Miniaturized Modular Click Chemistry-enabled Rapid Discovery of Unique SARS-CoV-2 Mpro Inhibitors With Robust Potency and Drug-like Profile.

The COVID-19 pandemic has required an expeditious advancement of innovative antiviral drugs. In this study, focused compound libraries are synthesized in 96- well plates utilizing modular click chemistry to rapidly discover potent inhibitors targeting the main protease (Mpro) of SARS-CoV-2. Subsequent direct biological screening identifies novel 1,2,3-triazole derivatives as robust Mpro inhibitors with high anti-SARS-CoV-2 activity. Notably, C5N17B demonstrates sub-micromolar Mpro inhibitory potency (IC50 = 0.12 µM) and excellent antiviral activity in Calu-3 cells determined in an immunofluorescence-based antiviral assay (EC50 = 0.078 µM, no cytotoxicity: CC50 > 100 µM). C5N17B shows superior potency to nirmatrelvir (EC50 = 1.95 µM) and similar efficacy to ensitrelvir (EC50 = 0.11 µM). Importantly, this compound displays high antiviral activities against several SARS-CoV-2 variants (Gamma, Delta, and Omicron, EC50 = 0.13 - 0.26 µM) and HCoV-OC43, indicating its broad-spectrum antiviral activity. It is worthy that C5N17B retains antiviral activity against nirmatrelvir-resistant strains with T21I/E166V and L50F/E166V mutations in Mpro (EC50 = 0.26 and 0.15 µM, respectively). Furthermore, C5N17B displays favorable pharmacokinetic properties. Crystallography studies reveal a unique, non-covalent multi-site binding mode. In conclusion, these findings substantiate the potential of C5N17B as an up-and-coming drug candidate targeting SARS-CoV-2 Mpro for clinical therapy.

Nonpeptidic Irreversible Inhibitors of SARS-CoV-2 Main Protease with Potent Antiviral Activity.

SARS-CoV-2 infections pose a high risk for vulnerable patients. In this study, we designed benzoic acid halopyridyl esters bearing a variety of substituents as irreversible inhibitors of the main viral protease (Mpro). Altogether, 55 benzoyl chloro/bromo-pyridyl esters were synthesized, with broad variation of the substitution pattern on the benzoyl moiety. A workflow was employed for multiparametric optimization, including Mpro inhibition assays of SARS-CoV-2 and related pathogenic coronaviruses, the duration of enzyme inhibition, the compounds' stability versus glutathione, cytotoxicity, and antiviral activity. Several compounds showed IC50 values in the low nanomolar range, kinact/Ki values of >100,000 M-1 s-1 and high antiviral activity. High-resolution X-ray cocrystal structures indicated an important role of ortho-fluorobenzoyl substitution, forming a water network that stabilizes the inhibitor-bound enzyme. The most potent antiviral compound was the p-ethoxy-o-fluorobenzoyl chloropyridyl ester (PSB-21110, 29b, MW 296 g/mol; EC50 2.68 nM), which may serve as a lead structure for broad-spectrum anticoronaviral therapeutics.

Structural Studies on the Binding Mode of Bisphenols to PPARγ.

Bisphenol A (BPA) and bisphenol B (BPB) are widely used in the production of plastics, and their potential adverse health effects, particularly on endocrine disruption and metabolic health, have raised concern. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a pivotal role in metabolic regulation and adipogenesis, making it a target of interest in understanding the development of obesity and associated health impacts. In this study, we employ X-ray crystallography and molecular dynamics (MD) simulations to study the interaction of PPARγ with BPA and BPB. Crystallographic structures reveal the binding of BPA and BPB to the ligand binding domain of PPARγ, next to C285, where binding of partial agonists as well as antagonists and inverse agonists of PPARγ signaling has been previously observed. However, no interaction of BPA and BPB with Y437 in the activation function 2 site is observed, showing that these ligands cannot stabilize the active conformation of helix 12 directly. Furthermore, free energy analyses of the MD simulations revealed that I341 has a large energetic contribution to the BPA and BPB binding modes characterized in this study.

Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics.

A titrant for the SARS-CoV-2 main protease (Mpro) was developed that enables, for the first time, the exact determination of the concentration of the enzymatically active Mpro by active-site titration. The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme-titrant complex. Four fluorogenic substrates of Mpro, including a prototypical, internally quenched Dabcyl-EDANS peptide, were compared in terms of solubility under typical assay conditions. By exploiting the new titrant, key kinetic parameters for the Mpro-catalyzed cleavage of these substrates were determined.

Structural Insights into Partial Activation of the Prototypic G Protein-Coupled Adenosine A2A Receptor.

