Development of a Second-Generation, Chemical Probe for PIKfyve.

We optimized our highly potent and cell-active chemical probe for phosphatidylinositol-3-phosphate 5-kinase (PIKfyve), SGC-PIKFYVE-1, resulting in compounds with improved potency and demonstrated stability. Use of an in-cell, kinome-wide selectivity panel allowed for confirmation of excellent in-cell selectivity of our lead compound, , and another promising analogue, . Evaluation of the pharmacokinetic (PK) profiles of these two compounds revealed that both are well tolerated systemically and orally bioavailable.

Targeting Na1.7 and Na1.8 with a PIKfyve inhibitor to reverse inflammatory and neuropathic pain.

PIKfyve (1-phosphatidylinositol 3-phosphate 5-kinase), a lipid kinase, plays an important role in generating phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P). SGC-PIKFYVE-1, a potent and selective inhibitor of PIKfyve, has been used as a chemical probe to explore pathways dependent on PIKfyve activity. Based on reported changes in membrane dynamics and ion transport in response to PIKfyve inhibition, we hypothesized that pharmacological inhibition of PIKfyve could modulate pain.

Strategic Fluorination to Achieve a Potent, Selective, Metabolically Stable, and Orally Bioavailable Inhibitor of CSNK2.

The host kinase casein kinase 2 (CSNK2) has been proposed to be an antiviral target against β-coronaviral infection. To pharmacologically validate CSNK2 as a drug target in vivo, potent and selective CSNK2 inhibitors with good pharmacokinetic properties are required. Inhibitors based on the pyrazolo[1,5-]pyrimidine scaffold possess outstanding potency and selectivity for CSNK2, but bioavailability and metabolic stability are often challenging.

Discovery and Characterization of a Chemical Probe for Cyclin-Dependent Kinase-Like 2.

Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound was selected as a CDKL2 chemical probe based on its potent inhibition of CDKL2 enzymatic activity, engagement of CDKL2 in cells, and excellent kinome-wide selectivity, especially when used in cells. Compound was designed as a negative control to be used alongside compound in experiments to interrogate CDKL2-mediated biology.

More than an Amide Bioisostere: Discovery of 1,2,4-Triazole-containing Pyrazolo[1,5-]pyrimidine Host CSNK2 Inhibitors for Combatting β-Coronavirus Replication.

The pyrazolo[1,5-]pyrimidine scaffold is a promising scaffold to develop potent and selective CSNK2 inhibitors with antiviral activity against β-coronaviruses. Herein, we describe the discovery of a 1,2,4-triazole group to substitute a key amide group for CSNK2 binding present in many potent pyrazolo[1,5-]pyrimidine inhibitors. Crystallographic evidence demonstrates that the 1,2,4-triazole replaces the amide in forming key hydrogen bonds with Lys68 and a water molecule buried in the ATP-binding pocket.

Development of SYK NanoBRET Cellular Target Engagement Assays for Gain-of-Function Variants.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK.

Discovery and Characterization of a Chemical Probe for Cyclin-Dependent Kinase-Like 2.

Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound was selected as a CDKL2 chemical probe based on its potent inhibition of CDKL2 enzymatic activity, engagement of CDKL2 in cells, and excellent kinome-wide selectivity, especially when used in cells. Compound was designed as a negative control to be used alongside compound in experiments to interrogate CDKL2-mediated biology.

Discovery and optimization of narrow spectrum inhibitors of Tousled like kinase 2 (TLK2) using quantitative structure activity relationships.

The oxindole scaffold has been the center of several kinase drug discovery programs, some of which have led to approved medicines. A series of two oxindole matched pairs from the literature were identified where TLK2 was potently inhibited as an off-target kinase. The oxindole has long been considered a promiscuous kinase inhibitor template, but across these four specific literature oxindoles TLK2 activity was consistent, while the kinome profile was radically different ranging from narrow to broad spectrum kinome coverage.

Synthesis of 5-Benzylamino and 5-Alkylamino-Substituted Pyrimido[4,5-c]quinoline Derivatives as CSNK2A Inhibitors with Antiviral Activity.

A series of 5-benzylamine-substituted pyrimido[4,5-c]quinoline derivatives of the CSNK2A chemical probe SGC-CK2-2 were synthesized with the goal of improving kinase inhibitor cellular potency and antiviral phenotypic activity while maintaining aqueous solubility. Among the range of analogs, those bearing electron-withdrawing ( and ) or donating () substituents on the benzyl ring as well as introduction of non-aromatic groups such as the cyclohexylmethyl () were shown to maintain CSNK2A activity. The CSNK2A activity was also retained with -methylation of SGC-CK2-2, but α-methyl substitution of the benzyl substituent led to a 10-fold reduction in potency.

Illumination of understudied ciliary kinases.

Cilia are cellular signaling hubs. Given that human kinases are central regulators of signaling, it is not surprising that kinases are key players in cilia biology. In fact, many kinases modulate ciliogenesis, which is the generation of cilia, and distinct ciliary pathways.

Discovery and Optimization of Narrow Spectrum Inhibitors of Tousled Like Kinase 2 (TLK2) Using Quantitative Structure Activity Relationships.

