Genetic Labeling of Drosophila Neurons

Flies of the genotype RN2-Flp, Tub-FRT-CD2-FRT-Gal4, UAS mCD8-GFP were used in our experiments to label the CSDn. The RN2-Flp transgene was constructed by fusing the regulatory region of the evenskipped (eve) locus necessary for expression in RP2, aCC and pCC neurons [26 (link)] to the Flippase coding region. The construct on the third chromosome was allowed to undergo recombination with a strain carrying the P-(Tub-FRT-CD2-FRT-Gal4) [62 (link)] cassette and also a UAS-mCD8-GFP transgene [63 (link)]. In flies of the RN2-Flp, Tub-FRT-CD2-FRT-Gal4, UAS-mCD8-GFP genotype, expression of Flippase in specific cells induces recombination at the FRT sites, hence inducing expression of GFP under Tub-Gal4 control. The UAS-EcR-B1-d655W650A stock (referred to as UAS-EcR-B1^W650A[31 (link),64 (link)]) was obtained from the Bloomington Drosophila Stock center, Indiana. UAS-TNT-G and UAS -IMPTNT-V stocks were kindly provided by Sean Sweeney [33 (link)]. The IMPTNT-V is mutated in LC2V233 to V237 mutation, rendering the toxin essentially ineffective [33 (link)]. Expression of the inactive toxin was used as a control in all experiments. The UAS-shi^ts1/UAS-shi^ts1; +/+;UAS-shi^ts1/UAS-shi^ts1stock [34 (link)], expresses a temperature sensitive form of Dynamin, under GAL4 control, at 29°C, which results in a block in endocytosis. The construction of the y, w; UAS-EGFP-Kir2.1 transformant has been described in [35 (link)]. Targeted expression leads to an inward flux of K⁺ions that hyperpolarizes the resting membrane, resulting in silencing of neuronal activity. The lz³stock was obtained from R Stocker [65 (link)]. Mushroom body marking was achieved using the mb247-DsRed stock kindly provided by Andre Fiala. UAS-nSybGFP was a gift from Mani Ramaswami, UAS-nod:lacZ from Clark et al. [23 (link)] and UAS-Dlg from Ulrich Thomas [25 (link)]. The UAS-Syt-GFP stock, Antennapaedia (Antp⁶), RN2-Gal4 and the temperature sensitive allele ddc^ts2were obtained from the Bloomington Stock Centre, Indiana.
All fly stocks were grown on standard cornmeal medium at 25°C. White prepupae (0 hours APF) were collected and allowed to develop on moist filter paper. RN2-Flp, Tub-FRT-CD2-FRT Gal4, UAS mCD8-GFP pupae take about 100 hours to eclose when grown in our laboratory. All perturbations were done at 25°C except for the experiments with shi^ts1ectopic expression, for which animals were reared at 29°C.

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Roy B., Singh A.P., Shetty C., Chaudhary V., North A., Landgraf M., VijayRaghavan K, & Rodrigues V. (2007). Metamorphosis of an identified serotonergic neuron in the Drosophila olfactory system. Neural Development, 2, 20.

Publication 2007

Allele Animals Cells Chromosome Drosophila Dynamin Ectopic expression Endocytosis Genotype Ions Lacz Membrane Mushroom body Mutation Neuronal Paper Pupae Recombination Regulatory region Toxin Transgene

Corresponding Organization :

Other organizations : National Centre for Biological Sciences, Tata Institute of Fundamental Research, University of Cambridge

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FRET-based Enzymatic Inhibition Assay for SARS-CoV-2 3CL Protease

