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.

(S)-ML-SA1 ACTIVATES AUTOPHAGY VIA TRPML1-TFEB PATHWAY.

Autophagic flux plays a crucial role in various diseases. Recently, the lysosomal ion channel TRPML1 has emerged as a promising target in lysosomal storage diseases, such as mucolipidosis. The discovery of mucolipin synthetic agonist-1 (ML-SA1) has expanded our understanding of TRPML1's function and its potential therapeutic uses. However, ML-SA1 is a racemate with limited cellular potency and poor water solubility. In this study, we synthetized rac-ML-SA1, separated the enantiomers by chiral liquid chromatography and determined their absolute configuration by vibrational circular dichroism (VCD). In addition, we focused on investigating the impact of each enantiomer of ML-SA1 on the TRPML1-TFEB axis. Our findings revealed that (S)-ML-SA1 acts as an agonist for TRPML1 at the lysosomal membrane. This activation prompts transcription factor EB (TFEB) to translocate from the cytosol to the nucleus in a dose-dependent manner within live cells. Consequently, this signaling pathway enhances the expression of coordinated lysosomal expression and regulation (CLEAR) genes and activates autophagic flux. Our study presents evidence for the potential use of (S)-ML-SA1 in the development of new therapies for lysosomal storage diseases that target TRPML1.

Novel Dihydropteridinone Derivatives As Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 1δ/ε.

Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Discovery of pyrazolo[3,4-d]pyrimidines as novel mitogen-activated protein kinase kinase 3 (MKK3) inhibitors.

The dual-specificity protein kinase MKK3 has been implicated in tumor cell proliferation and survival, yet its precise role in cancer remains inconclusive. A critical step in elucidating the kinase's involvement in disease biology is the identification of potent, cell-permeable kinase inhibitors. Presently, MKK3 lacks a dedicated tool compound for these purposes, along with validated methods for the facile screening, identification, and optimization of inhibitors. In this study, we have developed a TR-FRET-based enzymatic assay for the detection of MKK3 activity in vitro and a BRET-based assay to assess ligand binding to this enzyme within intact human cells. These assays were instrumental in identifying hit compounds against MKK3 that share a common chemical scaffold, sourced from a library of bioactive kinase inhibitors. Initial hits were subsequently expanded through the synthesis of novel analogs. The resulting structure-activity relationship (SAR) was rationalized using molecular dynamics simulations against a homology model of MKK3. We expect our findings to expedite the development of novel, potent, selective, and bioactive inhibitors, thus facilitating investigations into MKK3's role in various cancers.

Identification of potential inhibitors of casein kinase 2 alpha of Plasmodium falciparum with potent in vitro activity.

Drug resistance to commercially available antimalarials is a major obstacle in malaria control and elimination, creating the need to find new antiparasitic compounds with novel mechanisms of action. The success of kinase inhibitors for oncological treatments has paved the way for the exploitation of protein kinases as drug targets in various diseases, including malaria. Casein kinases are ubiquitous serine/threonine kinases involved in a wide range of cellular processes such as mitotic checkpoint signaling, DNA damage response, and circadian rhythm. In Plasmodium, it is suggested that these protein kinases are essential for both asexual and sexual blood-stage parasites, reinforcing their potential as targets for multi-stage antimalarials. To identify new putative PfCK2α inhibitors, we utilized an in silico chemogenomic strategy involving virtual screening with docking simulations and quantitative structure-activity relationship predictions. Our investigation resulted in the discovery of a new quinazoline molecule (542), which exhibited potent activity against asexual blood stages and a high selectivity index (>100). Subsequently, we conducted chemical-genetic interaction analysis on yeasts with mutations in casein kinases. Our chemical-genetic interaction results are consistent with the hypothesis that 542 inhibits yeast Cka1, which has a hinge region with high similarity to PfCK2α. This finding is in agreement with our in silico results suggesting that 542 inhibits PfCK2α via hinge region interaction.

Functionalization of 2,4-Dichloropyrimidines by 2,2,6,6-Tetramethylpiperidyl Zinc Base Enables Modular Synthesis of Antimalarial Diaminopyrimidine P218 and Analogues.

Two routes to the antimalarial diaminopyrimidine P218 were developed based on the C-6 metalation of suitable 2,4-dichloro-5-alkoxy pyrimidines using (TMP)2Zn·2MgCl2·2LiCl base. One approach involves a late-stage modification of the C-6 position, while the other allows for tail fragment modification of P218. Both routes have proven reliable in synthesizing P218, as well as eight analogues. These innovative strategies have the potential to contribute to the search for new antimalarial drugs.

Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation.

