On the origins of SARS-CoV-2 main protease inhibitors.

In order to address the world-wide health challenge caused by the COVID-19 pandemic, the 3CL protease/SARS-CoV-2 main protease (SARS-CoV-2-Mpro) coded by its nsp5 gene became one of the biochemical targets for the design of antiviral drugs. In less than 3 years of research, 4 inhibitors of SARS-CoV-2-Mpro have actually been authorized for COVID-19 treatment (nirmatrelvir, ensitrelvir, leritrelvir and simnotrelvir) and more such as EDP-235, FB-2001 and STI-1558/Olgotrelvir or five undisclosed compounds (CDI-988, ASC11, ALG-097558, QLS1128 and H-10517) are undergoing clinical trials. This review is an attempt to picture this quite unprecedented medicinal chemistry feat and provide insights on how these cysteine protease inhibitors were discovered. Since many series of covalent SARS-CoV-2-Mpro inhibitors owe some of their origins to previous work on other proteases, we first provided a description of various inhibitors of cysteine-bearing human caspase-1 or cathepsin K, as well as inhibitors of serine proteases such as human dipeptidyl peptidase-4 or the hepatitis C protein complex NS3/4A. This is then followed by a description of the results of the approaches adopted (repurposing, structure-based and high throughput screening) to discover coronavirus main protease inhibitors.

Illuminating the mechanism and allosteric behavior of NanoLuc luciferase.

NanoLuc, a superior ß-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O2 via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.

Biophysical analysis of Gaussia luciferase bioluminescence mechanisms using a non-oxidizable coelenterazine.

Gaussia luciferase (GLuc 18.2kDa; 168 residues) is a marine copepod luciferase that emits a bright blue light when oxidizing coelenterazine (CTZ). It is a helical protein where two homologous sequential repeats form two anti-parallel bundles, each made of four helices. We previously identified a hydrophobic cavity as a prime candidate for the catalytic site, but GLuc's fast bioluminescence reaction hampered a detailed analysis. Here, we used azacoelenterazine (Aza-CTZ), a non-oxidizable coelenterazine (CTZ) analog, as a probe to investigate its binding mode to GLuc. While analysing GLuc's activity, we unexpectedly found that salt and monovalent anions are absolutely required for Gluc's bioluminescence, which retrospectively appears reasonable for a sea-dwelling organism. The NMR-based investigation, using chemical shift perturbations monitored by 15N-1H HSQC, suggested that Aza-CTZ (and thus unoxidized CTZ) binds to residues in or near the hydrophobic cavity. These NMR data are in line with a recent structural prediction of GLuc, hypothesizing that large structural changes occur in regions remote from the hydrophobic cavity upon the addition of CTZ. Interestingly, these results point toward a unique mode of catalysis to achieve CTZ oxidative decarboxylation.

On drug discovery against infectious diseases and academic medicinal chemistry contributions.

This perspective is an attempt to document the problems that medicinal chemists are facing in drug discovery. It is also trying to identify relevant/possible, research areas in which academics can have an impact and should thus be the subject of grant calls. Accordingly, it describes how hit discovery happens, how compounds to be screened are selected from available chemicals and the possible reasons for the recurrent paucity of useful/exploitable results reported. This is followed by the successful hit to lead stories leading to recent and original antibacterials which are, or about to be, used in human medicine. Then, illustrated considerations and suggestions are made on the possible inputs of academic medicinal chemists. This starts with the observation that discovering a "good" hit in the course of a screening campaign still rely on a lot of luck - which is within the reach of academics -, that the hit to lead process requires a lot of chemistry and that if public-private partnerships can be important throughout these stages, they are absolute requirements for clinical trials. Concerning suggestions to improve the current hit success rate, one academic input in organic chemistry would be to identify new and pertinent chemical space, design synthetic accesses to reach these and prepare the corresponding chemical libraries. Concerning hit to lead programs on a given target, if no new hits are available, previously reported leads along with new structural data can be pertinent starting points to design, prepare and assay original analogues. In conclusion, this text is an actual plea illustrating that, in many countries, academic research in medicinal chemistry should be more funded, especially in the therapeutic area neglected by the industry. At the least, such funds would provide the intensive to secure series of hopefully relevant chemical entities which appears to often lack when considering the results of academic as well as industrial screening campaigns.

On Reuben G. Jones synthesis of 2-hydroxypyrazines.