The FRET-based enzymatic inhibition assay was utilized to measure the inhibitory activities of compounds against 3CL proteases. Primary screening was carried out to identify the inhibitors against SARS-CoV-2 3CL^pro using an FDA-approved drug library containing an array of 1,018 compounds obtained from Selleck Chemicals (#L1300) [30] (link). The fluorogenic substrate Dabcyl-KNSTLQSGLRKE-Edans (GenScript, Shanghai, China) was synthesized to measure the protease activity of SARS-CoV-2 3CL^pro or SARS-CoV 3CL^pro according to our previous method [30] (link), [37] (link). Dabcyl-KTSAVLQSGFRKME-Edans (GenScript, Shanghai, China) was used for enzymatic inhibition assay of MERS-CoV 3CL^pro and HCoV-229E 3CL^pro as described previously [38] (link), [39] (link). The enzymatic inhibition assays of different proteases were performed as follows. 120 nM of SARS-CoV-2 3CL^pro in the reaction buffer (0.1 M PBS, 1 mM EDTA, pH 7.4) incubated with varying concentrations of tested compounds for 1 h at 37 °C and then the substrate (final concentration 20 μM) was added to start the reaction. Fluorescence intensity was monitored continuously every 2 min for up to 20 min with an excitation wavelength of 340 nm and emission wavelength of 490 nm by using Spectramax® ID3 plate reader (Molecular Devices, California, USA). For the SARS-CoV 3CL^pro inhibition assay, 860 nM protease was mixed with 40 μM substrate in assay buffer.
To determine the effect of DTT or GSH on the enzymatic inhibition of SARS-CoV-2 3CL^pro by Thonzonium bromide, we performed the enzymatic inhibition assay according to the literature [40] (link). SARS-CoV-2 3CL^pro was preincubated in reaction buffer with Thonzonium bromide in the presence of 4 mM DTT or 1 mM GSH compared with in the absence of DTT or GSH buffer.
In the MERS-CoV 3CL^pro and HCoV-229E 3CL^pro enzymatic inhibition assay, the concentration of the proteases was 2 and 1 μM, corresponding to the final substrate concentration of 40 and 20 μM, respectively. The IC₅₀ values were calculated by fitting the curve of normalized inhibition ratio with the different concentration of test compounds.

Wang R., Zhai G., Zhu G., Wang M., Gong X., Zhang W., Ge G., Chen H, & Chen L. (2022). Discovery and mechanism of action of Thonzonium bromide from an FDA-approved drug library with potent and broad-spectrum inhibitory activity against main proteases of human coronaviruses. Bioorganic Chemistry, 130, 106264.

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Measuring 3CLpro Protease Inhibition

For assessing the ability to inhibit the main protease (3CLpro), the IC₅₀ was the half-maximal inhibitory concentration of the substance at which the fluorescence level reduced by 50% compared to the value obtained without adding the inhibitor [9 (link),10 (link),34 (link)]. Fluorescence occurred due to cleavage of the peptide substrate DabcylKTSAVLQ↓SGFRKME(Edans)NH2 by 3CLpro protease. ML188 inhibitor ((R)-N-(4-(tert-Butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Ambeed Inc, USA) was used as a positive control. In the study, the signal was recorded using the CLARIOstar Plus instrument (BMG Labtech) at 355 and 460 nm for excitation and radiation, respectively, in the kinetic scan mode. Reaction mixtures containing TrisHCl buffer, fluorogenic substrate, 3CLpro, and the compound being tested were prepared and incubated for 30 min in a 384-well plate at 30 °C. The measurement for each compound was carried out in triplicate. The instrument was calibrated using a solution of the peptide that had undergone complete hydrolysis. The accompanying MARS Data Analysis software was used to calculate IC₅₀ values.

Baev D.S., Blokhin M.E., Chirkova V.Y., Belenkaya S.V., Luzina O.A., Yarovaya O.I., Salakhutdinov N.F, & Shcherbakov D.N. (2022). Triterpenic Acid Amides as Potential Inhibitors of the SARS-CoV-2 Main Protease. Molecules, 28(1), 303.

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Inhibition of SARS-CoV-2 3CLpro Protease

To assess the ability to inhibit the main protease (3CLpro), the IC₅₀ is the semi-inhibitory concentration of the substance, at which the fluorescence level is reduced by 50% relative to the value obtained without adding the inhibitor [30 (link)]. Fluorescence occurs due to cleavage of the peptide substrate DabcylKTSAVLQ↓SGFRKME(Edans)NH2 by 3CLpro protease. In the study, the signal was recorded using the CLARIOstar Plus instrument (BMG Labtech) at 355 and 460 nm for excitation/radiation, respectively, in kinetic scan mode. Reaction mixtures containing TrisHCl buffer, fluorogenic substrate, 3CLpro, and the compound being tested were prepared and incubated for 30 min in a 384-well plate at 30 °C. The instrument was calibrated using a solution of the peptide that had undergone complete hydrolysis. The accompanying MARS Data Analysis software (https://www.selectscience.net/products/mars-data-analysis-software/?prodID=81306, accessed on 18 August 2022) was used to calculate IC₅₀.

