Theory and applications of differential scanning fluorimetry in early-stage drug discovery

Differential scanning fluorimetry (DSF) is an accessible, rapid, and economical biophysical technique that has seen many applications over the years, ranging from protein folding state detection to the identification of ligands that bind to the target protein. In this review, we discuss the theory, applications, and limitations of DSF, including the latest applications of DSF by ourselves and other researchers. We show that DSF is a powerful high-throughput tool in early drug discovery efforts. We place DSF in the context of other biophysical methods frequently used in drug discovery and highlight their benefits and downsides. We illustrate the uses of DSF in protein buffer optimization for stability, refolding, and crystallization purposes and provide several examples of each. We also show the use of DSF in a more downstream application, where it is used as an in vivo validation tool of ligand-target interaction in cell assays. Although DSF is a potent tool in buffer optimization and large chemical library screens when it comes to ligand-binding validation and optimization, orthogonal techniques are recommended as DSF is prone to false positives and negatives

Structural characterization and extended substrate scope analysis of two Mg<sup>2+</sup>-dependent O-methyltransferases from bacteria

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis in terms of their enzymatic properties and substrate scope and determined their crystal structures. Both OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, hydroxybenzoic acids and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether and 2-methoxyestradiol. Interestingly, certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering.Table of contents Two promiscuous O-methyltransferases from bacteria were found to methylate a panel of catechol substrates towards high-value medicinal compounds. Surprisingly, the non-catechol substrates 5-hydroxyflavonoids and o-hydroxybenzoic acids/aldehydes were also methylated at low conversion rates. The crystal structures reveal potential target sites for enzyme engineering for biocatalytic applications.Competing Interest StatementThe authors have declared no competing interest

Structural Characterization and Extended Substrate Scope Analysis of Two Mg²⁺-Dependent O-Methyltransferases from Bacteria**

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized the enzymatic properties and substrate scope of two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis and determined their crystal structures. Both OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, hydroxybenzoic acids, and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether, and 2-methoxyestradiol. Interestingly, certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering.</p

Engineering a Plant Polyketide Synthase for the Biosynthesis of Methylated Flavonoids

Homoeriodictyol and hesperetin are naturally occurring O-methylated flavonoids with many health-promoting properties. They are produced in plants in low abundance and as complex mixtures of similar compounds that are difficult to separate. Synthetic biology offers the opportunity to produce various flavonoids in a targeted, bottom-up approach in engineered microbes with high product titers. However, the production of O-methylated flavonoids is currently still highly inefficient. In this study, we investigated and engineered a combination of enzymes that had previously been shown to support homoeriodictyol and hesperetin production in Escherichia coli from fed O-methylated hydroxycinnamic acids. We determined the crystal structures of the enzyme catalyzing the first committed step of the pathway, chalcone synthase from Hordeum vulgare, in three ligand-bound states. Based on these structures and a multiple sequence alignment with other chalcone synthases, we constructed mutant variants and assessed their performance in E. coli toward producing methylated flavonoids. With our best mutant variant, HvCHS (Q232P, D234 V), we were able to produce homoeriodictyol and hesperetin at 2 times and 10 times higher titers than reported previously. Our findings will facilitate further engineering of this enzyme toward higher production of methylated flavonoids.</p

Engineering a plant polyketide synthase for the biosynthesis of methylated flavonoids

Homoeriodictyol and hesperetin are naturally occurring O-methylated flavonoids with many health-promoting properties. They are produced in plants in low abundance and as complex mixtures of similar compounds that are difficult to separate. Synthetic biology offers the opportunity to produce non-methylated flavonoids in a targeted, bottom-up approach in engineered microbes with high product titers. However, the production of these O-methylated flavonoids is currently still highly inefficient. In this study, we investigated and engineered a combination of enzymes that had previously been shown to support homoeriodictyol and hesperitin production from fed cinnamic acid precursors. We determined the crystal structures of the enzyme catalyzing the first committed step of the pathway, chalcone synthase from Hordeum vulgare in three ligand-bound states. Based on these structures and a multiple sequence alignment with other chalcone synthases, we constructed mutant variants and assessed their performance in Escherichia coli towards producing methylated flavonoids. With our best mutant variant, HvCHS (Q232P, D234V), we were able to produce homoeriodictyol and hesperetin at 2 times and 10 times higher titers than previously reported. Our findings will facilitate the further engineering of this enzyme towards higher production of methylated flavonoids

