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.

Analyzing the Potential Root Causes of Variability of Pharmacokinetics in Preclinical Species.

The purpose of this research was to assess variability in pharmacokinetic profiles (PK variability) in preclinical species and identify the risk factors associated with the properties of a drug molecule that contribute to the variability. Exposure data in mouse, rat, dog, and monkey for a total of 16,592 research compounds studied between 1999 and 2013 were included in the analysis. Both in vivo study parameters and in silico/experimental physicochemical properties of the molecules were analyzed. Areas under the plasma concentration vs time curves (AUC) were used to assess PK variability. PK variability was calculated as the ratio of the highest AUC within a defined set of AUC values (AUCmax) over the lowest AUC within that set (AUCmin). Both intra- and inter-animal variability were analyzed, with intra-animal exposures found to be more variable than inter-animal exposures. While several routes of administration were initially studied, the analysis was focused on the oral route, which corresponds to the large majority of data points and displays higher variability than the subcutaneous, intraperitoneal, or intravenous routes. The association between inter-animal PK variability and physical properties was studied, and low solubility, high administered dose, high preclinical dose number (PDo), and pH-dependent solubility were found to be associated with high variability in exposures. Permeability-as assessed by the measured permeability coefficient in the LLC-PK1 cell line-was also considered but appeared to only have a weak association with variability. Consistent with these findings, BCS class I and III compounds were found to be less prone to PK variability than BCS class II and IV compounds. A modest association of PK variability with clearance was observed while the association with bioavailability, a higher PK variability for compounds with lower bioavailability, appeared to be more pronounced. Finally, two case studies that highlight PK variability issues are described, and successful mitigation strategies are presented.

MARK inhibitors: Declaring a No-Go decision on a chemical series based on extensive DMPK experimentation.

Attempts to optimize pharmacokinetic properties in a promising series of pyrrolopyrimidinone MARK inhibitors for the treatment of Alzheimer's disease are described. A focus on physical properties and ligand efficiency while prosecuting this series afforded key tool compounds that revealed a large discrepancy in the rat in vitro-in vivo DMPK (Drug Metabolism/Pharmacokinetics) correlation. These differences prompted an in vivo rat disposition study employing a radiolabeled representative of the series, and the results from this experiment justified the termination of any further optimization efforts.

Structure guided design of a series of selective pyrrolopyrimidinone MARK inhibitors.

The initial structure activity relationships around an isoindoline uHTS hit will be described. Information gleaned from ligand co-crystal structures allowed for rapid refinements in both MARK potency and kinase selectivity. These efforts allowed for the identification of a compound with properties suitable for use as an in vitro tool compound for validation studies on MARK as a viable target for Alzheimer's disease.

Identification of quinazoline based inhibitors of IRAK4 for the treatment of inflammation.

Interleukin-1 receptor associated kinase 4 (IRAK4) has been implicated in IL-1R and TLR based signaling. Therefore selective inhibition of the kinase activity of this protein represents an attractive target for the treatment of inflammatory diseases. Medicinal chemistry optimization of high throughput screening (HTS) hits with the help of structure based drug design led to the identification of orally-bioavailable quinazoline based IRAK4 inhibitors with excellent pharmacokinetic profile and kinase selectivity. These highly selective IRAK4 compounds show activity in vivo via oral dosing in a TLR7 driven model of inflammation.

The discovery of reverse tricyclic pyridone JAK2 inhibitors. Part 2: lead optimization.

This communication discusses the discovery of novel reverse tricyclic pyridones as inhibitors of Janus kinase 2 (JAK2). By using a kinase cross screening approach coupled with molecular modeling, a unique inhibitor-water interaction was discovered to impart excellent broad kinase selectivity. Improvements in intrinsic potency were achieved by utilizing a rapid library approach, while targeted structural changes to lower lipophilicity led to improved rat pharmacokinetics. This multi-pronged approach led to the identification of 31, which demonstrated encouraging rat pharmacokinetics, in vivo potency, and excellent off-target kinase selectivity.

Inhibition of spleen tyrosine kinase attenuates allergen-mediated airway constriction.

