Development of potent and selective ULK1/2 inhibitors based on 7-azaindole scaffold with favorable in vivo properties.

The UNC-51-like kinase-1 (ULK1) is one of the central upstream regulators of the autophagy pathway, represents a key target for the development of molecular probes to abrogate autophagy and explore potential therapeutic avenues. Here we report the discovery, structure-activity and structure-property relationships of selective, potent, and cell-active ULK1/2 inhibitors based on a 7-azaindole scaffold. Using structure-based drug design, we have developed a series of analogs with excellent binding affinity and biochemical activity against ULK1/2 (IC50 < 25 nM). The validation of cellular target engagement for these compounds was achieved through the employment of the ULK1 NanoBRET intracellular kinase assay. Notably, we have successfully solved the crystal structure of the lead compound, MR-2088, bound to the active site of ULK1. Moreover, the combination treatment of MR-2088 with known KRAS→RAF→MEK→ERK pathway inhibitors, such as trametinib, showed promising synergistic effect in vitro using H2030 (KRASG12C) cell lines. Lastly, our findings underscore MR-2088's potential to inhibit starvation/stimuli-induced autophagic flux, coupled with its suitability for in vivo studies based on its pharmacokinetic properties.

Differential network analysis of ROS1 inhibitors reveals lorlatinib polypharmacology through co-targeting PYK2.

Multiple tyrosine kinase inhibitors (TKIs) are often developed for the same indication. However, their relative overall efficacy is frequently incompletely understood and they may harbor unrecognized targets that cooperate with the intended target. We compared several ROS1 TKIs for inhibition of ROS1-fusion-positive lung cancer cell viability, ROS1 autophosphorylation and kinase activity, which indicated disproportionately higher cellular potency of one TKI, lorlatinib. Quantitative chemical and phosphoproteomics across four ROS1 TKIs and differential network analysis revealed that lorlatinib uniquely impacted focal adhesion signaling. Functional validation using pharmacological probes, RNA interference, and CRISPR-Cas9 knockout uncovered a polypharmacology mechanism of lorlatinib by dual targeting ROS1 and PYK2, which form a multiprotein complex with SRC. Rational multi-targeting of this complex by combining lorlatinib with SRC inhibitors exhibited pronounced synergy. Taken together, we show that systems pharmacology-based differential network analysis can dissect mixed canonical/non-canonical polypharmacology mechanisms across multiple TKIs enabling the design of rational drug combinations.

The reversible inhibitor SR-4835 binds Cdk12/cyclin K in a noncanonical G-loop conformation.

Inhibition of cyclin-dependent kinases (CDKs) has evolved as an emerging anticancer strategy. In addition to the cell cycle-regulating CDKs, the transcriptional kinases Cdk12 and Cdk13 have become the focus of interest as they mediate a variety of functions, including the transition from transcription initiation to elongation and termination, precursor mRNA splicing, and intronic polyadenylation. Here, we determine the crystal structure of the small molecular inhibitor SR-4835 bound to the Cdk12/cyclin K complex at 2.68 Å resolution. The compound's benzimidazole moiety is embedded in a unique hydrogen bond network mediated by the kinase hinge region with flanking hydroxy groups of the Y815 and D819 side chains. Whereas the SR-4835 head group targets the adenine-binding pocket, the kinase's glycine-rich loop is shifted down toward the activation loop. Additionally, the αC-helix adopts an inward conformation, and the phosphorylated T-loop threonine interacts with all three canonical arginines, a hallmark of CDK activation that is altered in Cdk12 and Cdk13. Dose-response inhibition measurements with recombinant CMGC kinases show that SR-4835 is highly specific for Cdk12 and Cdk13 following a 10-fold lower potency for Cdk10. Whereas other CDK-targeting compounds exhibit tighter binding affinities and higher potencies for kinase inhibition, SR-4835 can be considered a selective transcription elongation antagonist. Our results provide the basis for a rational improvement of SR-4835 toward Cdk12 inhibition and a gain in selectivity over other transcription regulating CDKs.

CK1δ and CK1ε Signaling Sustains Mitochondrial Metabolism and Cell Survival in Multiple Myeloma.

