How many kinases are druggable? A review of our current understanding.

There are over 500 human kinases ranging from very well-studied to almost completely ignored. Kinases are tractable and implicated in many diseases, making them ideal targets for medicinal chemistry campaigns, but is it possible to discover a drug for each individual kinase? For every human kinase, we gathered data on their citation count, availability of chemical probes, approved and investigational drugs, PDB structures, and biochemical and cellular assays. Analysis of these factors highlights which kinase groups have a wealth of information available, and which groups still have room for progress. The data suggest a disproportionate focus on the more well characterized kinases while much of the kinome remains comparatively understudied. It is noteworthy that tool compounds for understudied kinases have already been developed, and there is still untapped potential for further development in this chemical space. Finally, this review discusses many of the different strategies employed to generate selectivity between kinases. Given the large volume of information available and the progress made over the past 20 years when it comes to drugging kinases, we believe it is possible to develop a tool compound for every human kinase. We hope this review will prove to be both a useful resource as well as inspire the discovery of a tool for every kinase.

Visible-Light-Induced Selective Defluoroborylation of Polyfluoroarenes, gem-Difluoroalkenes, and Trifluoromethylalkenes.

Fluorinated organoboranes serve as versatile synthetic precursors for the preparation of value-added fluorinated organic compounds. Recent progress has been mainly focused on the transition-metal catalyzed defluoroborylation. Herein, we report a photocatalytic defluoroborylation platform through direct B-H activation of N-heterocyclic carbene boranes, through the synergistic merger of a photoredox catalyst and a hydrogen atom transfer catalyst. This atom-economic and operationally simple protocol has enabled defluoroborylation of an extremely broad scope of multifluorinated substrates including polyfluoroarenes, gem-difluoroalkenes, and trifluoromethylalkenes in a highly selective fashion. Intriguingly, the defluoroborylation protocol can be transition-metal free, and the regioselectivity obtained is complementary to the reported transition-metal-catalysis in many cases.

Visible-Light-Mediated Metal-Free Difunctionalization of Alkenes with CO2 and Silanes or C(sp3 )-H Alkanes.

Catalytic alkene difunctionalization via Si-H and C-H activations represents an ideal atom- and step-economic pathway for quick assembly of molecular complexity. We herein developed a visible-light-promoted metal-free difunctionalization of alkenes using abundant CO2 and readily available Si-H and C(sp3 )-H bonds as feedstocks. Through the merger of photoredox and hydrogen-atom-transfer catalysis, a variety of value-added compounds, such as ß-silacarboxylic acids and acids bearing a γ-heteroatom (e.g., N, O, S) could be directly accessed from simple alkenes in a redox-neutral fashion.

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

The pyrazolo[1,5-a]pyrimidine scaffold is a promising scaffold to develop potent and selective CSNK2 inhibitors with antiviral activity against ß-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-a]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. This isosteric replacement improves potency and metabolic stability at a cost of solubility. Optimization for potency, solubility, and metabolic stability led to the discovery of the potent and selective CSNK2 inhibitor 53. Despite excellent in vitro metabolic stability, rapid decline in plasma concentration of 53 in vivo was observed and may be attributed to lung accumulation, although in vivo pharmacological effect was not observed. Further optimization of this novel chemotype may validate CSNK2 as an antiviral target in vivo.

Parasite and host kinases as targets for antimalarials.

INTRODUCTION: The deployment of Artemisinin-based combination therapies and transmission control measures led to a decrease in the global malaria burden over the recent decades. Unfortunately, this trend is now reversing, in part due to resistance against available treatments, calling for the development of new drugs against untapped targets to prevent cross-resistance. AREAS COVERED: In view of their demonstrated druggability in noninfectious diseases, protein kinases represent attractive targets. Kinase-focussed antimalarial drug discovery is facilitated by the availability of kinase-targeting scaffolds and large libraries of inhibitors, as well as high-throughput phenotypic and biochemical assays. We present an overview of validated Plasmodium kinase targets and their inhibitors, and briefly discuss the potential of host cell kinases as targets for host-directed therapy. EXPERT OPINION: We propose priority research areas, including (i) diversification of Plasmodium kinase targets (at present most efforts focus on a very small number of targets); (ii) polypharmacology as an avenue to limit resistance (kinase inhibitors are highly suitable in this respect); and (iii) preemptive limitation of resistance through host-directed therapy (targeting host cell kinases that are required for parasite survival) and transmission-blocking through targeting sexual stage-specific kinases as a strategy to protect curative drugs from the spread of resistance.

Discovery of potent Plasmodium falciparum protein kinase 6 (PfPK6) inhibitors with a type II inhibitor pharmacophore.

Malaria is a devastating disease that causes significant global morbidity and mortality. The rise of drug resistance against artemisinin-based combination therapy demonstrates the necessity to develop alternative antimalarials with novel mechanisms of action. We report the discovery of Ki8751 as an inhibitor of essential kinase PfPK6. 79 derivatives were designed, synthesized and evaluated for PfPK6 inhibition and antiplasmodial activity. Using group efficiency analyses, we established the importance of key groups on the scaffold consistent with a type II inhibitor pharmacophore. We highlight modifications on the tail group that contribute to antiplasmodial activity, cumulating in the discovery of compound 67, a PfPK6 inhibitor (IC50 = 13 nM) active against the P. falciparum blood stage (EC50 = 160 nM), and compound 79, a PfPK6 inhibitor (IC50 < 5 nM) with dual-stage antiplasmodial activity against P. falciparum blood stage (EC50 = 39 nM) and against P. berghei liver stage (EC50 = 220 nM).