Spleen tyrosine kinase (SYK) is a key activator of signaling pathways downstream of multiple surface receptors implicated in asthma. SYK function has been extensively studied in mast cells downstream of the high-affinity IgE receptor, FcεR1. Preclinical studies have demonstrated a role for SYK in models of allergic inflammation, but a role in airway constriction has not been demonstrated. Here, we have used a potent and selective pharmacological inhibitor of SYK to determine the role of SYK in allergen-mediated inflammation and airway constriction in preclinical models. Attenuation of allergic airway responses was evaluated in a rat passive anaphylaxis model and rat and sheep inhaled allergen challenge models, as well as an ex vivo model of allergen-mediated airway constriction in rats and cynomolgus monkeys. Pharmacological inhibition of SYK dose-dependently blocked IgE-mediated tracheal plasma extravasation in rats. In a rat ovalbumin-sensitized airway challenge model, oral dosing with an SYK inhibitor led to a dose-dependent reduction in lung inflammatory cells. Ex vivo analysis of allergen-induced airway constriction in ovalbumin-sensitized brown Norway rats showed a complete attenuation with treatment of a SYK inhibitor, as well as a complete block of allergen-induced serotonin release. Similarly, allergen-mediated airway constriction was attenuated in ex vivo studies from nonhuman primate lungs. Intravenous administration of an SYK inhibitor attenuated both early- and late-phase allergen-induced increases in airway resistance in an Ascaris-sensitive sheep allergen challenge model. These data support a key role for SYK signaling in mediating allergic airway responses.

Genetic and pharmacological inhibition of PDK1 in cancer cells: characterization of a selective allosteric kinase inhibitor.

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

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.

Discovery of PDK1 kinase inhibitors with a novel mechanism of action by ultrahigh throughput screening.

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

Carboxamide Spleen Tyrosine Kinase (Syk) Inhibitors: Leveraging Ground State Interactions To Accelerate Optimization.

Optimization of a series of highly potent and kinome selective carbon-linked carboxamide spleen tyrosine kinase (Syk) inhibitors with favorable drug-like properties is described. A pervasive Ames liability in an analogous nitrogen-linked carboxamide series was obviated by replacement with a carbon-linked moiety. Initial efforts lacked on-target potency, likely due to strain induced between the hinge binding amide and solvent front heterocycle. Consideration of ground state and bound state energetics allowed rapid realization of improved solvent front substituents affording subnanomolar Syk potency and high kinome selectivity. These molecules were also devoid of mutagenicity risk as assessed via the Ames test using the TA97a Salmonella strain.

Inhibition of spleen tyrosine kinase attenuates IgE-mediated airway contraction and mediator release in human precision cut lung slices.

BACKGROUND AND PURPOSE: Asthma presents as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyper-reactivity (AHR). Spleen tyrosine kinase (Syk) mediates allergen-induced mast cell degranulation, a central component of allergen-induced inflammation and AHR. However, the role of Syk in IgE-mediated constriction of human small airways remains unknown. In this study, we addressed whether selective inhibition of Syk attenuates IgE-mediated constriction and mast cell mediator release in human small airways. EXPERIMENTAL APPROACH: Human precision cut lung slices (hPCLS) ex vivo derived from non-asthmatic donors were incubated overnight with human IgE, dexamethasone, montelukast, antihistamines or a selective Syk inhibitor (SYKi). High-affinity IgE receptor (FcεRI) activation by anti-IgE cross-linking was performed, and constriction and mediator release measured. Airway constriction was normalized to that induced by maximal carbachol stimulation. Syk expression (determined by qPCR and immunoblot) was also evaluated in human primary airway smooth muscle (HASM) cells to determine whether Syk directly modulates HASM function. KEY RESULTS: While dexamethasone had little effect on FcεR-mediated contraction, montelukast or antihistamines partially attenuated the response. SYKi abolished anti-IgE-mediated contraction and suppressed the release of mast cell or basophil mediators from the IgE-treated hPCLS. In contrast, SYKi had little effect on the non-allergic contraction induced by carbachol. Syk mRNA and protein were undetectable in HASM cells. CONCLUSIONS AND IMPLICATIONS: A selective Syk inhibitor, but not corticosteroids, abolished FcεR-mediated contraction in human small airways ex vivo. The mechanism involved FcεRI receptor activation on mast cells or basophils that degranulate causing airway constriction, rather than direct actions on HASM.