Filimonov A.S., Yarovaya O.I., Zaykovskaya A.V., Rudometova N.B., Shcherbakov D.N., Chirkova V.Y., Baev D.S., Borisevich S.S., Luzina O.A., Pyankov O.V., Maksyutov R.A, & Salakhutdinov N.F. (2022). (+)-Usnic Acid and Its Derivatives as Inhibitors of a Wide Spectrum of SARS-CoV-2 Viruses. Viruses, 14(10), 2154.

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Repurposing Drugs for COVID-19 Treatment

The first step involved the curation and preparation of the experimental screening data. We downloaded the data on SARS-CoV-2 cytopathic effect (CPE) from the NCATS COVID-19 portal. This assay measures the ability of a compound to reverse the cytopathic effect induced by the virus in Vero E6 host cells (one of the most commonly used cell lines for studying this family of virus). The assay provides valuable basic information about the ability of a compound to restore the cells’ function but does not provide indication of the possible mechanism of action(s) of the small chemical compound under study. We also explored using the same Vero E6 cells the cytotoxic effect of these molecules in a counterscreen since the observed effect of some compounds may be mediated through cell viability and could lead to erroneous interpretation of the CPE results. Such information on cell viability is evidently important in the applicability of such compounds in clinical pharmacological interventions. Some additional data are important to take into account in the present analysis and involve the effect of the chemical compounds on human fibroblasts. This assay measures the host cell ATP content as a readout for cytotoxicity. When available, we annotated further the CPE data with the results of this assay. To be able to quickly repurpose a compound, we focused essentially on molecules that have been approved or are in clinical trials (in some instances, drugs are only approved for veterinary use). We thus cross-linked the CPE sample ID numbers to drug names (eg, international nonproprietary names), PubChem SID or CID numbers23 (link) or to ChEMBL ID numbers24 (link) and to molecules that possess ATC (Anatomical Therapeutic Chemical Classification system) codes and/or have documented therapeutic indications as found in DrugBank,25 (link) DrugCentral26 (link) and eDrug3d.27 (link) Molecules that are at the preclinical stage (eg, chemical probes) were essentially excluded via this procedure. Additionally, the salts, when present in the chemical file, were deleted using the Maya chemistry toolkit,28 (link) and the chemistry of the molecules was standardized using our FAF-Drugs4 web server.29 (link) The drugs in duplicate were removed. Further curation of the CPE data also involved the following: i) removal of a few molecules without potency values (AC50 values are reported for this dataset, ie, concentration for half-maximal activity30 (link)), ii) investigation of the curve class (ie, the evaluation of the quality of the dose–response curve and thus the quality of the measurements) in the CPE data (a curve class of type 4 is a flat curve indicating no activity) and maximum response (ie, maximum response value detected in the experiment). In such assay, compounds are usually considered active when the maximum response (absolute) value is above 30.31 Further, we removed molecules that were found highly cytotoxic in the Vero E6 counterscreen assay. From the initial CPE data containing 10,721 assessed samples (some molecules have been screened several times and most are chemical probes), we ended up with a list of 198 bioactive drugs. In this list of drugs, 93 were not tested for toxicity on human fibroblast and for the remaining 105 molecules, only one was found cytotoxic (Disulfiram, used for alcohol addiction, this molecule was also removed not only because of its cytotoxic nature in these assays but also as found to be a promiscuous SARS-CoV–2 main protease inhibitor32 (link)). At this stage of the analysis, we decided to retain all the compounds including those not annotated for human fibroblast toxicity. We then focused our attention on families of molecules that contain several members and removed some molecules with highly specific disease indication such as Deferasirox (an oral iron chelator given to patients receiving long-term blood transfusions). In this last step of data curation, we grouped the 198 compounds according to chemistry and disease indications and removed molecules that did not fulfill our selection criteria. This step led to the selection of a final list of 161 molecules reported in Table 1. Numerous approaches can be used to investigate low molecular weight chemical compounds in silico (eg,33–35 (link)) and here we decided to analyze these molecules with DataWarrior.36 (link) In this final list, very few molecules are only chemical probes, like Calpeptin (moderate activity against SARS-CoV-2 main protease M^pro). They were kept because they are often used to assess some enzymatic activities. The structural classification of chemical entities was then performed using the ClassyFire application.37 (link)Table 1