The adenosine A2A receptor (A2AAR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family, which constitutes the largest class of GPCRs. Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist. Here, we determined an X-ray crystal structure of the partial A2AAR agonist 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-p-hydroxyphenyl-3,5-pyridinedicarbonitrile (LUF5834) in complex with the A2AAR construct A2A-PSB2-bRIL, stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket. The determined high-resolution structure (2.43 Å) resolved water networks and crucial binding pocket interactions. A direct hydrogen bond of the p-hydroxy group of LUF5834 with T883.36 was observed, an amino acid that was mutated to alanine in the most frequently used A2AAR crystallization constructs thus preventing the discovery of its interactions in most of the previous A2AAR co-crystal structures. G protein dissociation studies confirmed partial agonistic activity of LUF5834 as compared to that of the full agonist N-ethylcarboxamidoadenosine (NECA). In contrast to NECA, the partial agonist was still able to bind to the receptor construct locked in its inactive conformation by an S913.39K mutation, although with an affinity lower than that at the native receptor. This could explain the compound's partial agonistic activity: while full A2AAR agonists bind exclusively to the active conformation, likely following conformational selection, partial agonists bind to active as well as inactive conformations, showing higher affinity for the active conformation. This might be a general mechanism of partial agonism also applicable to other GPCRs.

Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV-2 Main Protease Inhibitors as Antiviral Drugs.

Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M-1s-1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents.

Design, Synthesis, and Biological Evaluation of Trisubstituted Piperazine Derivatives as Noncovalent Severe Acute Respiratory Syndrome Coronavirus 2 Main Protease Inhibitors with Improved Antiviral Activity and Favorable Druggability.

The ongoing transmission of SARS-CoV-2 necessitates the development of additional potent antiviral agents capable of combating the current highly infectious variants and future coronaviruses. Here, we present the discovery of potent nonpeptide main protease (Mpro) inhibitors with prominent antiviral activity and improved pharmacokinetic properties. Three series of 1,2,4-trisubstituted piperazine derivatives were designed and synthesized, and the optimal GC-78-HCl demonstrated high enzyme-inhibitory potency (IC50 = 0.19 µM) and exhibited excellent antiviral activity (EC50 = 0.40 µM), reaching the same level as Nirmatrelvir (EC50 = 0.38 µM). Additionally, GC-78-HCl displayed potent antiviral activities against various SARS-CoV-2 variants as well as HCoV-OC43 and HCoV-229E, indicating its potential broad-spectrum anticoronaviral activity. Notably, the pharmacokinetic properties of GC-78-HCl were somewhat enhanced compared to those of the lead compound. Furthermore, the cocrystal and molecular docking elucidated the mechanism of action. In conclusion, we discovered a novel nonpeptidic Mpro inhibitor with promising antiviral activity and a favorable pharmacokinetic profile.

The dimerized pentraxin-like domain of the adhesion G protein-coupled receptor 112 (ADGRG4) suggests function in sensing mechanical forces.

Adhesion G protein-coupled receptors (aGPCRs) feature large extracellular regions with modular domains that often resemble protein classes of various function. The pentraxin (PTX) domain, which is predicted by sequence homology within the extracellular region of four different aGPCR members, is well known to form pentamers and other oligomers. Oligomerization of GPCRs is frequently reported and mainly driven by interactions of the seven-transmembrane region and N or C termini. While the functional importance of dimers is well-established for some class C GPCRs, relatively little is known about aGPCR multimerization. Here, we showcase the example of ADGRG4, an orphan aGPCR that possesses a PTX-like domain at its very N-terminal tip, followed by an extremely long stalk containing serine-threonine repeats. Using X-ray crystallography and biophysical methods, we determined the structure of this unusual PTX-like domain and provide experimental evidence for a homodimer equilibrium of this domain which is Ca2+-independent and driven by intermolecular contacts that differ vastly from the known soluble PTXs. The formation of this dimer seems to be conserved in mammalian ADGRG4 indicating functional relevance. Our data alongside of theoretical considerations lead to the hypothesis that ADGRG4 acts as an in vivo sensor for shear forces in enterochromaffin and Paneth cells of the small intestine.

Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction.

The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations.

PTK7 is a positive allosteric modulator of GPR133 signaling in glioblastoma.