Overcoming mutagenicity and ion channel activity: optimization of selective spleen tyrosine kinase inhibitors.

Development of a series of highly kinome-selective spleen tyrosine kinase (Syk) inhibitors with favorable druglike properties is described. Early leads were discovered through X-ray crystallographic analysis, and a systematic survey of cores within a selected chemical space focused on ligand binding efficiency. Attenuation of hERG ion channel activity inherent within the initial chemotype was guided through modulation of physicochemical properties including log D, PSA, and pKa. PSA proved most effective for prospective compound design. Further profiling of an advanced compound revealed bacterial mutagenicity in the Ames test using TA97a Salmonella strain, and subsequent study demonstrated that this mutagenicity was pervasive throughout the series. Identification of intercalation as a likely mechanism for the mutagenicity-enabled modification of the core scaffold. Implementation of a DNA binding assay as a prescreen and models in DNA allowed resolution of the mutagenicity risk, affording molecules with favorable potency, selectivity, pharmacokinetic, and off-target profiles.

Discovery of 1-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]-N-(pyridin-2-ylmethyl)methanesulfonamide (MK-8033): A Specific c-Met/Ron dual kinase inhibitor with preferential affinity for the activated state of c-Met.

This report documents the first example of a specific inhibitor of protein kinases with preferential binding to the activated kinase conformation: 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one 11r (MK-8033), a dual c-Met/Ron inhibitor under investigation as a treatment for cancer. The design of 11r was based on the desire to reduce time-dependent inhibition of CYP3A4 (TDI) by members of this structural class. A novel two-step protocol for the synthesis of benzylic sulfonamides was developed to access 11r and analogues. We provide a rationale for the observed selectivity based on X-ray crystallographic evidence and discuss selectivity trends with additional examples. Importantly, 11r provides full inhibition of tumor growth in a c-Met amplified (GTL-16) subcutaneous tumor xenograft model and may have an advantage over inactive form kinase inhibitors due to equal potency against a panel of oncogenic activating mutations of c-Met in contrast to c-Met inhibitors without preferential binding to the active kinase conformation.

Identification of direct target engagement biomarkers for kinase-targeted therapeutics.

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3'-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1(Ser410) and p-PDK1(Thr513) are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1(Ser241)), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.

PDK1 attenuation fails to prevent tumor formation in PTEN-deficient transgenic mouse models.

PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target.

Efficient identification of novel leads by dynamic focused screening: PDK1 case stud.

A dynamic, focused screening strategy that utilized a limited but diversified set of target-specific compounds was explored as an efficient means for the identification of inhibitors of the protein kinase PDK1. Approximately 21,500 compounds, including a 19,000 molecule kinase-focused compound collection (KFCC), were screened at two concentrations to identify initial leads. The KFCC included several empirically-derived, general kinase libraries and molecules chosen by PDK1-specific virtual screens. As was expected, this initial screen mostly identified potent leads with limited novelty. In order to overcome this limitation, the data from the screen were used to drive several rounds of a customized iterative focused screening (IFS) campaign. A machine-learning technique was used to build a predictive model to identify compounds to be screened in subsequent rounds. Molecules deemed not to be novel were removed from the training set for the next round, which allowed this campaign to progressively walk away from the chemical space covered by the KFCC. This resulted in the identification of PDK1 inhibitors which are uniquely different from publicly known chemotypes after just three rounds of screenings. A retrospective analysis of this IFS approach against an ultra-high throughput screen (uHTS) indicated that while uHTS is still the most prolific paradigm for lead identification, this dynamic, focused screening approach was successful in discovering novel scaffolds for a medicinal chemistry effort. Finally, a theoretical optimization suggested the dynamic, focused screening approaches could provide either a complementary or alternative approach to uHTS for the efficient and rapid lead identification.