In 1949, Reuben G. Jones disclosed an original synthesis of 2-hydroxypyrazines involving a double condensation between 1,2-dicarbonyls and α-aminoamides upon treatment with sodium hydroxide at low temperature. This discovery turned out to be of importance as even today there are no simple alternatives to this preparation. Across the years, it was employed to prepare 2-hydroxypyrazines but some of its limits, notably regioselectivity issues when starting from α-ketoaldehydes, certainly hampered a full-fledged generation of pyrazine-containing new chemical entities of potential interest in medicinal chemistry. The present text describes some insights and improvements, such as the unprecedented use of tetraalkylammonium hydroxide, in the reaction parameters affecting the regioselectivity and yield when starting from phenylglyoxal and two α-aminoamides. We also suggest a mechanism explaining the counterintuitive occurrence of 3,5-substituted-2-hydroxypyrazine as the major reaction product.

A Bioluminescent 3CLPro Activity Assay to Monitor SARS-CoV-2 Replication and Identify Inhibitors.

Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CLPro of coronaviruses, so that the luminescent biosensor is turned on upon 3CLPro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.

Measuring the subcellular compartmentalization of viral infections by protein complementation assay.

The recent emergence and reemergence of viruses in the human population has highlighted the need to develop broader panels of therapeutic molecules. High-throughput screening assays opening access to untargeted steps of the viral replication cycle will provide powerful leverage to identify innovative antiviral molecules. We report here the development of an innovative protein complementation assay, termed αCentauri, to measure viral translocation between subcellular compartments. As a proof of concept, the Centauri fragment was either tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment (<16 amino acids) was attached to the integrase proteins of infectious HIV-1. The translocation of viral ribonucleoproteins from the cytoplasm to the nuclear envelope or to the nucleoplasm efficiently reconstituted superfolder green fluorescent protein or NanoLuc αCentauri reporters. These fluorescence- or bioluminescence-based assays offer a robust readout of specific steps of viral infection in a multiwell format that is compatible for high-throughput screening and is validated by a short hairpin RNA-based prototype screen.

High seroprevalence but short-lived immune response to SARS-CoV-2 infection in Paris.

Although the COVID-19 pandemic peaked in March/April 2020 in France, the prevalence of infection is barely known. Using high-throughput methods, we assessed herein the serological response against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of 1847 participants working in three sites of an institution in Paris conurbation. In May-July 2020, 11% (95% confidence interval [CI]: 9.7-12.6) of serums were positive for IgG against the SARS-CoV-2 N and S proteins, and 9.5% (95% CI: 8.2-11.0) were neutralizer in pseudo-typed virus assays. The prevalence of seroconversion was 11.6% (95% CI: 10.2-13.2) when considering positivity in at least one assay. In 5% of RT-qPCR positive individuals, no systemic IgGs were detected. Among immune individuals, 21% had been asymptomatic. Anosmia (loss of smell) and ageusia (loss of taste) occurred in 52% of the IgG-positive individuals and in 3% of the negative ones. In contrast, 30% of the anosmia-ageusia cases were seronegative, suggesting that the true prevalence of infection may have reached 16.6%. In sera obtained 4-8 weeks after the first sampling, anti-N and anti-S IgG titers and neutralization activity in pseudo-virus assay declined by 31%, 17%, and 53%, resulting thus in half-life of 35, 87, and 28 days, respectively. The population studied is representative of active workers in Paris. The short lifespan of the serological systemic responses suggests an underestimation of the true prevalence of infection.

Core-Modified Coelenterazine Luciferin Analogues: Synthesis and Chemiluminescence Properties.

In this work on the design and studies of luciferins related to the blue-hued coelenterazine, the synthesis of heterocyclic analogues susceptible to produce a photon, possibly at a different wavelength, is undertaken. Here, the synthesis of O-acetylated derivatives of imidazo[1,2-b]pyridazin-3(5 H)-one, imidazo[2,1-f][1,2,4]triazin-7(1 H)-one, imidazo[1,2-a]pyridin-3-ol, imidazo[1,2-a]quinoxalin-1(5 H)-one, benzo[f]imidazo[1,2-a]quinoxalin-3(11 H)-one, imidazo[1',2':1,6]pyrazino[2,3-c]quinolin-3(11 H)-one, and 5,11-dihydro-3 H-chromeno[4,3-e]imidazo[1,2-a]pyrazin-3-one is described thanks to extensive use of the Buchwald-Hartwig N-arylation reaction. The acidic hydrolysis of these derivatives then gave solutions of the corresponding luciferin analogues, which were studied. Not too unexpectedly, even if these were "dressed" with substituents found in actual substrates of the nanoKAZ/NanoLuc luciferase, no bioluminescence was observed with these compounds. However, in a phosphate buffer, all produced a light signal, by chemiluminescence, with extensive variations in their respective intensity and this could be increased by adding a quaternary ammonium salt in the buffer. This aspect was actually instrumental to determine the emission spectra of many of these luciferin analogues.