Multiple myeloma remains an incurable malignancy due to acquisition of intrinsic programs that drive therapy resistance. Here we report that casein kinase-1δ (CK1δ) and CK1ε are therapeutic targets in multiple myeloma that are necessary to sustain mitochondrial metabolism. Specifically, the dual CK1δ/CK1ε inhibitor SR-3029 had potent in vivo and ex vivo anti-multiple myeloma activity, including against primary multiple myeloma patient specimens. RNA sequencing (RNA-seq) and metabolic analyses revealed inhibiting CK1δ/CK1ε disables multiple myeloma metabolism by suppressing genes involved in oxidative phosphorylation (OxPhos), reducing citric acid cycle intermediates, and suppressing complexes I and IV of the electron transport chain. Finally, sensitivity of multiple myeloma patient specimens to SR-3029 correlated with elevated expression of mitochondrial genes, and RNA-seq from 687 multiple myeloma patient samples revealed that increased CSNK1D, CSNK1E, and OxPhos genes correlate with disease progression and inferior outcomes. Thus, increases in mitochondrial metabolism are a hallmark of multiple myeloma progression that can be disabled by targeting CK1δ/CK1ε. SIGNIFICANCE: CK1δ and CK1ε are attractive therapeutic targets in multiple myeloma whose expression increases with disease progression and connote poor outcomes, and that are necessary to sustain expression of genes directing OxPhos.

Going beyond Binary: Rapid Identification of Protein-Protein Interaction Modulators Using a Multifragment Kinetic Target-Guided Synthesis Approach.

Kinetic target-guided synthesis (KTGS) is a powerful screening approach that enables identification of small molecule modulators for biomolecules. While many KTGS variants have emerged, a majority of the examples suffer from limited throughput and a poor signal/noise ratio, hampering reliable hit detection. Herein, we present our optimized multifragment KTGS screening strategy that tackles these limitations. This approach utilizes selected reaction monitoring liquid chromatography tandem mass spectrometry for hit detection, enabling the incubation of 190 fragment combinations per screening well. Consequentially, our fragment library was expanded from 81 possible combinations to 1710, representing the largest KTGS screening library assembled to date. The expanded library was screened against Mcl-1, leading to the discovery of 24 inhibitors. This work unveils the true potential of KTGS with respect to the rapid and reliable identification of hits, further highlighting its utility as a complement to the existing repertoire of screening methods used in drug discovery.

Chemical Proteomics with Novel Fully Functionalized Fragments and Stringent Target Prioritization Identifies the Glutathione-Dependent Isomerase GSTZ1 as a Lung Cancer Target.

Photoreactive fragment-like probes have been applied to discover target proteins that constitute novel cellular vulnerabilities and to identify viable chemical hits for drug discovery. Through forming covalent bonds, functionalized probes can achieve stronger target engagement and require less effort for on-target mechanism validation. However, the design of probe libraries, which directly affects the biological target space that is interrogated, and effective target prioritization remain critical challenges of such a chemical proteomic platform. In this study, we designed and synthesized a diverse panel of 20 fragment-based probes containing natural product-based privileged structural motifs for small-molecule lead discovery. These probes were fully functionalized with orthogonal diazirine and alkyne moieties and used for protein crosslinking in live lung cancer cells, target enrichment via "click chemistry," and subsequent target identification through label-free quantitative liquid chromatography-tandem mass spectrometry analysis. Pair-wise comparison with a blunted negative control probe and stringent prioritization via individual cross-comparisons against the entire panel identified glutathione S-transferase zeta 1 (GSTZ1) as a specific and unique target candidate. DepMap database query, RNA interference-based gene silencing, and proteome-wide tyrosine reactivity profiling suggested that GSTZ1 cooperated with different oncogenic alterations by supporting survival signaling in refractory non-small cell lung cancer cells. This finding may form the basis for developing novel GSTZ1 inhibitors to improve the therapeutic efficacy of oncogene-directed targeted drugs. In summary, we designed a novel fragment-based probe panel and developed a target prioritization scheme with improved stringency, which allows for the identification of unique target candidates, such as GSTZ1 in refractory lung cancer.

Aminoalkoxycarbonyloxymethyl Ether Prodrugs with a pH-Triggered Release Mechanism: A Case Study Improving the Solubility, Bioavailability, and Efficacy of Antimalarial 4(1H)-Quinolones with Single Dose Cures.