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 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 2h in mice. A double codosing protocol, using a combination of ethacrynic acid and irreversible P450 inhibitor 1-aminobenzotriazole, increased the blood level of 2h by 40-fold at a 5 h time point.

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 2h in mice. A double co-dosing protocol, using a combination of ethacrynic acid and irreversible P450 inhibitor 1-aminobenzotriazole increased the blood level of 2h by 40-fold at a 5 h time point.

Characterization of 2,4-Dianilinopyrimidines Against Five P. falciparum Kinases PfARK1, PfARK3, PfNEK3, PfPK9, and PfPKB.

Plasmodium kinases are increasingly recognized as potential novel antiplasmodial targets for the treatment of malaria, but only a small subset of these kinases have had structure-activity relationship (SAR) campaigns reported. Herein we report the discovery of CZC-54252 (1) as an inhibitor of five P. falciparum kinases PfARK1, PfARK3, PfNEK3, PfPK9, and PfPKB. 39 analogues were evaluated against all five kinases to establish SAR at three regions of the kinase active site. Nanomolar inhibitors of each kinase were discovered. We identified common and divergent SAR trends across all five kinases, highlighting substituents in each region that improve potency and selectivity for each kinase. Potent analogues were evaluated against the P. falciparum blood stage. Eight submicromolar inhibitors were discovered, of which 37 demonstrated potent antiplasmodial activity (EC50 = 0.16 µM). Our results provide an understanding of features needed to inhibit each individual kinase and lay groundwork for future optimization efforts toward novel antimalarials.

PROTAC Linkerology Leads to an Optimized Bivalent Chemical Degrader of Polycomb Repressive Complex 2 (PRC2) Components.

Bivalent chemical degraders, otherwise known as proteolysis-targeting chimeras (PROTACs), have proven to be an efficient strategy for targeting overexpressed or mutated proteins in cancer. PROTACs provide an alternative approach to small-molecule inhibitors, which are restricted by occupancy-driven pharmacology, often resulting in acquired inhibitor resistance via compensatory increases in protein expression. Despite the advantages of bivalent chemical degraders, they often have suboptimal physicochemical properties and optimization for efficient degradation remains highly unpredictable. Herein, we report the development of a potent EED-targeted PRC2 degrader, UNC7700. UNC7700 contains a unique cis-cyclobutane linker and potently degrades PRC2 components EED (DC50 = 111 nM; Dmax = 84%), EZH2WT/EZH2Y641N (DC50 = 275 nM; Dmax = 86%), and to a lesser extent SUZ12 (Dmax = 44%) after 24 h in a diffuse large B-cell lymphoma DB cell line. Characterization of UNC7700 and related compounds for ternary complex formation and cellular permeability to provide a rationale for the observed improvement in degradation efficiency remained challenging. Importantly, UNC7700 dramatically reduces H3K27me3 levels and is anti-proliferative in DB cells (EC50 = 0.79 ± 0.53 µM).

A Potent and Selective CDKL5/GSK3 Chemical Probe 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. We prepared analogs of AT-7519, a known inhibitor of several cyclin dependent and cyclin-dependent kinase-like kinases that has been advanced into Phase II clinical trials. We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/ß affinity. As confirmation that our chemical probe is a high-quality tool to use in directed biological studies, we demonstrated inhibition of downstream CDKL5 and GSK3α/ß signaling and solved a co-crystal structure of analog 2 bound to CDKL5. A structurally similar analog ( 4 ) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/ß. Finally, we used our chemical probe pair ( 2 and 4 ) to demonstrate that inhibition of CDKL5 and/or GSK3α/ß promotes the survival of human motor neurons exposed to endoplasmic reticulum (ER) stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.

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. We prepared analogs of AT-7519, a compound that has advanced to phase II clinical trials and is a known inhibitor of several cyclin-dependent kinases (CDKs) and cyclin-dependent kinase-like kinases (CDKLs). We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/ß affinity. We next demonstrated the inhibition of downstream CDKL5 and GSK3α/ß signaling and solved a co-crystal structure of analog 2 bound to human CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/ß, making it a suitable negative control. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3α/ß promotes the survival of human motor neurons exposed to endoplasmic reticulum stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.

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 (CDKL5) 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. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3ß, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3ß activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3ß activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3ß. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces postsynaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity, and human neuropathology.

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 ( CDKL5 ) 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. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3b, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3b activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3b activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3b. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces post-synaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated, key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity and human neuropathology.

Identification of Novel 2,4,5-Trisubstituted Pyrimidines as Potent Dual Inhibitors of Plasmodial PfGSK3/PfPK6 with Activity against Blood Stage Parasites In Vitro.

Essential plasmodial kinases PfGSK3 and PfPK6 are considered novel drug targets to combat rising resistance to traditional antimalarial therapy. Herein, we report the discovery of IKK16 as a dual PfGSK3/PfPK6 inhibitor active against blood stage Pf3D7 parasites. To establish structure-activity relationships for PfPK6 and PfGSK3, 52 analogues were synthesized and assessed for the inhibition of PfGSK3 and PfPK6, with potent inhibitors further assessed for activity against blood and liver stage parasites. This culminated in the discovery of dual PfGSK3/PfPK6 inhibitors 23d (PfGSK3/PfPK6 IC50 = 172/11 nM) and 23e (PfGSK3/PfPK6 IC50 = 97/8 nM) with antiplasmodial activity (23d Pf3D7 EC50 = 552 ± 37 nM and 23e Pf3D7 EC50 = 1400 ± 13 nM). However, both compounds exhibited significant promiscuity when tested in a panel of human kinase targets. Our results demonstrate that dual PfPK6/PfGSK3 inhibitors with antiplasmodial activity can be identified and can set the stage for further optimization efforts.