Metabolic RNA labeling in non-engineered cells following spontaneous uptake of fluorescent nucleoside phosphate analogues.

RNA and its building blocks play central roles in biology and have become increasingly important as therapeutic agents and targets. Hence, probing and understanding their dynamics in cells is important. Fluorescence microscopy offers live-cell spatiotemporal monitoring but requires labels. We present two fluorescent adenine analogue nucleoside phosphates which show spontaneous uptake and accumulation in cultured human cells, likely via nucleoside transporters, and show their potential utilization as cellular RNA labels. Upon uptake, one nucleotide analogue, 2CNqAXP, localizes to the cytosol and the nucleus. We show that it could then be incorporated into de novo synthesized cellular RNA, i.e. it was possible to achieve metabolic fluorescence RNA labeling without using genetic engineering to enhance incorporation, uptake-promoting strategies, or post-labeling through bio-orthogonal chemistries. By contrast, another nucleotide analogue, pAXP, only accumulated outside of the nucleus and was rapidly excreted. Consequently, this analogue did not incorporate into RNA. This difference in subcellular accumulation and retention results from a minor change in nucleobase chemical structure. This demonstrates the importance of careful design of nucleoside-based drugs, e.g. antivirals to direct their subcellular localization, and shows the potential of fine-tuning fluorescent base analogue structures to enhance the understanding of the function of such drugs.

An automated positive selection screen in yeast provides support for boron-containing compounds as inhibitors of SARS-CoV-2 main protease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus continues to cause severe disease and deaths in many parts of the world, despite massive vaccination efforts. Antiviral drugs to curb an ongoing infection remain a priority. The virus-encoded 3C-like main protease (MPro; nsp5) is seen as a promising target. Here, with a positive selection genetic system engineered in Saccharomyces cerevisiae using cleavage and release of MazF toxin as an indicator, we screened in a robotized setup small molecule libraries comprising ~2,500 compounds for MPro inhibitors. We detected eight compounds as effective against MPro expressed in yeast, five of which are characterized proteasome inhibitors. Molecular docking indicates that most of these bind covalently to the MPro catalytically active cysteine. Compounds were confirmed as MPro inhibitors in an in vitro enzymatic assay. Among those were three previously only predicted in silico; the boron-containing proteasome inhibitors bortezomib, delanzomib, and ixazomib. Importantly, we establish reaction conditions in vitro preserving the MPro-inhibitory activity of the boron-containing drugs. These differ from the standard conditions, which may explain why boron compounds have gone undetected in screens based on enzymatic in vitro assays. Our screening system is robust and can find inhibitors of a specific protease that are biostable, able to penetrate a cell membrane, and are not generally toxic. As a cellular assay, it can detect inhibitors that fail in a screen based on an in vitro enzymatic assay using standardized conditions, and now give support for boron compounds as MPro inhibitors. This method can also be adapted for other viral proteases.IMPORTANCEThe coronavirus disease 2019 (COVID-19) pandemic triggered the realization that we need flexible approaches to find treatments for emerging viral threats. We implemented a genetically engineered platform in yeast to detect inhibitors of the virus's main protease (MPro), a promising target to curb severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Screening molecule libraries, we identified candidate inhibitors and verified them in a biochemical assay. Moreover, the system detected boron-containing molecules as MPro inhibitors. Those were previously predicted computationally but never shown effective in a biochemical assay. Here, we demonstrate that they require a non-standard reaction buffer to function as MPro inhibitors. Hence, our cell-based method detects protease inhibitors missed by other approaches and provides support for the boron-containing molecules. We have thus demonstrated that our platform can screen large numbers of chemicals to find potential inhibitors of a viral protease. Importantly, the platform can be modified to detect protease targets from other emerging viruses.

All-photonic kinase inhibitors: light-controlled release-and-report inhibition.