Preclinical and clinical development of numerous small molecules is prevented by their poor aqueous solubility, limited absorption, and oral bioavailability. Herein, we disclose a general prodrug approach that converts promising lead compounds into aminoalkoxycarbonyloxymethyl (amino AOCOM) ether-substituted analogues that display significantly improved aqueous solubility and enhanced oral bioavailability, restoring key requirements typical for drug candidate profiles. The prodrug is completely independent of biotransformations and animal-independent because it becomes an active compound via a pH-triggered intramolecular cyclization-elimination reaction. As a proof-of-concept, the utility of this novel amino AOCOM ether prodrug approach was demonstrated on an antimalarial compound series representing a variety of antimalarial 4(1H)-quinolones, which entered and failed preclinical development over the last decade. With the amino AOCOM ether prodrug moiety, the 3-aryl-4(1H)-quinolone preclinical candidate was shown to provide single-dose cures in a rodent malaria model at an oral dose of 3 mg/kg, without the use of an advanced formulation technique.

The promise and current status of CDK12/13 inhibition for the treatment of cancer.

CDK12 and CDK13 are Ser/Thr protein kinases that regulate transcription and co-transcriptional processes. Genetic silencing of CDK12 is associated with genomic instability in a variety of cancers, including difficult-to-treat breast, ovarian, colorectal, brain and pancreatic cancers, and is synthetic lethal with PARP, MYC or EWS/FLI inhibition. CDK13 is amplified in hepatocellular carcinoma. Consequently, selective CDK12/13 inhibitors constitute powerful research tools as well as promising anti-cancer therapeutics, either alone or in combination therapy. Herein the authors discuss the role of CDK12 and CDK13 in normal and cancer cells, describe their utility as a biomarker and therapeutic target, review the medicinal chemistry optimization of existing CDK12/13 inhibitors and outline strategies for the rational design of CDK12/13 selective inhibitors.

The dual PI3Kδ/CK1ε inhibitor umbralisib exhibits unique immunomodulatory effects on CLL T cells.

The in-clinic phosphatidylinositol 3-kinase (PI3K) inhibitors idelalisib (CAL-101) and duvelisib (IPI-145) have demonstrated high rates of response and progression-free survival in clinical trials of B-cell malignancies, such as chronic lymphocytic leukemia (CLL). However, a high incidence of adverse events has led to frequent discontinuations, limiting the clinical development of these inhibitors. By contrast, the dual PI3Kδ/casein kinase-1-ε (CK1ε) inhibitor umbralisib (TGR-1202) also shows high rates of response in clinical trials but has an improved safety profile with fewer severe adverse events. Toxicities typical of this class of PI3K inhibitors are largely thought to be immune mediated, but they are poorly characterized. Here, we report the effects of idelalisib, duvelisib, and umbralisib on regulatory T cells (Tregs) on normal human T cells, T cells from CLL patients, and T cells in an Eµ-TCL1 adoptive transfer mouse CLL model. Ex vivo studies revealed differential effects of these PI3K inhibitors; only umbralisib treatment sustained normal and CLL-associated FoxP3+ human Tregs. Further, although all 3 inhibitors exhibit antitumor efficacy in the Eµ-TCL1 CLL model, idelalisib- or duvelisib-treated mice displayed increased immune-mediated toxicities, impaired function, and reduced numbers of Tregs, whereas Treg number and function were preserved in umbralisib-treated CLL-bearing mice. Finally, our studies demonstrate that inhibition of CK1ε can improve CLL Treg number and function. Interestingly, CK1ε inhibition mitigated impairment of CLL Tregs by PI3K inhibitors in combination treatment. These results suggest that the improved safety profile of umbralisib is due to its role as a dual PI3Kδ/CK1ε inhibitor that preserves Treg number and function.

Targeting Casein Kinase 1 Delta Sensitizes Pancreatic and Bladder Cancer Cells to Gemcitabine Treatment by Upregulating Deoxycytidine Kinase.

Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. This study evaluated casein kinase 1 delta (CK1δ) as a potential therapeutic target for PDA and bladder cancer, in which CK1δ is frequently overexpressed. We assessed the antitumor effects of genetically silencing or pharmacologically inhibiting CK1δ using our in-house CK1δ small-molecule inhibitor SR-3029, either alone or in combination with gemcitabine, on the proliferation and survival of pancreatic and bladder cancer cell lines and orthotopic mouse models. Genetic studies confirmed that silencing CK1δ or treatment with SR-3029 induced a significant upregulation of deoxycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine metabolite activation. The combination of SR-3029 with gemcitabine induced synergistic antiproliferative activity and enhanced apoptosis in both pancreatic and bladder cancer cells. Furthermore, in an orthotopic pancreatic tumor model, we observed improved efficacy with combination treatment concomitant with increased dCK expression. This study demonstrates that CK1δ plays a role in gemcitabine metabolism, and that the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic PDA as well as other cancers with upregulated CK1δ expression.

Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer.

Epigenetic regulation enables tumors to respond to changing environments during tumor progression and metastases and facilitates treatment resistance. Targeting chromatin modifiers or catalytic effectors of transcription is an emerging anti-cancer strategy. The cyclin-dependent kinases (CDKs) 12 and 13 phosphorylate the C-terminal domain of RNA polymerase II, regulating transcription and co-transcriptional processes. Here we report the development of SR-4835, a highly selective dual inhibitor of CDK12 and CDK13, which disables triple-negative breast cancer (TNBC) cells. Mechanistically, inhibition or loss of CDK12/CDK13 triggers intronic polyadenylation site cleavage that suppresses the expression of core DNA damage response proteins. This provokes a "BRCAness" phenotype that results in deficiencies in DNA damage repair, promoting synergy with DNA-damaging chemotherapy and PARP inhibitors.

Development of dual casein kinase 1δ/1ε (CK1δ/ε) inhibitors for treatment of breast cancer.

Casein kinase 1δ/ε have been identified as promising therapeutic target for oncology application, including breast and brain cancer. Here, we described our continued efforts in optimization of a lead series of purine scaffold inhibitors that led to identification of two new CK1δ/ε inhibitors 17 and 28 displaying low nanomolar values in antiproliferative assays against the human MDA-MB-231 triple negative breast cancer cell line and have physical, in vitro and in vivo pharmacokinetic properties suitable for use in proof of principle animal xenograft studies against human cancers.

Design and Synthesis of Orally Bioavailable Piperazine Substituted 4(1H)-Quinolones with Potent Antimalarial Activity: Structure-Activity and Structure-Property Relationship Studies.

Malaria deaths have been decreasing over the last 10-15 years, with global mortality rates having fallen by 47% since 2000. While the World Health Organization (WHO) recommends the use of artemisinin-based combination therapies (ACTs) to combat malaria, the emergence of artemisinin resistant strains underscores the need to develop new antimalarial drugs. Recent in vivo efficacy improvements of the historical antimalarial ICI 56,780 have been reported, however, with the poor solubility and rapid development of resistance, this compound requires further optimization. A series of piperazine-containing 4(1H)-quinolones with greatly enhanced solubility were developed utilizing structure-activity relationship (SAR) and structure-property relationship (SPR) studies. Furthermore, promising compounds were chosen for an in vivo scouting assay to narrow selection for testing in an in vivo Thompson test. Finally, two piperazine-containing 4(1H)-quinolones were curative in the conventional Thompson test and also displayed in vivo activity against the liver stages of the parasite.

Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors.

The development of a new series of apoptosis signal-regulating kinase 1 (ASK1) inhibitors is described. Starting from purine, pyrimidine and quinazoline scaffolds identified by high throughput screening, we used tools of structure-based drug design to develop a series of potent kinase inhibitors, including 2-arylquinazoline derivatives 12 and 23, with submicromolar inhibitory activities against ASK1. Kinetic analysis demonstrated that the 2-arylquinazoline scaffold ASK1 inhibitors described herein are ATP competitive.

Metal-free arylation of ethyl acetoacetate with hypervalent diaryliodonium salts: an immediate access to diverse 3-aryl-4(1H)-quinolones.

A clean arylation protocol of ethyl acetoacetate was developed using hypervalent diaryliodonium salts under mild and metal-free conditions. The scope of the reaction, using symmetric and unsymmetric iodonium salts with varying sterics and electronics, was examined. Further, this method has been applied for the synthesis of antimalarial compound ELQ-300, which is currently in preclinical development.

Orally bioavailable 6-chloro-7-methoxy-4(1H)-quinolones efficacious against multiple stages of Plasmodium.