Investigated Chemical Probes and Approved Drugs

Name	Indication	AC50 (uM)	Fibroblast-Toxicity	ClinicalTrials.gov (Feb 24, 2021)	cLogP	Simple Rule-Based Estimation of Basic Nitrogen Atoms
Abemaciclib	Cancer	1.412537545	Not determined (ND)	No	4.1154	4
Acetopromazine	Psychotropic	12.58925412	No (N)	No	3.8721	1
Acolbifene	Cancer	11.22018454	ND	No	4.9846	1
Amiodarone	Cardiovascular	8.912509381	N	Yes	6.2801	1
Amitriptyline	Psychotropic	12.58925412	N	No	4.4056	1
Amlodipine	Cardiovascular	12.58925412	ND	Yes	2.071	1
Amodiaquine	Infectious	1.584893193	ND	Yes	4.2018	2
Amoxapine	Psychotropic	8.912509381	N	No	3.3035	2
Andrographolide	Infectious	11.22018454	ND	No	1.883	0
Anidulafungin	Infectious	5.623413252	ND	No	−0.4951	0
Apilimod	Infectious	12.58925412	ND	Yes	5.1942	2
Aprindine	Cardiovascular	8.912509381	N	No	4.5009	1
Arbidol (or Umifenovir)	Infectious	8.912509381	ND	Yes	4.1689	1
Aripiprazole	Psychotropic	12.58925412	N	No	4.4351	1
Asenapine	Psychotropic	10	ND	No	4.9405	1
Ataciguat (experimental)	Cardiovascular	12.58925412	ND	No	1.8036	0
Azelastine	Antihistamine	11.22018454	N	No	4.3744	1
Azithromycin	Infectious	11.22018454	N	Yes	1.6569	2
Bazedoxifene	Bone problems and possibly Cancer	3.981071706	N	No	5.6968	1
Benazepril	Cardiovascular	10	N	Yes	0.8715	1
Bencyclane	Cardiovascular	12.58925412	N	No	3.8639	1
Bepridil	Cardiovascular	8.912509381	N	No	4.4044	1
Berbamine (experimental)	Cancer	1.412537545	ND	No	6.2253	2
Berzosertib (experimental)	Cancer	2.238721139	ND	No	1.669	1
Bexarotene	Cancer	12.58925412	N	No	4.799	0
Bifemelane (approved Japan)	Psychotropic	12.58925412	N	No	3.7476	1
Blonanserin (Japan)	Psychotropic	5.623413252	N	No	5.3682	2
Brexpiprazole	Psychotropic	11.22018454	ND	No	3.8983	1
Bromodiphenhydramine	Antihistamine	12.58925412	N	No	3.631	1
Buclizine	Antihistamine	12.58925412	N	No	6.6827	2
Calpeptin (experimental)	Cancer	0.316227766	ND	No	2.8358	0
Carvedilol	Cardiovascular	11.22018454	ND	Yes	3.1668	1
Cenicriviroc	Infectious	8.912509381	ND	Yes	7.2606	1
Ceritinib	Cancer	10	ND	No	5.5817	3
Chlorcyclizine	Antihistamine	12.58925412	N	No	3.6827	2
Chloroquine	Infectious	3.981071706	N	Yes	4.0091	2
Chloroxine	Infectious	12.58925412	N	No	2.8419	0
Chlorpromazine	Psychotropic	8.912509381	N	Yes	4.6069	1
Chlorprothixene	Psychotropic	3.548133892	N	No	5.1216	1
Ciclopirox	Infectious	5.011872336	N	No	1.4285	0
Cilnidipine	Cardiovascular	11.22018454	N	No	3.6409	0
Clemastine	Antihistamine	11.22018454	N	No	4.5988	1
Clioquinol (withdrawn)	Infectious	10	N	No	2.673	0
Clofazimine	Infectious	6.309573445	ND	Yes	6.2781	1
Clomipramine	Psychotropic	7.943282347	N	No	4.4969	1
Closantel (veterinary drug, likely toxic in humans)	Infectious	11.22018454	N	No	6.4242	0
Clozapine	Psychotropic	12.58925412	N	No	3.2447	2
Cyclobenzaprine	Initially used as Psychotropic	10	N	No	4.3534	1
Cyproheptadine	Antihistamine	3.981071706	N	No	4.5999	1
Cysmethynil (experimental)	Cancer	10	ND	No	5.9027	0
Dabrafenib	Cancer	12.58925412	ND	No	3.666	0