Bioluminescence Profiling of NanoKAZ/NanoLuc Luciferase Using a Chemical Library of Coelenterazine Analogues.

We describe here an extensive structure-bioluminescence relationship study of a chemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, a mutated luciferase originated from the catalytic subunit of the deep-sea shrimp Oplophorus gracilirostris. Out of the 135 O-acetylated precursors that were prepared by using our recently reported synthesis and following their hydrolysis to give solutions of the corresponding luciferins, notable bioluminescence improvements were achieved in comparison with furimazine, which is currently amongst the best substrates of nanoKAZ/NanoLuc. For instance, the rather more lipophilic analogue 8-(2,3-difluorobenzyl)-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one provided a 1.5-fold improvement of the total light output over a 2 h period, a close to threefold increase of the initial signal intensity and a signal-to-background ratio five times greater than furimazine. The kinetic parameters for the enzymatic reaction were obtained for a selection of luciferin analogues and provided unexpected insights into the luciferase activity. Most prominently, along with a general substrate-dependent and irreversible inactivation of this enzyme, in the case of the optimized luciferin mentioned above, the consumption of 2664 molecules was found to be required for the detection of a single Relative Light Unit (RLU; a luminometer-dependent fraction of a photon).

Maximizing binary interactome mapping with a minimal number of assays.

Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes.

Gram-scale synthesis of luciferins derived from coelenterazine and original insights into their bioluminescence properties.

An original gram-scale synthesis of O-acetylated forms of coelenterazine, furimazine or hydroxy-bearing analogues of luciferins is described. The comparison over two hours of their bioluminescence, using the nanoKAZ/NanoLuc luciferase, provides remarkable insights useful for the selection of a substrate adapted for a given application.

Synthesis of unnatural α-amino esters using ethyl nitroacetate and condensation or cycloaddition reactions.

We report here on the use of ethyl nitroacetate as a glycine template to produce α-amino esters. This started with a study of its condensation with various arylacetals to give ethyl 3-aryl-2-nitroacrylates followed by a reduction (NaBH4 and then zinc/HCl) into α-amino esters. The scope of this method was explored as well as an alternative with arylacylals instead. We also focused on various [2 + 3] cycloadditions, one leading to a spiroacetal, which led to the undesired ethyl 5-(benzamidomethyl)isoxazole-3-carboxylate. The addition of ethyl nitroacetate on a 5-methylene-4,5-dihydrooxazole using cerium(IV) ammonium nitrate was also explored and the synthesis of other oxazole-bearing α-amino esters was achieved using gold(I) chemistry.

Unnatural α-amino ethyl esters from diethyl malonate or ethyl ß-bromo-α-hydroxyiminocarboxylate.

We have explored here the scope of the age-old diethyl malonate-based accesses to α-amino esters involving Knoevenagel condensations of diethyl malonate on aldehydes, reductions of the resulting alkylidenemalonates, the preparation of the corresponding α-hydroxyimino esters and their final reduction. This synthetic pathway turned out to be general although some unexpected limitations were encountered. The synthetic modifications of some of the intermediates - using Suzuki-Miyaura coupling or cycloadditions - before undertaking the oximation step - provided accesses to further α-amino esters. Moreover, other pathways to α-hydroxyimino esters were explored including an attempt to improve the cycloadditions between ethyl ß-bromo-α-hydroxyiminocarboxylate and various alkylfuranes.

Checkpoint kinase 1 inhibition sensitises transformed cells to dihydroorotate dehydrogenase inhibition.

Reduction in nucleotide pools through the inhibition of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) has been demonstrated to effectively reduce cancer cell proliferation and tumour growth. The current study sought to investigate whether this antiproliferative effect could be enhanced by combining Chk1 kinase inhibition. The pharmacological activity of DHODH inhibitor teriflunomide was more selective towards transformed mouse embryonic fibroblasts than their primary or immortalised counterparts, and this effect was amplified when cells were subsequently exposed to PF477736 Chk1 inhibitor. Flow cytometry analyses revealed substantial accumulations of cells in S and G2/M phases, followed by increased cytotoxicity which was characterised by caspase 3-dependent induction of cell death. Associating PF477736 with teriflunomide also significantly sensitised SUM159 and HCC1937 human triple negative breast cancer cell lines to dihydroorotate dehydrogenase inhibition. The main characteristic of this effect was the sustained accumulation of teriflunomide-induced DNA damage as cells displayed increased phospho serine 139 H2AX (γH2AX) levels and concentration-dependent phosphorylation of Chk1 on serine 345 upon exposure to the combination as compared with either inhibitor alone. Importantly a similar significant increase in cell death was observed upon dual siRNA mediated depletion of Chk1 and DHODH in both murine and human cancer cell models. Altogether these results suggest that combining DHODH and Chk1 inhibitions may be a strategy worth considering as a potential alternative to conventional chemotherapies.

