DNA-encoded chemistry technology yields expedient access to SARS-CoV-2 Mpro inhibitors

Significance SARS-CoV-2 has had a crippling impact on human life globally. Vaccine development has been used as a first-line strategy for COVID-19 prevention and mitigation; however, small-molecule drugs are still vitally needed to extend treatment options. Traditional screening methods for identifying biologically active small molecules are sluggish and often sample an insufficient number of compounds to identify suitable hits. Here, we applied a screening method known as DNA-encoded chemistry technology (DEC-Tec) to screen billions of compounds against a critical viral protein, Mpro. In rapid fashion, we identified the compound CDD-1713 as a potent and selective Mpro inhibitor. This study illuminates DEC-Tec as a highly expeditious strategy toward generating small molecules against critical targets of infectious agents.

. Enzymatic activity data, a Less than 50% inhibition observed with 25 µM compound added for initial screening; Inactive= Compounds that inhibited M pro activity by less than 90% with 25 µM compound were considered inactive Timeline of DEL process Protein expression and purification 1-2 weeks DEL Selection against M pro and Data analysis 1-2 weeks Off DNA synthesis and validation 2-3 weeks Antiviral testing 2 weeks Table S2. Approximate time line of entire process from protein production to antiviral testing Figure S6. Potential off-target inhibition of major proteases, i.e. cathepsin B (a cysteine protease like M pro ), thrombin (a serine protease), renin (an aspartic protease), and matrix metallopeptidase 1 (MMP-1), was tested with all active compounds shown here with CDD-1713 and CDD-1976 and control inhibitors as indicated. The best inhibition was observed by CDD-1713 of renin with an estimated Kiapp of 53 uM, calculated as described in methods section.  The HepG2 cells were incubated with CDD-1713 and CDD-1976 (0-100 µM) for 24 hours at 37 °C. Cell viability was measured with XTT assay. XTT readings were normalized by the control group (DMSO) to give the normalized viabilities. The IC50 values were expressed as ">100 µM" for CDD-1713 and CDD-1976, as the cell viabilities were larger than 80% in the 100 µM group for both compounds.

General Information of DECL
The general materials, procedures and equipment utilized in this study referenced the related DECL work in our group reported previously 1,2,3,4,5,6,7,8 or other DECL publications. 9,10,11 Materials and equipment used for the DNA-encoded chemical libraries. The starting unit dsDNA oligonucleotide with modified phosphates with PEG4 linker and terminal amine (DEC-Tec Starting Unit/DTSU, S1) and encoding 5'-phosphorylated oligonucleotides were purchased from LGC Biosearch Technologies. A "spike-in" with 10-mer DNA oligonucleotide featuring a Structure of DTSU S1 (5'-Phos-CTGCAT-Spacer 9-Amino C7 plus AOP-Spacer 9-ATGCAGGT

3').
General procedure for the analysis of DNA oligonucleotides. DNA sample or reaction mixture were diluted to 10 μM final concentration and injected in amounts of 5-10 µL on a Vanquish/LTQ system. reporting software using ZNova novel algorithm to produce artifact-free mass spectra.

Synthesis of a DNA-Encoded Chemical Library (DECL)
Architecture of the main library build and Building block diversity analysis. Similar strategy for the two aspects were adopted from previous reported literature. Suzuki coupling and Sonogashira coupling need to be removed by centrifugation for further ligation to be proceeded successfully.

Preparation of amplifiable DECL samples for further selection experiments.
After completion of the main library builds, the entire library material was ligated with a duplexed pair of 12-mer DNA oligonucleotides (library ID) to encode the overall library construct. After ethanol precipitation, the DECL material underwent sequential ligation with DNA oligonucleotides, containing a region to encode selection experiment, a degenerate region as molecular identifier during amplification, and a reverse primer region for post-selection PCR amplification (the purposes/design of these components are discussed in our previous publication).