The TcK2 protein kinase of Trypanosoma cruzi, the causative agent of Chagas disease, is structurally similar to the human kinase PERK, which phosphorylates the initiation factor eIF2α and, in turn, inhibits translation initiation. We have previously shown that absence of TcK2 kinase impairs parasite proliferation within mammalian cells, positioning it as a potential target for treatment of Chagas disease. To better understand its role in the parasite, here we initially confirmed the importance of TcK2 in parasite proliferation by generating CRISPR/Cas9 TcK2-null cells, albeit they more efficiently differentiate into infective forms. Proteomics indicates that the TcK2 knockout of proliferative forms expresses proteins including trans-sialidases, normally restricted to infective and nonproliferative trypomastigotes explaining decreased proliferation and better differentiation. TcK2 knockout cells lost phosphorylation of eukaryotic initiation factor 3 and cyclic AMP responsive-like element, recognized to promote growth, likely explaining both decreased proliferation and augmented differentiation. To identify specific inhibitors, a library of 379 kinase inhibitors was screened by differential scanning fluorimetry using a recombinant TcK2 encompassing the kinase domain and selected molecules were tested for kinase inhibition. Only Dasatinib and PF-477736, inhibitors of Src/Abl and ChK1 kinases, showed inhibitory activity with IC50 of 0.2 ± 0.02 mM and 0.8 ± 0.1, respectively. In infected cells Dasatinib inhibited growth of parental amastigotes (IC50 = 0.6 ± 0.2 mM) but not TcK2 of depleted parasites (IC50 > 34 mM) identifying Dasatinib as a potential lead for development of therapeutics for Chagas disease targeting TcK2.

A novel BRET-based assay to investigate binding and residence time of unmodified ligands to the human lysosomal ion channel TRPML1 in intact cells.

Here, we report a bioluminescence resonance energy transfer (BRET) assay as a novel way to investigate the binding of unlabeled ligands to the human transient receptor potential mucolipin 1 (hTRPML1), a lysosomal ion channel involved in several genetic diseases and cancer progression. This novel BRET assay can be used to determine equilibrium and kinetic binding parameters of unlabeled compounds to hTRPML1 using intact human-derived cells, thus complementing the information obtained using functional assays based on ion channel activation. We expect this new BRET assay to expedite the identification and optimization of cell-permeable ligands that interact with hTRPML1 within the physiologically relevant environment of lysosomes.

SGC-CAMKK2-1: A Chemical Probe for CAMKK2.

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

An open source plant kinase chemogenomics set.

One hundred twenty-nine protein kinases, selected to represent the diversity of the rice (Oryza sativa) kinome, were cloned and tested for expression in Escherichia coli. Forty of these rice kinases were purified and screened using differential scanning fluorimetry (DSF) against 627 diverse kinase inhibitors, with a range of structures and activities targeting diverse human kinases. Thirty-seven active compounds were then tested for their ability to modify primary root development in Arabidopsis. Of these, 14 compounds caused a significant reduction of primary root length compared with control plants. Two of these inhibitory compounds bind to the predicted orthologue of Arabidopsis PSKR1, one of two receptors for PSK, a small sulfated peptide that positively controls root development. The reduced root length phenotype could not be rescued by the exogenous addition of the PSK peptide, suggesting that chemical treatment may inhibit both PSKR1 and its closely related receptor PSKR2. Six of the compounds acting as root growth inhibitors in Arabidopsis conferred the same effect in rice. Compound RAF265 (CHIR-265), previously shown to bind the human kinase BRAF (B-Raf proto-oncogene, serine/threonine kinase), also binds to nine highly conserved rice kinases tested. The binding of human and rice kinases to the same compound suggests that human kinase inhibitor sets will be useful for dissecting the function of plant kinases.

A Target Engagement Assay to Assess Uptake, Potency, and Retention of Antibiotics in Living Bacteria.

New antibiotics are urgently needed to counter the emergence of antimicrobial-resistant pathogenic bacteria. A major challenge in antibiotic drug discovery is to turn potent biochemical inhibitors of essential bacterial components into effective antimicrobials. This difficulty is underpinned by a lack of methods to investigate the physicochemical properties needed for candidate antibiotics to permeate the bacterial cell envelope and avoid clearance by the action of bacterial efflux pumps. To address these issues, here we used a target engagement assay to measure the equilibrium and kinetic binding parameters of antibiotics targeting dihydrofolate reductase (DHFR) in live bacteria. We also used this assay to identify novel DHFR ligands having antimicrobial activity. We validated this approach using the Gram-negative bacteria Escherichia coli and the emerging human pathogen Mycobacterium abscessus. We expect the use of target engagement assays in bacteria to expedite the discovery and progression of novel, cell-permeable antibiotics with on-target activity.

Discovery of novel benzothiophene derivatives as potent and narrow spectrum inhibitors of DYRK1A and DYRK1B.

The discovery of potent and selective inhibitors for understudied kinases can provide relevant pharmacological tools to illuminate their biological functions. DYRK1A and DYRK1B are protein kinases linked to chronic human diseases. Current DYRK1A/DYRK1B inhibitors also antagonize the function of related protein kinases, such as CDC2-like kinases (CLK1, CLK2, CLK4) and DYRK2. Here, we reveal narrow spectrum dual inhibitors of DYRK1A and DYRK1B based on a benzothiophene scaffold. Compound optimization exploited structural differences in the ATP-binding sites of the DYRK1 kinases and resulted in the discovery of 3n, a potent and cell-permeable DYRK1A/DYRK1B inhibitor. This compound has a different scaffold and a narrower off-target profile compared to current DYRK1A/DYRK1B inhibitors. We expect the benzothiophene derivatives described here to aid establishing DYRK1A/DYRK1B cellular functions and their role in human pathologies.

