of the CORE domain, and Asp-41 belongs to the nucleotide monophosphate binding domain. Upon ligand binding the nucleotide monophosphate binding domain rotates relative to the CORE domain, causing a conformational change that might be expected to affect alkanol binding. Indeed, the substrate-mimicking inhibitor adenosine-(5')-pentaphospho-(5')-adenosine (Ap5A) reduced the photoincorporation of 3-[(3)H]azioctanol by 75%.