Light-responsive molecular tools targeting kinases affords one the opportunity to study the underlying cellular function of selected kinases. In efforts to externally control lymphocyte-specific protein tyrosine kinase (LCK) activity, the development of release-and-report LCK inhibitors is described, in which (i) the release of the active kinase inhibitor can be controlled externally with light; and (ii) fluorescence is employed to report both the release and binding of the active kinase inhibitor. This introduces an unprecedented all-photonic method for users to both control and monitor real-time inhibitory activity. A functional cellular assay demonstrated light-mediated LCK inhibition in natural killer cells. The use of coumarin-derived caging groups resulted in rapid cellular uptake and non-specific intracellular localisation, while a BODIPY-derived caging group predominately localised in the cellular membrane. This concept of release-and-report inhibitors has the potential to be extended to other biorelevant targets where both spatiotemporal control in a cellular setting and a reporting mechanism would be beneficial.

Exploring Subsite Selectivity within Plasmodium vivax N-Myristoyltransferase Using Pyrazole-Derived Inhibitors.

N-myristoyltransferase (NMT) is a promising antimalarial drug target. Despite biochemical similarities between Plasmodium vivax and human NMTs, our recent research demonstrated that high selectivity is achievable. Herein, we report PvNMT-inhibiting compounds aimed at identifying novel mechanisms of selectivity. Various functional groups are appended to a pyrazole moiety in the inhibitor to target a pocket formed beneath the peptide binding cleft. The inhibitor core group polarity, lipophilicity, and size are also varied to probe the water structure near a channel. Selectivity index values range from 0.8 to 125.3. Cocrystal structures of two selective compounds, determined at 1.97 and 2.43 Å, show that extensions bind the targeted pocket but with different stabilities. A bulky naphthalene moiety introduced into the core binds next to instead of displacing protein-bound waters, causing a shift in the inhibitor position and expanding the binding site. Our structure-activity data provide a conceptual foundation for guiding future inhibitor optimizations.

Sulfone-based human liver pyruvate kinase inhibitors - Design, synthesis and in vitro bioactivity.

Non-alcoholic fatty liver disease (NAFLD) is a prevalent pathological condition characterised by the accumulation of fat in the liver. Almost one-third of the global population is affected by NAFLD, making it a significant health concern. However, despite its prevalence, there is currently no approved drug specifically designed for the treatment of NAFLD. To address this critical gap, researchers have been investigating potential targets for NAFLD drug development. One promising candidate is the liver isoform of pyruvate kinase (PKL). In recent studies, Urolithin C, an allosteric inhibitor of PKL, has emerged as a potential lead compound for therapeutic intervention. Building upon this knowledge, our team has conducted a comprehensive structure-activity relationship of Urolithin C. In this work, we have employed a scaffold-hopping approach, modifying the urolithin structure by replacing the urolithin carbonyl with a sulfone moiety. Our structure-activity relationship analysis has identified the sulfone group as particularly favourable for potent PKL inhibition. Additionally, we have found that the presence of catechol moieties on the two aromatic rings further improves the inhibitory activity. The most promising inhibitor from this new series displayed nanomolar inhibition, boasting an IC50 value of 0.07 µM. This level of potency rivals that of urolithin D and significantly surpasses the effectiveness of urolithin C by an order of magnitude. To better understand the molecular interactions underlying this inhibition, we obtained the crystal structure of one of the inhibitors complexed with PKL. This structural insight served as a valuable reference point, aiding us in the design of inhibitors.

Identification of potent and selective N-myristoyltransferase inhibitors of Plasmodium vivax liver stage hypnozoites and schizonts.

Drugs targeting multiple stages of the Plasmodium vivax life cycle are needed to reduce the health and economic burdens caused by malaria worldwide. N-myristoyltransferase (NMT) is an essential eukaryotic enzyme and a validated drug target for combating malaria. However, previous PvNMT inhibitors have failed due to their low selectivity over human NMTs. Herein, we apply a structure-guided hybridization approach combining chemical moieties of previously reported NMT inhibitors to develop the next generation of PvNMT inhibitors. A high-resolution crystal structure of PvNMT bound to a representative selective hybrid compound reveals a unique binding site architecture that includes a selective conformation of a key tyrosine residue. The hybridized compounds significantly decrease P. falciparum blood-stage parasite load and consistently exhibit dose-dependent inhibition of P. vivax liver stage schizonts and hypnozoites. Our data demonstrate that hybridized NMT inhibitors can be multistage antimalarials, targeting dormant and developing forms of liver and blood stage.

