Discovery of 3-Amino-1H-pyrazole-Based Kinase Inhibitors to Illuminate the Understudied PCTAIRE Family.

The PCTAIRE subfamily belongs to the CDK (cyclin-dependent kinase) family and represents an understudied class of kinases of the dark kinome. They exhibit a highly conserved binding pocket and are activated by cyclin Y binding. CDK16 is targeted to the plasma membrane after binding to N-myristoylated cyclin Y and is highly expressed in post-mitotic tissues, such as the brain and testis. Dysregulation is associated with several diseases, including breast, prostate, and cervical cancer. Here, we used the N-(1H-pyrazol-3-yl)pyrimidin-4-amine moiety from the promiscuous inhibitor 1 to target CDK16, by varying different residues. Further optimization steps led to 43d, which exhibited high cellular potency for CDK16 (EC50 = 33 nM) and the other members of the PCTAIRE and PFTAIRE family with 20-120 nM and 50-180 nM, respectively. A DSF screen against a representative panel of approximately 100 kinases exhibited a selective inhibition over the other kinases. In a viability assessment, 43d decreased the cell count in a dose-dependent manner. A FUCCI cell cycle assay revealed a G2/M phase cell cycle arrest at all tested concentrations for 43d, caused by inhibition of CDK16.

CETSA MS Profiling for a Comparative Assessment of FDA-Approved Antivirals Repurposed for COVID-19 Therapy Identifies TRIP13 as a Remdesivir Off-Target.

The reuse of preexisting small molecules for a novel emerging disease threat is a rapid measure to discover unknown applications for previously validated therapies. A pertinent and recent example where such a strategy could be employed is in the fight against coronavirus disease 2019 (COVID-19). Therapies designed or discovered to target viral proteins also have off-target effects on the host proteome when employed in a complex physiological environment. This study aims to assess these host cell targets for a panel of FDA-approved antiviral compounds including remdesivir, using the cellular thermal shift assay (CETSA) coupled with mass spectrometry (CETSA MS) in noninfected cells. CETSA MS is a powerful method to delineate direct and indirect interactions between small molecules and protein targets in intact cells. Biologically active compounds can induce changes in thermal stability, in their primary binding partners, and in proteins that in turn interact with the direct targets. Such engagement of host targets by antiviral drugs may contribute to the clinical effect against the virus but can also constitute a liability. We present here a comparative study of CETSA molecular target engagement fingerprints of antiviral drugs to better understand the link between off-targets and efficacy.

Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1.

Methods to measure cellular target engagement are increasingly being used in early drug discovery. The Cellular Thermal Shift Assay (CETSA) is one such method. CETSA can investigate target engagement by measuring changes in protein thermal stability upon compound binding within the intracellular environment. It can be performed in high-throughput, microplate-based formats to enable broader application to early drug discovery campaigns, though high-throughput forms of CETSA have only been reported for a limited number of targets. CETSA offers the advantage of investigating the target of interest in its physiological environment and native state, but it is not clear yet how well this technology correlates to more established and conventional cellular and biochemical approaches widely used in drug discovery. We report two novel high-throughput CETSA (CETSA HT) assays for B-Raf and PARP1, demonstrating the application of this technology to additional targets. By performing comparative analyses with other assays, we show that CETSA HT correlates well with other screening technologies and can be applied throughout various stages of hit identification and lead optimization. Our results support the use of CETSA HT as a broadly applicable and valuable methodology to help drive drug discovery campaigns to molecules that engage the intended target in cells.

Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site.

The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the crystal structure of inhibitor-bound hFEN1, which shows a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein-substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the necessary unpairing of substrate DNA. Other compounds were more competitive with substrate. Cellular thermal shift data showed that both inhibitor types engaged with hFEN1 in cells, and activation of the DNA damage response was evident upon treatment with inhibitors. However, cellular EC50 values were significantly higher than in vitro inhibition constants, and the implications of this for exploitation of hFEN1 as a drug target are discussed.

The Cellular Thermal Shift Assay: A Novel Biophysical Assay for In Situ Drug Target Engagement and Mechanistic Biomarker Studies.

A drug must engage its intended target to achieve its therapeutic effect. However, conclusively measuring target engagement (TE) in situ is challenging. This complicates preclinical development and is considered a key factor in the high rate of attrition in clinical trials. Here, we discuss a recently developed, label-free, biophysical assay, the cellular thermal shift assay (CETSA), which facilitates the direct assessment of TE in cells and tissues at various stages of drug development. CETSA also reveals biochemical events downstream of drug binding and therefore provides a promising means of establishing mechanistic biomarkers. The implementation of proteome-wide CETSA using quantitative mass spectrometry represents a novel strategy for defining off-target toxicity and polypharmacology and for identifying downstream mechanistic biomarkers. The first year of CETSA applications in the literature has focused on TE studies in cell culture systems and has confirmed the broad applicability of CETSA to many different target families. The next phase of CETSA applications will likely encompass comprehensive animal and patient studies, and CETSA will likely serve as a very valuable tool in many stages of preclinical and clinical drug development.