Development of dihydropyrrolopyridinone-based PKN2/PRK2 chemical tools to enable drug discovery.

The Protein Kinase N proteins (PKN1, PKN2 and PKN3) are Rho GTPase effectors. They are involved in several biological processes such as cytoskeleton organization, cell mobility, adhesion, and cell cycle. Recently PKNs have been reported as essential for survival in several tumor cell lines, including prostate and breast cancer. Here, we report the development of dihydropyrrolopyridinone-based inhibitors for PKN2 and its closest homologue, PKN1, and their associated structure-activity relationship (SAR). Our studies identified a range of molecules with high potency exemplified by compound 8 with Ki = 8 nM for PKN2 and 14x selectivity over PKN1. Membrane permeability and target engagement for PKN2 were assessed by a NanoBRET cellular assay. Importantly, good selectivity across the wider human kinome and other kinase family members was achieved. These compounds provide strong starting points for lead optimization to PKN1/2 development compounds.

Development of the First Covalent Monopolar Spindle Kinase 1 (MPS1/TTK) Inhibitor.

Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint and clinically evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer. While long drug-target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors have been reported. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor, RMS-07, targeting a poorly conserved cysteine in the kinase's hinge region. RMS-07 shows potent MPS1 inhibitory activity and selectivity against all protein kinases with an equivalent cysteine but also in a broader kinase panel. We demonstrate potent cellular target engagement and pronounced activity against various cancer cell lines. The covalent binding mode was validated by mass spectrometry and an X-ray crystal structure. This proof of MPS1 covalent ligandability may open new avenues for the design of MPS1-specific chemical probes or drugs.

Structure of the Mycobacterium tuberculosis cPknF and conformational changes induced in forkhead-associated regulatory domains.

Mycobacterium tuberculosis (Mtb) has 11 Serine-Threonine Protein Kinases (STPK) that control numerous physiological processes, including cell growth, cell division, metabolic flow, and transcription. PknF is one of the 11 Mtb STPKs that has, among other substrates, two FHA domains (FHA-1 and FHA-2) of the ATP-Binding Cassette (ABC) transporter Rv1747. Phosphorylation in T152 and T210 located in a non-structured linker that connects Rv1747 FHA domains is considerate to be the regulatory mechanism of the transporter. In this work, we resolved the three-dimensional structure of the PknF catalytic domain (cPknF) in complex with the human kinase inhibitor IKK16. cPknF is conserved when compared to other STPKs but shows specific residues in the binding site where the inhibitor is positioned. In addition, using Small Angle X-Ray Scattering analysis we monitored the behavior of the wild type and three FHA-phosphomimetic mutants in solution, and measured the cPknF affinity for these domains. The kinase showed higher affinity for the non-phosphorylated wild type domain and preference for phosphorylation of T152 inducing the rapprochement of the domains and significant structural changes. The results shed some light on the process of regulating the transporter's activity by phosphorylation and arises important questions about evolution and importance of this mechanism for the bacillus.

Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes.

CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934, a total of 32 compounds, composed of single-ring, 5,6-, and 6,6-fused heteroaromatic cores, were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. Compared to GSK650394 and STO-609, 13 compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge-binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs.

Identification of P218 as a potent inhibitor of Mycobacterium ulcerans DHFR.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a debilitating chronic disease that mainly affects the skin. Current treatments for Buruli ulcer are efficacious, but rely on the use of antibiotics with severe side effects. The enzyme dihydrofolate reductase (DHFR) plays a critical role in the de novo biosynthesis of folate species and is a validated target for several antimicrobials. Here we describe the biochemical and structural characterization of M. ulcerans DHFR and identified P218, a safe antifolate compound in clinical evaluation for malaria, as a potent inhibitor of this enzyme. We expect our results to advance M. ulcerans DHFR as a target for future structure-based drug discovery campaigns.

Raising the Bar(-seq) in Leishmania Genetic Screens.

Our understanding of regulatory factors in Leishmania differentiation has long been restricted by the available genetic tools, but the availability of CRISPR/Cas9 has changed the landscape forever. Recently, Baker and Catta-Preta et al. applied Cas9 editing and kinome-wide bar-seq to dissect the function of 204 kinases in the Leishmania mexicana life cycle.

Chemical Space Exploration of Oxetanes.

This paper focuses on new derivatives bearing an oxetane group to extend accessible chemical space for further identification of kinase inhibitors. The ability to modulate kinase activity represents an important therapeutic strategy for the treatment of human illnesses. Known as a nonclassical isoster of the carbonyl group, due to its high polarity and great ability to function as an acceptor of hydrogen bond, oxetane seems to be an attractive and underexplored structural motif in medicinal chemistry.