A Covalent Chemical Probe for nsP2 Cysteine Protease with Antialphaviral Activity and Proteome-wide Selectivity.

RA-0003022 () was identified as a high-quality covalent chemical probe for nsP2 cysteine protease (nsP2pro). Isoxazole covalently captured the active site C478 and inactivated the enzyme with a / ratio of 6000 Ms. A negative control analog RA-0025453 () retained the covalent warhead but demonstrated >100-fold decrease in enzyme inhibition.

Rational Exploration of 2,4-Diaminopyrimidines as DHFR Inhibitors Active against and , Two Emerging Human Pathogens.

Nontuberculous mycobacteria (NTM) are emerging human pathogens linked to severe pulmonary diseases. Current treatments involve the prolonged use of multiple drugs and are often ineffective. Bacterial dihydrofolate reductase (DHFR) is a key enzyme targeted by antibiotics in Gram-negative bacterial infections.

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.

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.

(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.

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.

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.

Identification of potential inhibitors of casein kinase 2 alpha of with potent 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.

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 were developed based on the C-6 metalation of suitable 2,4-dichloro-5-alkoxy pyrimidines using (TMP)Zn·2MgCl·2LiCl base. One approach involves a late-stage modification of the C-6 position, while the other allows for tail fragment modification of . Both routes have proven reliable in synthesizing , as well as eight analogues.

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.

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.

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.

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, , targeting a poorly conserved cysteine in the kinase's hinge region.

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.

Structure of the PknF and conformational changes induced in forkhead-associated regulatory domains.

() 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.

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.

Identification of P218 as a potent inhibitor of DHFR.

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 biosynthesis of folate species and is a validated target for several antimicrobials.

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.

Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major.

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively.

Mitochondrial uncoupling protein-dependent signaling in plant bioenergetics and stress response.

The biological function of plant mitochondrial uncoupling proteins (pUCPs) has been a matter of considerable controversy. For example, the pUCP capacity to uncouple respiration from ATP synthesis in vivo has never been fully acknowledged, in contrast to the mammalian UCP1 (mUCP1) role in uncoupling respiration-mediated thermogenesis. Interestingly, both pUCPs and mUCPs have been associated with stress response and metabolic perturbations.