Exploration of Novel Urolithin C Derivatives as Non-Competitive Inhibitors of Liver Pyruvate Kinase.

The inhibition of liver pyruvate kinase could be beneficial to halt or reverse non-alcoholic fatty liver disease (NAFLD), a progressive accumulation of fat in the liver that can lead eventually to cirrhosis. Recently, urolithin C has been reported as a new scaffold for the development of allosteric inhibitors of liver pyruvate kinase (PKL). In this work, a comprehensive structure-activity analysis of urolithin C was carried out. More than 50 analogues were synthesized and tested regarding the chemical features responsible for the desired activity. These data could pave the way to the development of more potent and selective PKL allosteric inhibitors.

Ellagic Acid and Its Metabolites as Potent and Selective Allosteric Inhibitors of Liver Pyruvate Kinase.

Liver pyruvate kinase (PKL) has recently emerged as a new target for non-alcoholic fatty liver disease (NAFLD), and inhibitors of this enzyme could represent a new therapeutic option. However, this breakthrough is complicated by selectivity issues since pyruvate kinase exists in four different isoforms. In this work, we report that ellagic acid (EA) and its derivatives, present in numerous fruits and vegetables, can inhibit PKL potently and selectively. Several polyphenolic analogues of EA were synthesized and tested to identify the chemical features responsible for the desired activity. Molecular modelling studies suggested that this inhibition is related to the stabilization of the PKL inactive state. This unique inhibition mechanism could potentially herald the development of new therapeutics for NAFLD.

Tuning liver pyruvate kinase activity up or down with a new class of allosteric modulators.

The liver isoform of pyruvate kinase (PKL) has gained interest due to its potential capacity to regulate fatty acid synthesis involved in the progression of non-alcoholic fatty liver disease (NAFLD). Here we describe a novel series of PKL modulators that can either activate or inhibit the enzyme allosterically, from a cryptic site at the interface of two protomers in the tetrameric enzyme. Starting from urolithin D, we designed and synthesised 42 new compounds. The effect of these compounds on PKL enzymatic activity was assessed after incubation with cell lysates obtained from a liver cell line. Pronounced activation of PKL activity, up to 3.8-fold, was observed for several compounds at 10 µM, while other compounds were prominent PKL inhibitors reducing its activity to 81% at best. A structure-activity relationship identified linear-shaped sulfone-sulfonamides as activators and non-linear compounds as inhibitors. Crystal structures revealed the conformations of these modulators, which were used as a reference for designing new modulators.

Serendipitous Identification of a Covalent Activator of Liver Pyruvate Kinase.

Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50 =0.29 µM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.

Development of Dicationic Bisguanidine-Arylfuran Derivatives as Potent Agents against Gram-Negative Bacteria.

Antibiotic resistance among bacteria is a growing global challenge. A major reason for this is the limited progress in developing new classes of antibiotics active against Gram-negative bacteria. Here, we investigate the antibacterial activity of a dicationic bisguanidine-arylfuran, originally developed as an antitrypanosomal agent, and new derivatives thereof. The compounds showed good activity (EC50 2-20 µM) against antibiotic-resistant isolates of the Gram-negative members of the ESKAPE group (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Escherichia coli with different antibiotic susceptibility patterns, including ESBL isolates. Cytotoxicity was moderate, and several of the new derivatives were less cytotoxic than the lead molecule, offering better selectivity indices (40-80 for several ESKAPE isolates). The molecular mechanism for the antibacterial activity of these molecules is unknown, but sensitivity profiling against human ESKAPE isolates and E. coli collections with known susceptibility patterns against established antibiotics indicates that it is distinct from lactam and quinolone antibiotics.

Design and development of photoswitchable DFG-Out RET kinase inhibitors.

REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase that is required for development of multiple human tissues, but which is also an important contributor to human cancers. RET activation through rearrangement or point mutations occurs in thyroid and lung cancers. Furthermore, activation of wild type RET is an increasingly recognized mechanism promoting tumor growth and dissemination of a much broader group of cancers. RET is therefore an attractive therapeutic target for small-molecule kinase inhibitors. Non-invasive control of RET signaling with light offers the promise of unveiling its complex spatiotemporal dynamics in vivo. In this work, photoswitchable DFG-out RET kinase inhibitors based on heterocycle-derived azobenzenes were developed, enabling photonic control of RET activity. Based on the binding mode of DFG-out kinase inhibitors and using RET kinase as the test model, we developed a photoswitchable inhibitor with a quinoline "head" constituting the azoheteroarene. This azo compound was further modified by three different strategies to increase the difference in biological activity between the E-isomer and the light enriched Z-isomer. Stilbene-based derivatives were used as model compounds to guide in the selection of substituents that could eventually be introduced to the corresponding azo compounds. The most promising quinoline-based compound showed more than a 15-fold difference in bioactivity between the two isomers in a biochemical assay. However, the same compound showed a decreased Z/E (IC50) ratio in the cellular assay, tentatively assigned to stability issues. The corresponding stilbene compound gave a Z/E (IC50) ratio well above 100, consistent with that measured in the biochemical assay. Ultimately, a 7-azaindole based photoswitchable DFG-out kinase inhibitor was shown to display more than a 10-fold difference in bioactivity between the two isomers, in both a biochemical and a cell-based assay, as well as excellent stability even under reducing conditions.

Anthraquinone derivatives as ADP-competitive inhibitors of liver pyruvate kinase.

Liver pyruvate kinase (PKL) is a major regulator of metabolic flux and ATP production during liver cell glycolysis and is considered a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD). In this study, we report the first ADP-competitive PKL inhibitors and identify several starting points for the further optimization of these inhibitors. Modeling and structural biology guided the optimization of a PKL-specific anthraquinone-based compound. A structure-activity relationship study of 47 novel synthetic derivatives revealed PKL inhibitors with half-maximal inhibitory concentration (IC50) values in the 200 nM range. Despite the difficulty involved in studying a binding site as exposed as the ADP site, these derivatives feature expanded structural diversity and chemical spaces that may be used to improve their inhibitory activities against PKL. The obtained results expand the knowledge of the structural requirements for interactions with the ADP-binding site of PKL.

A Genetic Trap in Yeast for Inhibitors of SARS-CoV-2 Main Protease.

The ongoing COVID-19 pandemic urges searches for antiviral agents that can block infection or ameliorate its symptoms. Using dissimilar search strategies for new antivirals will improve our overall chances of finding effective treatments. Here, we have established an experimental platform for screening of small molecule inhibitors of the SARS-CoV-2 main protease in Saccharomyces cerevisiae cells, genetically engineered to enhance cellular uptake of small molecules in the environment. The system consists of a fusion of the Escherichia coli toxin MazF and its antitoxin MazE, with insertion of a protease cleavage site in the linker peptide connecting the MazE and MazF moieties. Expression of the viral protease confers cleavage of the MazEF fusion, releasing the MazF toxin from its antitoxin, resulting in growth inhibition. In the presence of a small molecule inhibiting the protease, cleavage is blocked and the MazF toxin remains inhibited, promoting growth. The system thus allows positive selection for inhibitors. The engineered yeast strain is tagged with a fluorescent marker protein, allowing precise monitoring of its growth in the presence or absence of inhibitor. We detect an established main protease inhibitor by a robust growth increase, discernible down to 1 µM. The system is suitable for robotized large-scale screens. It allows in vivo evaluation of drug candidates and is rapidly adaptable for new variants of the protease with deviant site specificities. IMPORTANCE The COVID-19 pandemic may continue for several years before vaccination campaigns can put an end to it globally. Thus, the need for discovery of new antiviral drug candidates will remain. We have engineered a system in yeast cells for the detection of small molecule inhibitors of one attractive drug target of SARS-CoV-2, its main protease, which is required for viral replication. The ability to detect inhibitors in live cells brings the advantage that only compounds capable of entering the cell and remain stable there will score in the system. Moreover, because of its design in yeast cells, the system is rapidly adaptable for tuning the detection level and eventual modification of the protease cleavage site in the case of future mutant variants of the SARS-CoV-2 main protease or even for other proteases.