Danazol	Cancer and endometriosis	12.58925412	N	No	3.4638	0
Dapoxetine	Initially psychotropic agent but now premature ejaculation	12.58925412	N	No	4.2367	1
Deserpidine	Cardiovascular	12.58925412	ND	No	3.6454	1
Desipramine	Psychotropic	8.912509381	N	No	3.6244	1
Desloratadine	Antihistamine	11.22018454	N	No	4.0583	1
Desmethylclozapine (metabolite of clozapine)	Psychotropic	8.912509381	ND	No	2.9918	2
Dexanabinol (experimental)	Cancer	10	ND	No	6.9355	0
Difeterol	Antihistamine	12.58925412	N	No	4.0432	1
Doramectin (veterinary drug)	Infectious	12.58925412	ND	No	5.3516	0
Dosulepin	Psychotropic	12.58925412	ND	No	3.8201	1
Doxazosin	Cancer and hypertension	12.58925412	N	Yes	2.1379	2
Duloxetine	Psychotropic	8.912509381	N	No	3.8368	1
Efavirenz	Infectious	10	N	No	3.6614	0
Emodepside (veterinary drug)	Infectious	7.079457844	ND	No	5.237	0
Enzastaurin (experimental)	Cancer	12.58925412	N	No	3.1099	1
Flumatinib (experimental)	Cancer	7.943282347	ND	No	3.8397	2
Flunarizine	Antihistamine	11.22018454	N	No	5.4576	2
Fluoxetine	Psychotropic	4.466835922	N	Yes	3.6241	1
Fluphenazine-decanoate	Psychotropic	12.58925412	N	No	8.527	2
Fonazine (Japan and Europe)	Antihistamine	28.18382931	ND	No	3.1244	1
Halofantrine	Infectious	11.22018454	N	No	8.1762	1
Haloperidol	Psychotropic	25.11886432	N	No	4.3049	1
Hesperadin (experimental)	Cancer	12.58925412	ND	No	2.3768	1
Hexachlorophene	Infectious	0.223872114	N	No	6.4201	0
Hexetidine	Infectious	3.548133892	N	No	5.2587	1
Homochlorcyclizine (Japan)	Antihistamine	5.011872336	N	No	4.0247	2
Hycanthone	Infectious	12.58925412	ND	No	3.3212	1
Hydroquinidine	Cardiovascular	12.58925412	ND	No	2.7959	1
Icaritin	Cancer	12.58925412	ND	No	4.1341	0
Iloperidone	Psychotropic	12.58925412	N	No	4.3044	1
Imatinib	Cancer	5.623413252	N	Yes	3.9383	2
Imipramine	Psychotropic	8.912509381	N	No	3.8909	1
Lapatinib	Cancer	10	N	No	4.7281	1
Lemildipine (experimental)	Cardiovascular	12.58925412	N	No	3.3887	0
Lercanidipine	Cardiovascular	11.22018454	N	No	4.7102	1
Letermovir	Infectious	10	ND	No	4.8615	1
Lomerizine	Antihistamine (treatment of migraines)	12.58925412	N	No	4.4864	2
Lopinavir	Infectious	12.58925412	N	Yes	4.847	0
Loratadine	Antihistamine	10	N	No	4.9774	0
Loxapine	Psychotropic	10	N	No	3.5564	1
Manassantin-A (experimental)	Cancer	3.16227766	ND	No	5.8318	0
Manidipine	Cardiovascular	8.912509381	N	No	2.6132	2
Maprotiline	Psychotropic	8.912509381	N	No	3.7029	1
Masitinib	Cancer	11.22018454	ND	Yes	4.7573	3
Mefloquine	Infectious	11.22018454	ND	Yes	3.5076	1
Melitracen	Psychotropic	11.22018454	ND	No	4.6647	1
Merimepodib	Infectious	12.58925412	ND	Yes	2.7196	0
Mesoridazine	Psychotropic	10	N	No	4.5579	1
Methdilazine	Antihistamine	7.079457844	ND	No	4.2005	1
Methotrimeprazine	Psychotropic	12.58925412	ND	No	4.1492	1
Momelotinib (experimental)	Cancer	12.58925412	ND	No	2.5062	0
Monatepil (experimental)	Cardiovascular	12.58925412	N	No	4.7269	1
Nafronyl	Psychotropic	12.58925412	ND	No	4.2981	1
Naftopidil	Cancer	12.58925412	N	No	3.42	1
Nicardipine	Cardiovascular	10	N	No	1.5582	1