Structure of Mycobacterium tuberculosis 1-Deoxy-D-Xylulose 5-Phosphate Synthase in Complex with Butylacetylphosphonate

Stagnation in the development of new antibiotics emphasizes the need for the discovery of drugs with novel modes of action that can tackle antibiotic resistance. Contrary to humans, most bacteria use the methylerythritol phosphate (MEP) pathway to synthesize crucial isoprenoid precursors. 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) catalyzes the first and rate-limiting step of the pathway, making it an attractive target. Alkylacetylphosphonates (alkylAPs) are a class of pyruvate mimicking DXPS inhibitors that react with thiamin diphosphate (ThDP) to form a stable phosphonolactyl (PLThDP) adduct. Here, we present the first M. tuberculosis DXPS crystal structure in complex with an inhibitor (butylacetylphosphonate (BAP)) using a construct with improved crystallization properties. The 1.6 Å structure shows that the BAP adduct interacts with catalytically important His40 and several other conserved residues of the active site. In addition, a glycerol molecule, present in the D-glyceraldehyde 3-phosphate (D-GAP) binding site and within 4 Å of the BAP adduct, indicates that there is space to extend and develop more potent alkylAPs. The structure reveals the BAP binding mode and provides insights for enhancing the activity of alkylAPs against M. tuberculosis, aiding in the development of novel antibiotics.</p

Hypericin Inhibit Alpha-Coronavirus Replication by Targeting 3CL Protease

The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections

Combining High-Throughput Synthesis and High-Throughput Protein Crystallography for Accelerated Hit Identification

Protein crystallography (PX) is widely used to drive advanced stages of drug optimization or to discover medicinal chemistry starting points by fragment soaking. However, recent progress in PX could allow for a more integrated role into early drug discovery. Here, we demonstrate for the first time the interplay of high throughput synthesis and high throughput PX. We describe a practical multicomponent reaction approach to acrylamides and ‐esters from diverse building blocks suitable for mmol scale synthesis on 96‐well format and on a high‐throughput nanoscale format in a highly automated fashion. High‐throughput PX of our libraries efficiently yielded potent covalent inhibitors of the main protease of the COVID‐19 causing agent, SARS‐CoV‐2. Our results demonstrate, that the marriage of in situ HT synthesis of (covalent) libraires and HT PX has the potential to accelerate hit finding and to provide meaningful strategies for medicinal chemistry projects

Repurposing the HCV NS3-4A protease drug boceprevir as COVID-19 therapeutics

The rapid growth of COVID-19 cases is causing an increasing death toll and also paralyzing the world economy. De novo drug discovery takes years to move from idea and/or pre-clinic to market, and it is not a short-term solution for the current SARS-CoV-2 pandemic. Drug repurposing is perhaps the only short-term solution, while vaccination is a middle-term solution. Here, we describe the discovery path of the HCV NS3–4A protease inhibitors boceprevir and telaprevir as SARS-CoV-2 main protease (3CLpro) inhibitors. Based on our hypothesis that α-ketoamide drugs can covalently bind to the active site cysteine of the SARS-CoV-2 3CLpro, we performed docking studies, enzyme inhibition and co-crystal structure analyses and finally established that boceprevir, but not telaprevir, inhibits replication of SARS-CoV-2 and mouse hepatitis virus (MHV), another coronavirus, in cell culture. Based on our studies, the HCV drug boceprevir deserves further attention as a repurposed drug for COVID-19 and potentially other coronaviral infections as well

First crystal structures of 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) from Mycobacterium tuberculosis indicate a distinct mechanism of intermediate stabilization

The development of drug resistance by Mycobacterium tuberculosis and other pathogenic bacteria emphasizes the need for new antibiotics. Unlike animals, most bacteria synthesize isoprenoid precursors through the MEP pathway. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the frst reaction of the MEP pathway and is an attractive target for the development of new antibiotics. We report here the successful use of a loop truncation to crystallize and solve the frst DXPS structures of a pathogen, namely M. tuberculosis (MtDXPS). The main diference found to other DXPS structures is in the active site where a highly coordinated water was found, showing a new mechanism for the enamine-intermediate stabilization. Unlike other DXPS structures, a “fork-like” motif could be identifed in the enamine structure, using a diferent residue for the interaction with the cofactor, potentially leading to a decrease in the stability of the intermediate. In addition, electron density suggesting a phosphate group could be found close to the active site, provides new evidence for the D-GAP binding site. These results provide the opportunity to improve or develop new inhibitors specifc for MtDXPS through structure-based drug design