Respiratory syncytial virus infection in macaques is not suppressed by intranasal sprays of pyrimidine biosynthesis inhibitors.

There is imperious need for efficient therapies against ubiquitous and life-threatening respiratory viruses, foremost among them being the human respiratory syncytial virus (hRSV). Several research groups who performed functional screens for broad-spectrum antivirals identified compounds targeting the de novo pyrimidine biosynthesis pathway. Despite their strong antiviral activity in vitro, whether such antimetabolites are effective in vivo remains highly controversial. Here, we evaluated two potent pyrimidine biosynthesis inhibitors developed in our laboratory, IPPA17-A04 and GAC50, in a model of mild hRSV-infection in cynomolgus macaques. In this model, hRSV replication is restricted to the epithelium of the upper respiratory tract, and is compatible with a topical treatment by intranasal sprays. The local administration of palivizumab, a neutralizing anti-hRSV antibody used in clinics, significantly reduced virus replication. In contrast, pyrimidine biosynthesis inhibitors did not show any inhibitory effect on hRSV growth when delivered topically as experimented in our model. Our results should help to better define the potential applications of this class of antimetabolites in the treatment of viral infections.

Synthetic Routes to Coelenterazine and Other Imidazo[1,2-a]pyrazin-3-one Luciferins: Essential Tools for Bioluminescence-Based Investigations.

In the last few decades, bioluminescent systems based on the expression of a luciferase and the addition of a luciferin to monitor the emission of light have become very important tools for biological investigations. A growing proportion of these systems use coelenterazine or analogues of imidazo[1,2-a]pyrazine luciferins along with photoproteins or luciferases from sea creatures such as Aequorea, Renilla, Gaussia or Oplophorus. Central to the success of these tools are the synthetic pathways developed not only to prepare the naturally occurring luciferins, but also to design altered compounds that exhibit improved bioluminescence. Current work is indeed focused on the design of systems exhibiting extended luminescence ("glow" systems) or redshifted wavelengths, as well as constructions better adapted to conditions in cells or in vivo. This review describes the synthetic pathways used to prepare imidazo[1,2-a]pyrazine luciferins along with the research efforts aimed at preparing analogues even better suited to the design of assays.

Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH).

Following our discovery of human dihydroorotate dehydrogenase (DHODH) inhibition by 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidine derivatives as well as 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)-5-methylpyridine, we describe here the syntheses and evaluation of an array of azine-bearing analogues. As in our previous report, the structure-activity study of this series of human DHODH inhibitors was based on a phenotypic assay measuring measles virus replication. Among other inhibitors, this round of syntheses and biological evaluation iteration led to the highly active 5-cyclopropyl-2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-3-fluoropyridine. Inhibition of DHODH by this compound was confirmed in an array of in vitro assays, including enzymatic tests and cell-based assays for viral replication and cellular growth. This molecule was found to be more active than the known inhibitors of DHODH, brequinar and teriflunomide, thus opening perspectives for its use as a tool or for the design of an original series of immunosuppressive agent. Moreover, because other series of inhibitors of human DHODH have been found to also affect Plasmodium falciparum DHODH, all the compounds were assayed for their effect on P. falciparum growth. However, the modest in vitro inhibition solely observed for two compounds did not correlate with their inhibition of P. falciparum DHODH.

Original 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidine derivatives as inhibitors of human dihydroorotate dehydrogenase (DHODH).

From a research program aimed at the design of new chemical entities followed by extensive screening on various models of infectious diseases, an original series of 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidines endowed with notable antiviral properties were found. Using a whole cell measles virus replication assay, we describe here some aspects of the iterative process that, from 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)pyrimidine, led to 2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-5-ethylpyrimidine and a 4000-fold improvement of antiviral activity with a subnanomolar level of inhibition. Moreover, recent precedents in the literature describing antiviral derivatives acting at the level of the de novo pyrimidine biosynthetic pathway led us to determine that the mode of action of this series is based on the inhibition of the cellular dihydroorotate dehydrogenase (DHODH), the fourth enzyme of this pathway. Biochemical studies with recombinant human DHODH led us to measure IC50 as low as 13 nM for the best example of this original series when using 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone (coenzyme Q1) as a surrogate for coenzyme Q10, the cofactor of this enzyme.