The adhesion G-protein-coupled receptor GPR133 (ADGRD1) supports growth of the brain malignancy glioblastoma. How the extracellular interactome of GPR133 in glioblastoma modulates signaling remains unknown. Here, we use affinity proteomics to identify the transmembrane protein PTK7 as an extracellular binding partner of GPR133 in glioblastoma. PTK7 binds the autoproteolytically generated N-terminal fragment of GPR133 and its expression in trans increases GPR133 signaling. This effect requires the intramolecular cleavage of GPR133 and PTK7's anchoring in the plasma membrane. PTK7's allosteric action on GPR133 signaling is additive with but topographically distinct from orthosteric activation by soluble peptide mimicking the endogenous tethered Stachel agonist. GPR133 and PTK7 are expressed in adjacent cells in glioblastoma, where their knockdown phenocopies each other. We propose that this ligand-receptor interaction is relevant to the pathogenesis of glioblastoma and possibly other physiological processes in healthy tissues.

Structure and function of the metagenomic plastic-degrading polyester hydrolase PHL7 bound to its product.

The recently discovered metagenomic-derived polyester hydrolase PHL7 is able to efficiently degrade amorphous polyethylene terephthalate (PET) in post-consumer plastic waste. We present the cocrystal structure of this hydrolase with its hydrolysis product terephthalic acid and elucidate the influence of 17 single mutations on the PET-hydrolytic activity and thermal stability of PHL7. The substrate-binding mode of terephthalic acid is similar to that of the thermophilic polyester hydrolase LCC and deviates from the mesophilic IsPETase. The subsite I modifications L93F and Q95Y, derived from LCC, increased the thermal stability, while exchange of H185S, derived from IsPETase, reduced the stability of PHL7. The subsite II residue H130 is suggested to represent an adaptation for high thermal stability, whereas L210 emerged as the main contributor to the observed high PET-hydrolytic activity. Variant L210T showed significantly higher activity, achieving a degradation rate of 20 µm h-1 with amorphous PET films.

Structural basis of the activation of PPARγ by the plasticizer metabolites MEHP and MINCH.

Di-2-ethylhexyl phthalate (DEHP) and its substitute 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) are widely used as plasticizers but may have adverse health effects. Via hydrolysis of one of the two ester bonds in the human body, DEHP and DINCH form the monoesters MEHP and MINCH, respectively. Previous studies demonstrated binding of these metabolites to PPARγ and the induction of adipogenesis via this pathway. Detailed structural understanding of how these metabolites interact with PPARγ and thereby affect human health is lacking until now. We therefore characterized the binding modes of MINCH and MEHP to the ligand binding domain of PPARγ by X-ray crystallography and molecular dynamics (MD) simulations. Both compounds bind to the activating function-2 (AF-2) binding site via an interaction of the free carboxylates with the histidines 323 and 449, tyrosine 473 and serine 289. The alkyl chains form hydrophobic interactions with the tunnel next to cysteine 285. These binding modes are generally stable as demonstrated by the MD simulations and they resemble the complexation of fatty acids and their metabolites to the AF-2 site of PPARγ. Similar to the situation for these natural PPARγ agonists, the interaction of the free carboxylate groups of MEHP and MINCH with tyrosine 473 and surrounding residues stabilizes the AF-2 helix in the upward conformation. This state promotes binding of coactivator proteins and thus formation of the active complex for transcription of the specific target genes. Moreover, a comparison of the residues involved in binding of the plasticizer metabolites in vertebrate PPARγ orthologs shows that these compounds likely have similar effects in other species.

Structural and Mechanistic Studies on Substrate and Stereoselectivity of the Indole Monooxygenase VpIndA1: New Avenues for Biocatalytic Epoxidations and Sulfoxidations.

Flavoprotein monooxygenases are a versatile group of enzymes for biocatalytic transformations. Among these, group E monooxygenases (GEMs) catalyze enantioselective epoxidation and sulfoxidation reactions. Here, we describe the crystal structure of an indole monooxygenase from the bacterium Variovorax paradoxus EPS, a GEM designated as VpIndA1. Complex structures with substrates reveal productive binding modes that, in conjunction with force-field calculations and rapid mixing kinetics, reveal the structural basis of substrate and stereoselectivity. Structure-based redesign of the substrate cavity yielded variants with new substrate selectivity (for sulfoxidation of benzyl phenyl sulfide) or with greatly enhanced stereoselectivity (from 35.1 % to 99.8 % ee for production of (1S,2R)-indene oxide). This first determination of the substrate binding mode of GEMs combined with structure-function relationships opens the door for structure-based design of these powerful biocatalysts.

Discovery and Crystallographic Studies of Nonpeptidic Piperazine Derivatives as Covalent SARS-CoV-2 Main Protease Inhibitors.