Design and development of a photoswitchable DFG-out kinase inhibitor.

We report the synthesis and characterisation of a photoswitchable DFG-out kinase inhibitor. Photocontrol of the target kinase in both enzymatic and living cell assays is demonstrated.

Interbase-FRET binding assay for pre-microRNAs.

The aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA-target binding studies.

A Small Molecule Targeting Human MEK1/2 Enhances ERK and p38 Phosphorylation under Oxidative Stress or with Phenothiazines.

Small molecules are routinely used to inhibit protein kinases, but modulators capable of enhancing kinase activity are rare. We have previously shown that the small molecule INR119, designed as an inhibitor of MEK1/2, will enhance the activity of its fission yeast homologue, Wis1, under oxidative stress. To investigate the generality of these findings, we now study the effect of INR119 in human cells under similar conditions. Cells of the established breast cancer line MCF-7 were exposed to H2O2 or phenothiazines, alone or combined with INR119. In line with the previous results in fission yeast, the phosphorylation of the MAPKs ERK and p38 increased substantially more with the combination treatment than by H2O2 or phenothiazines, whereas INR119 alone did not affect phosphorylation. We also measured the mRNA levels of TP53 and BAX, known to be affected by ERK and p38 activity. Similarly, the combination of INR119 and phenothiazines increased both mRNAs to higher levels than for phenothiazines alone. In conclusion, the mechanism of action of INR119 on its target protein kinase may be conserved between yeast and humans.

Discovery of Functional Alternatively Spliced PKM Transcripts in Human Cancers.

Pyruvate kinase muscle type (PKM) is a key enzyme in glycolysis and plays an important oncological role in cancer. However, the association of PKM expression and the survival outcome of patients with different cancers is controversial. We employed systems biology methods to reveal prognostic value and potential biological functions of PKM transcripts in different human cancers. Protein products of transcripts were shown and detected by western blot and mass spectrometry analysis. We focused on different transcripts of PKM and investigated the associations between their mRNA expression and the clinical survival of the patients in 25 different cancers. We find that the transcripts encoding PKM2 and three previously unstudied transcripts, namely ENST00000389093, ENST00000568883, and ENST00000561609, exhibited opposite prognostic indications in different cancers. Moreover, we validated the prognostic effect of these transcripts in an independent kidney cancer cohort. Finally, we revealed that ENST00000389093 and ENST00000568883 possess pyruvate kinase enzymatic activity and may have functional roles in metabolism, cell invasion, and hypoxia response in cancer cells. Our study provided a potential explanation to the controversial prognostic indication of PKM, and could invoke future studies focusing on revealing the biological and oncological roles of these alternative spliced variants of PKM.

Peroxiredoxin promotes longevity and H2O2-resistance in yeast through redox-modulation of protein kinase A.

Peroxiredoxins are H2O2 scavenging enzymes that also carry out H2O2 signaling and chaperone functions. In yeast, the major cytosolic peroxiredoxin, Tsa1 is required for both promoting resistance to H2O2 and extending lifespan upon caloric restriction. We show here that Tsa1 effects both these functions not by scavenging H2O2, but by repressing the nutrient signaling Ras-cAMP-PKA pathway at the level of the protein kinase A (PKA) enzyme. Tsa1 stimulates sulfenylation of cysteines in the PKA catalytic subunit by H2O2 and a significant proportion of the catalytic subunits are glutathionylated on two cysteine residues. Redox modification of the conserved Cys243 inhibits the phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity, and preventing Thr241 phosphorylation can overcome the H2O2 sensitivity of Tsa1-deficient cells. Results support a model of aging where nutrient signaling pathways constitute hubs integrating information from multiple aging-related conduits, including a peroxiredoxin-dependent response to H2O2.

Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor.

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Förster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.