Niraparib	Cancer	12.58925412	ND	No	2.0619	1
Nirogacestat	Cancer	12.58925412	ND	No	4.3631	3
Nitazoxanide	Infectious	3.16227766	N	Yes	1.6871	0
Nitroxoline	Infectious	12.58925412	ND	No	0.7083	0
Oxatomide	Antihistamine	10	N	No	4.2761	2
Pamelor	Psychotropic	12.58925412	ND	No	4.1391	1
Parthenolide	Cancer	10	ND	No	2.7525	0
Pazopanib	Cancer	12.58925412	ND	No	0.9125	2
Periciazine	Psychotropic	10	ND	No	4.1174	1
Perphenazine	Antihistamine and psychotropic	12.58925412	N	No	4.1649	2
Pipequaline (experimental)	Psychotropic	12.58925412	ND	No	4.9763	1
Piperacetazine (prodrug)	Psychotropic	12.58925412	N	No	4.8021	1
Piroctone (experimental)	Infectious and Cancer	7.943282347	ND	No	2.9044	0
Pizotifen	Psychotropic and antihistamine	10	N	No	4.5187	1
Pluripotin (experimental)	Cancer	12.58925412	ND	No	4.2874	0
Prochlorperazine	Psychotropic	8.912509381	N	No	4.6853	2
Promazine	Psychotropic	10	N	No	4.0009	1
Promethazine	Antihistamine	8.912509381	N	Yes	3.9058	1
Propafenone	Cardiovascular	12.58925412	N	No	3.0837	1
Propionylpromazine (veterinary drug)	Antihistamine and neuroleptic	10	N	No	4.3265	1
Protriptyline	Psychotropic	12.58925412	N	No	3.6848	1
Pyrimethamine	Infectious	4.466835922	ND	No	2.5414	2
Quinidine	Cardiovascular	12.58925412	ND	No	2.6104	1
Quizartinib (experimental)	Cancer	2.511886432	ND	No	4.7979	2
Refametinib (experimental)	Cancer	11.22018454	ND	No	1.88	0
Remdesivir	Infectious	7.943282347	ND	Yes	0.3048	0
Rescimetol (Japan)	Cardiovascular	12.58925412	N	No	3.6989	1
Reserpine	Cardiovascular	11.22018454	N	Yes	3.5754	1
Retapamulin	Infectious	12.58925412	ND	No	5.218	1
Sarpogrelate	Cardiovascular	12.58925412	N	No	1.8567	1
Serdemetan (experimental)	Cancer	8.912509381	ND	No	3.7237	1
Siccanin (experimental)	Infectious	12.58925412	ND	No	4.3637	0
Spiclomazine (experimental)	Cancer	12.58925412	ND	No	5.2169	1
Spiperone (Japan)	Psychotropic	7.943282347	N	No	3.0219	1
Spiramycin-II	Infectious	12.58925412	N	No	2.4608	2
Sulfatinib (experimental)	Cancer	12.58925412	ND	No	2.0918	1
Teicoplanin	Infectious	14.12537545	ND	Yes	−3.4194	1
Thioproperazine	Psychotropic	11.22018454	N	No	3.2979	2
Thiothixene	Psychotropic	12.58925412	ND	No	2.937	2
Tilorone	Infectious	10	ND	No	4.0607	2
Timiperone (Japan)	Psychotropic	10	ND	No	3.7407	1
Tioguanine	Cancer	10	ND	No	−0.9683	1
Tipifarnib (experimental)	Cancer	12.58925412	ND	No	4.03	2
Tivantinib (experimental)	Cancer	12.58925412	ND	No	4.1837	0
Tizoxanide	Infectious	3.16227766	ND	Yes	1.3547	0
Triamterene	Cardiovascular	7.079457844	N	Yes	0.6075	2
Trifluomeprazine	Psychotropic	10	ND	No	5.0675	1
Trifluoperazine	Psychotropic	12.58925412	ND	No	4.9276	2
Triflupromazine	Psychotropic	11.22018454	N	No	4.8492	1
Trimeprazine (or Alimemazine)	Psychotropic	8.912509381	N	No	4.2192	1
Trimetrexate	Cancer	10	ND	No	1.9786	2
Trimipramine	Psychotropic	10	N	No	4.1092	1
Vilazodone	Psychotropic	10	ND	No	3.6629	1
Vorapaxar	Cardiovascular	8.912509381	ND	No	4.7865	0
Zotepine	Psychotropic	12.58925412	N	No	4.6741	1