The spread of SARS-CoV-2 keeps threatening human life and health, and small-molecule antivirals are in demand. The main protease (Mpro) is an effective and highly conserved target for anti-SARS-CoV-2 drug design. Herein, we report the discovery of potent covalent non-peptide-derived Mpro inhibitors. A series of covalent compounds with a piperazine scaffold containing different warheads were designed and synthesized. Among them, GD-9 was identified as the most potent compound with a significant enzymatic inhibition of Mpro (IC50 = 0.18 µM) and good antiviral potency against SARS-CoV-2 (EC50 = 2.64 µM), similar to that of remdesivir (EC50 = 2.27 µM). Additionally, GD-9 presented favorable target selectivity for SARS-CoV-2 Mpro versus human cysteine proteases. The X-ray co-crystal structure confirmed our original design concept showing that GD-9 covalently binds to the active site of Mpro. Our nonpeptidic covalent inhibitors provide a basis for the future development of more efficient COVID-19 therapeutics.

Discovery and Crystallographic Studies of Trisubstituted Piperazine Derivatives as Non-Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity and Low Toxicity.

The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (Mpro) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide Mpro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits Mpro with high potency (IC50 = 0.40 µM) and displays excellent antiviral activity (EC50 = 1.1 µM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC50 > 100 µM) and excellent target selectivity for SARS-CoV-2 Mpro (IC50 > 50 µM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.

Analysis of double-emulsion droplets with ESI mass spectrometry for monitoring lipase-catalyzed ester hydrolysis at nanoliter scale.

Microfluidic double-emulsion droplets allow the realization and study of biphasic chemical processes such as chemical reactions or extractions on the nanoliter scale. Double emulsions of the rare type (o1/w/o2) are used here to realize a lipase-catalyzed reaction in the non-polar phase. The surrounding aqueous phase induces the transfer of the hydrophilic product from the core oil phase, allowing on-the-fly MS analysis in single double droplets. A microfluidic two-step emulsification process is developed to generate the (o1/w/o2) double-emulsion droplets. In this first example of microfluidic double-emulsion MS coupling, we show in proof-of-concept experiments that the chemical composition of the water layer can be read online using ESI-MS. Double-emulsion droplets were further employed as two-phase micro-reactors for the hydrolysis of the lipophilic ester p-nitrophenyl palmitate catalyzed by the Candida antarctica lipase B (CalB). Finally, the formation of the hydrophilic reaction product p-nitrophenol within the double-emulsion droplet micro-reactors is verified by subjecting the double-emulsion droplets to online ESI-MS analysis.

Structure-Activity Relationship of 3-Methylcytidine-5'-α,ß-methylenediphosphates as CD73 Inhibitors.

We recently reported N4-substituted 3-methylcytidine-5'-α,ß-methylenediphosphates as CD73 inhibitors, potentially useful in cancer immunotherapy. We now expand the structure-activity relationship of pyrimidine nucleotides as human CD73 inhibitors. 4-Chloro (MRS4598 16; Ki = 0.673 nM) and 4-iodo (MRS4620 18; Ki = 0.436 nM) substitution of the N4-benzyloxy group decreased Ki by ∼20-fold. Primary alkylamine derivatives coupled through a p-amido group with a varying methylene chain length (24 and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. X-ray structures of hCD73 with two inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal and N-terminal domains) as N4-benzyl groups in adenine inhibitors. Molecular dynamics identified stabilizing interactions and predicted conformational diversity. Thus, by N4-benzyloxy substitution, we have greatly enhanced the inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in tumor tissues in situ.

Mono-ADP-ribosylation sites of human CD73 inhibit its adenosine-generating enzymatic activity.

CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.

Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester Hydrolase.

Earth is flooded with plastics and the need for sustainable recycling strategies for polymers has become increasingly urgent. Enzyme-based hydrolysis of post-consumer plastic is an emerging strategy for closed-loop recycling of polyethylene terephthalate (PET). The polyester hydrolase PHL7, isolated from a compost metagenome, completely hydrolyzes amorphous PET films, releasing 91 mg of terephthalic acid per hour and mg of enzyme. Vertical scanning interferometry shows degradation rates of the PET film of 6.8 µm h-1 . Structural analysis indicates the importance of leucine at position 210 for the extraordinarily high PET-hydrolyzing activity of PHL7. Within 24 h, 0.6 mgenzyme gPET -1 completely degrades post-consumer thermoform PET packaging in an aqueous buffer at 70 °C without any energy-intensive pretreatments. Terephthalic acid recovered from the enzymatic hydrolysate is then used to synthesize virgin PET, demonstrating the potential of polyester hydrolases as catalysts in sustainable PET recycling processes with a low carbon footprint.

Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5'-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives.

Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5'-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the Ki value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.