Tracking cancer drugs in living cells by thermal profiling of the proteome.

The thermal stability of proteins can be used to assess ligand binding in living cells. We have generalized this concept by determining the thermal profiles of more than 7000 proteins in human cells by means of mass spectrometry. Monitoring the effects of small-molecule ligands on the profiles delineated more than 50 targets for the kinase inhibitor staurosporine. We identified the heme biosynthesis enzyme ferrochelatase as a target of kinase inhibitors and suggest that its inhibition causes the phototoxicity observed with vemurafenib and alectinib. Thermal shifts were also observed for downstream effectors of drug treatment. In live cells, dasatinib induced shifts in BCR-ABL pathway proteins, including CRK/CRKL. Thermal proteome profiling provides an unbiased measure of drug-target engagement and facilitates identification of markers for drug efficacy and toxicity.

Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay.

The efficacy of therapeutics is dependent on a drug binding to its cognate target. Optimization of target engagement by drugs in cells is often challenging, because drug binding cannot be monitored inside cells. We have developed a method for evaluating drug binding to target proteins in cells and tissue samples. This cellular thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. Using this assay, we validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects and drug resistance in cancer cell lines, as well as drug distribution in tissues. CETSA is likely to become a valuable tool for the validation and optimization of drug target engagement.

Arginine 104 is a key catalytic residue in leukotriene C4 synthase.

Human leukotriene C(4) synthase (hLTC(4)S) is an integral membrane enzyme that conjugates leukotriene (LT) A(4) with glutathione to form LTC(4), a precursor to the cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) that are involved in the pathogenesis of human bronchial asthma. From the crystal structure of hLTC(4)S, Arg-104 and Arg-31 have been implicated in the conjugation reaction. Here, we used site-directed mutagenesis, UV spectroscopy, and x-ray crystallography to examine the catalytic role of Arg-104 and Arg-31. Exchange of Arg-104 with Ala, Ser, Thr, or Lys abolished 94.3-99.9% of the specific activity against LTA(4). Steady-state kinetics of R104A and R104S revealed that the K(m) for GSH was not significantly affected. UV difference spectra of the binary enzyme-GSH complex indicated that GSH ionization depends on the presence of Arg-104 because no thiolate signal, with λ(max) at 239 nm, could be detected using R104A or R104S hLTC(4)S. Apparently, the interaction of Arg-104 with the thiol group of GSH reduces its pK(a) to allow formation of a thiolate anion and subsequent nucleophilic attack at C6 of LTA(4). On the other hand, exchange of Arg-31 with Ala or Glu reduced the catalytic activity of hLTC(4)S by 88 and 70%, respectively, without significantly affecting the k(cat)/K(m) values for GSH, and a crystal structure of R31Q hLTC(4)S (2.1 Å) revealed a Gln-31 side chain pointing away from the active site. We conclude that Arg-104 plays a critical role in the catalytic mechanism of hLTC(4)S, whereas a functional role of Arg-31 seems more elusive. Because Arg-104 is a conserved residue, our results pertain to other homologous membrane proteins and represent a structure-function paradigm probably common to all microsomal GSH transferases.

Catalysis within the lipid bilayer-structure and mechanism of the MAPEG family of integral membrane proteins.

Integral membrane enzymes of the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) family catalyze glutathione-dependent transformations of lipophilic substrates harvested from the lipid bilayer. Recent studies of members of this family have yielded extensive insights into the structural basis for their substrate binding and catalytic activity. Most informative are the structural studies of leukotriene C4 synthase, revealing a narrow hydrophobic substrate binding pocket allowing extensive recognition of the aliphatic chain of the LTA(4) substrate. A key feature of the pocket is a tryptophan residue that pins down the omega-end of the aliphatic chain into the active site. Since MAPEG members cannot utilize a hydrophobic effect for substrate binding, this novel mode of substrate recognition appears well suited for harvesting lipophilic substrates from the membrane. The binding mode also allows for the specific alignment of the substrate in the active site, positioning the C6 of the substrate for conjugation with glutathione. The glutathione is in turn bound in a polar pocket submerged into the protein core. Structure-based sequence alignments of human MAPEG members support the notion that the glutathione binding site is highly conserved among MAPEG enzymes and that they use a similar mechanism for glutathione activation.

Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase.

Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase: this reaction is the key step in cysteinyl leukotriene formation. Here we present the crystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 A resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a horseshoe-shaped conformation on GSH, and effectively positions the thiol group for activation by a nearby arginine at the membrane-enzyme interface. In addition, the structure provides a model for how the omega-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular 'ruler' to align the reactive epoxide at the thiol of glutathione. This provides new structural insights into the mechanism of LTC4 formation, and also suggests that the observed binding and activation of GSH might be common for a family of homologous proteins important for inflammatory and detoxification responses.