The oxindole scaffold has been the center of several kinase drug discovery programs, some of which have led to approved medicines. A series of two oxindole matched pairs from the literature were identified where TLK2 was a potent off-target kinase. The oxindole has long been considered a promiscuous inhibitor template, but across these 4 specific literature oxindoles TLK2 activity was consistent, while the kinome profile was radically different from narrow to broad spectrum coverage.

Illuminating function of the understudied druggable kinome.

The human kinome, with more than 500 proteins, is crucial for cell signaling and disease. Yet, about one-third of kinases lack in-depth study. The Data and Resource Generating Center for Understudied Kinases has developed multiple resources to address this challenge including creation of a heavy amino acid peptide library for parallel reaction monitoring and quantitation of protein kinase expression, use of understudied kinases tagged with a miniTurbo-biotin ligase to determine interaction networks by proximity-dependent protein biotinylation, NanoBRET probe development for screening chemical tool target specificity in live cells, characterization of small molecule chemical tools inhibiting understudied kinases, and computational tools for defining kinome architecture.

Optimization of 3-Cyano-7-cyclopropylamino-pyrazolo[1,5-]pyrimidines toward the Development of an In Vivo Chemical Probe for CSNK2A.

3-Cyano-7-cyclopropylamino-pyrazolo[1,5-]pyrimidines, including the chemical probe SGC-CK2-1, are potent and selective inhibitors of CSNK2A in cells but have limited utility in animal models due to their poor pharmacokinetic properties. While developing analogues with reduced intrinsic clearance and the potential for sustained exposure in mice, we discovered that phase II conjugation by GST enzymes was a major metabolic transformation in hepatocytes. A protocol for codosing with ethacrynic acid, a covalent reversible GST inhibitor, was developed to improve the exposure of analogue in mice.

Fused Tetrahydroquinolines Are Interfering with Your Assay.

Tricyclic tetrahydroquinolines (THQs) have been repeatedly reported as hits across a diverse range of high-throughput screening (HTS) campaigns. The activities of these compounds, however, are likely due to reactive byproducts that interfere with the assay. As a lesser studied class of pan-assay interference compounds, the mechanism by which fused THQs react with protein targets remains largely unknown.

Discovery of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer's disease.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer's disease (AD) traits and microglia. These proteins are more abundant in Alzheimer's patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP) and the cytoplasmic tail of CD44.

Characterization of covalent inhibitors that disrupt the interaction between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2).

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin-dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology.

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 () cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual, and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD has indicated that CDKL5 kinase function is central to disease pathology. encodes a serine-threonine kinase with significant homology to GSK3β, which has also been linked to synaptic function.

Development of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic strategy for Alzheimer's disease.

Recent genome-wide association studies have revealed genetic risk factors for Alzheimer's disease (AD) that are exclusively expressed in microglia within the brain. A proteomics approach identified moesin (MSN), a FERM (four-point-one ezrin radixin moesin) domain protein, and the receptor CD44 as hub proteins found within a co-expression module strongly linked to AD clinical and pathological traits as well as microglia. The FERM domain of MSN interacts with the phospholipid PIP and the cytoplasmic tails of receptors such as CD44.

Optimization of 3-Cyano-7-cyclopropylamino-pyrazolo[1,5-a]pyrimidines Toward the Development of an In Vivo Chemical Probe for CSNK2A.

3-cyano-7-cyclopropylamino-pyrazolo[1,5-a]pyrimidines, including the chemical probe SGC-CK2-1, are potent and selective inhibitors of CSNK2A in cells but have limited utility in animal models due to their poor pharmacokinetic properties. While developing analogs with reduced intrinsic clearance and the potential for sustained exposure in mice, we discovered that Phase II conjugation by GST enzymes was a major metabolic transformation in hepatocytes. A protocol for co-dosing with ethacrynic acid, a covalent reversible GST inhibitor, was developed to improve the exposure of analog in mice.

Open drug discovery in Alzheimer's disease.

Alzheimer's disease (AD) drug discovery has focused on a set of highly studied therapeutic hypotheses, with limited success. The heterogeneous nature of AD processes suggests that a more diverse, systems-integrated strategy may identify new therapeutic hypotheses. Although many target hypotheses have arisen from systems-level modeling of human disease, in practice and for many reasons, it has proven challenging to translate them into drug discovery pipelines.

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology.

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 ( ) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD have indicated that CDKL5 kinase function is central to disease pathology. encodes a serine-threonine kinase with significant homology to GSK3b, which has also been linked to synaptic function.

Discovery of a Potent and Selective CDKL5/GSK3 Chemical Probe That Is Neuroprotective.

Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation.

Discovery of a Potent and Selective Naphthyridine-Based Chemical Probe for Casein Kinase 2.

Naphthyridine-based inhibitors were synthesized to yield a potent and cell-active inhibitor of casein kinase 2 (CK2). Compound selectively inhibits CK2α and CK2α' when profiled broadly, thereby making it an exquisitely selective chemical probe for CK2. A negative control that is structurally related but lacks a key hinge-binding nitrogen () was designed on the basis of structural studies.