Notes: The names of the 161 selected bioactive molecules are reported in the first column. For each compound, the main indication, the experimental AC50 values (see text), the experimental fibroblast toxicity (ND, not determined, N, No), the computed log P (cLogP), and a simple estimation of the number of basic nitrogen atoms are shown. Also, molecules that are in clinical trials for COVID-19 treatments indicated; Experimental and investigational drugs are all labelled in this Table experimental.

Villoutreix B.O., Krishnamoorthy R., Tamouza R., Leboyer M, & Beaune P. (2021). Chemoinformatic Analysis of Psychotropic and Antihistaminic Drugs in the Light of Experimental Anti-SARS-CoV-2 Activities. Advances and Applications in Bioinformatics and Chemistry : AABC, 14, 71-85.

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Fluorescence Polarization Assay for Mpro Inhibitor Screening

In this FP screening assay, 29 µL sample of 400 nM Mpro diluted in the FP assay buffer was mixed with 1 µL of natural product (1 mg/mL in DMSO) in a black 384-well microplate, and the mixture was further incubated for 35 min at RT before adding 20 µL sample of 60 nM FP tracer. After proceeding for 20 min at RT, the reaction was quenched by addition of 10 µL sample of 300 nM avidin, and the mP values were measured by a microplate reader. In each assay plate, GC-376 (1 µM) and DMSO were used as a positive and negative control, respectively. The well containing only 60 µL sample of 20 nM FITC-AVLQ peptide was used to assess the background noise. The inhibitory activity of screening compound was calculated using Eq. (1), and the candidate compounds (> 50% inhibition) were analyzed in triplicate to generate the dose-response curves in the second screening.
The inhibitory activity of dieckol in this FP screening assay was carried out as mentioned above. The initial concentration of dieckol was 100 µM, and 8 two-fold dilutions were prepared for the determination of IC₅₀ value. To remove the DTT effect on the enzymatic assay [26 (link)], the IC₅₀ value was also obtained in the absence of DTT in a FP screening assay.

Yan G., Li D., Lin Y., Fu Z., Qi H., Liu X., Zhang J., Si S, & Chen Y. (2021). Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors. Cell & Bioscience, 11, 199.

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Variable analysis

independent variables

Expression of Flippase in specific cells
Expression of UAS-EcR-B1-d655W650A
Expression of UAS-TNT-G
Expression of UAS-IMPTNT-V
Expression of UAS-shi^ts1
Expression of UAS-EGFP-Kir2.1

dependent variables

Recombination at the FRT sites
Expression of GFP under Tub-Gal4 control
Silencing of neuronal activity

control variables

Genotype of flies (RN2-Flp, Tub-FRT-CD2-FRT-Gal4, UAS-mCD8-GFP)
Developmental stage of flies (white prepupae, 0 hours APF)
Temperature (25°C, except for shi^ts1 experiments which were at 29°C)

positive controls

Expression of inactive toxin IMPTNT-V as a control for UAS-TNT-G experiments

negative controls

None mentioned

Annotations

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