The continued proliferation of malaria throughout temperate and tropical regions of the world has promoted a push for more efficacious treatments to combat the disease. Unfortunately, more recent remedies such as artemisinin combination therapies have been rendered less effective due to developing parasite resistance, and new drugs are required that target the parasite in the liver to support the disease elimination efforts. Research was initiated to revisit antimalarials developed in the 1940s and 1960s that were deemed unsuitable for use as therapeutic agents as a result of poor understanding of both physicochemical properties and parasitology. Structure-activity and structure-property relationship studies were conducted to generate a set of compounds with the general 6-chloro-7-methoxy-2-methyl-4(1H)-quinolone scaffold which were substituted at the 3-position with a variety of phenyl moieties possessing various properties. Extensive physicochemical evaluation of the quinolone series was carried out to downselect the most promising 4(1H)-quinolones, 7, 62, 66, and 67, which possessed low-nanomolar EC50 values against W2 and TM90-C2B as well as improved microsomal stability. Additionally, in vivo Thompson test results using Plasmodium berghei in mice showed that these 4(1H)-quinolones were efficacious for the reduction of parasitemia at >99% after 6 days.

4(1H)-pyridone and 4(1H)-quinolone derivatives as antimalarials with erythrocytic, exoerythrocytic, and transmission blocking activities.

Infectious diseases are the second leading cause of deaths in the world with malaria being responsible for approximately the same amount of deaths as cancer in 2012. Despite the success in malaria prevention and control measures decreasing the disease mortality rate by 45% since 2000, the development of single-dose therapeutics with radical cure potential is required to completely eradicate this deadly condition. Targeting multiple stages of the malaria parasite is becoming a primary requirement for new candidates in antimalarial drug discovery and development. Recently, 4(1H)- pyridone, 4(1H)-quinolone, 1,2,3,4-tetrahydroacridone, and phenoxyethoxy-4(1H)-quinolone chemotypes have been shown to be antimalarials with blood stage activity, liver stage activity, and transmission blocking activity. Advancements in structure-activity relationship and structure-property relationship studies, biological evaluation in vitro and in vivo, as well as pharmacokinetics of the 4(1H)-pyridone and 4(1H)-quinolone chemotypes are discussed.

4(1H)-Quinolones with liver stage activity against Plasmodium berghei.

With the exception of primaquine, tafenoquine, and atovaquone, there are very few antimalarials that target liver stage parasites. In this study, a transgenic Plasmodium berghei parasite (1052Cl1; PbGFP-Luc(con)) that expresses luciferase was used to assess the anti-liver stage parasite activity of ICI 56,780, a 7-(2-phenoxyethoxy)-4(1H)-quinolone (PEQ), as well as two 3-phenyl-4(1H)-quinolones (P4Q), P4Q-146 and P4Q-158, by using bioluminescent imaging (BLI). Results showed that all of the compounds were active against liver stage parasites; however, ICI 56,780 and P4Q-158 were the most active, with low nanomolar activity in vitro and causal prophylactic activity in vivo. This potent activity makes these compounds ideal candidates for advancement as novel antimalarials.

Endochin optimization: structure-activity and structure-property relationship studies of 3-substituted 2-methyl-4(1H)-quinolones with antimalarial activity.

Since the 1940s endochin and analogues thereof were known to be causal prophylactic and potent erythrocytic stage agents in avian models. Preliminary screening in a current in vitro assay identified several 4(1H)-quinolones with nanomolar EC(50) against erythrocytic stages of multidrug resistant W2 and TM90-C2B isolates of Plasmodium falciparum. Follow-up structure-activity relationship (SAR) studies on 4(1H)-quinolone analogues identified several key features for biological activity. Nevertheless, structure-property relationship (SPR) studies conducted in parallel revealed that 4(1H)-quinolone analogues are limited by poor solubilities and rapid microsomal degradations. To improve the overall efficacy, multiple 4(1H)-quinolone series with varying substituents on the benzenoid quinolone ring and/or the 3-position were synthesized and tested for in vitro antimalarial activity. Several structurally diverse 6-chloro-2-methyl-7-methoxy-4(1H)-quinolones with EC(50) in the low nanomolar range against the clinically relevant isolates W2 and TM90-C2B were identified with improved physicochemical properties while maintaining little to no cross-resistance with atovaquone.