General Methods for off DNA Synthesis:
All starting materials and reagents were purchased from commercial sources and used without further purification. Solvents were purchased as either anhydrous grade products in sealed containers or reagent grade and used as received. All reactions were carried out in dry glassware under a nitrogen atmosphere using standard disposable or gastight syringes, disposable or stainless steel needles, and septa. Stirring was achieved with magnetic stir bars. Flash column chromatography was performed with SiO2 (230-400 mesh) or by using an automated chromatography instrument with an appropriately sized column. Thin layer chromatography was performed on silica gel 60F254 plates (E. Merck). Non-UV active compounds were visualized on TLC using one of the following stains: KMnO4, ninhydrin, p-anisaldehyde. 1 H and 13 C NMR spectra were recorded on an instrument operating at either 600MHz, and 151MHz respectively. LCMS data were collected using an HPLC instrument coupled to a low resolution mass spectrometer with single quadrupole ionization operating in either positive or negative ion mode. The analytical method utilized a C18 column (2.1 × 50 mm, 1.8 μm) eluting with a linear gradient of 95%/5% water/CH3CN (modified with 0.05% formic acid; T = 0 min flow = 0.35 mL/min) to 95%/5% CH3CN/water (T = 3.5 min flow = 0.5 mL/min) then 95%/5% CH3CN/water to T = 5min (0.5 mL/min). Peak detection was done at 254 nm and 230 nm for UV active compounds. Highresolution mass spectrometry (HRMS) spectra were obtained on a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer equipped with a HESI source and using lock masses for correction. Samples were introduced into the HRMS via reversed phase HPLC on an Accucore Vanquish C18+ column (2.1 × 100 mm, 1.5 μm) eluting with a linear gradient of 95%/5% water/acetonitrile (modified with 0.1% formic acid) to 10%/90% water/acetonitrile over 8 min.

Experimental procedures and NMR data:
General procedure-1 (amidation); into a round bottom flask equipped with magnetic stir bar and septum under nitrogen, the acid compound (1 equiv.) was dissolved in DMF, amine (1.1 equiv.) was added followed by DIPEA (1.5 equiv.) and HATU (1.1 equiv.) at room temperature. The reaction was allowed to stir for 16h, after which time TLC and LCMS indicated complete consumption of starting material. The reaction was worked up by diluting with ethyl acetate and washed with Sat. aq NaHCO3 and brine. The organic phase was collected and dried over anhydrous Na2SO4. Filtered and the solvent was removed under reduced pressure to give the crude product, which was used in next reaction without further purification.
General procedure-2 (-Boc-removal); 12 into a round bottom flask equipped with magnetic stir bar and septum, the Boc-compound (1 equiv.) was dissolved in dichloromethane and TFA (10% TFA in DCM, V/V, 10 mL/mmol) was added and allowed to stir at room temperature under nitrogen. After 2h the starting material was consumed according to TLC and LCMS. The volatiles were evaporated under reduced pressure and then redissolved in toluene and evaporated to remove excess TFA. The crude product as TFA salt was used in next reaction without further purification.
General procedure-3 (O-alkylation); 13 into a microwave vial equipped with magnetic stir bar and septum under nitrogen, the phenol compound (1 equiv.) was dissolved in acetone and 2-bromo-N,N-dimethylacetamide (1.1 equiv.) was added followed by K2CO3 (1.5 equiv.) and tetrabutylammonium iodide (TBAI; 0.1 equiv.). The reaction vial was sealed and allowed to heat at 65 °C for 2h, after which time TLC and LCMS indicated complete consumption of starting material. The reaction was worked up by diluting with ethyl acetate and washed with water and brine. The organic phase was collected and dried over anhydrous Na2SO4. Filtered and the solvent was removed under reduced pressure to give the crude residue. Purification by silica gel chromatography (ethyl acetate/ hexanes) provided the pure product.
General procedure-4 (hydroxy ketone synthesis); 14 into a round bottom flask equipped with magnetic stir bar and septum, the methyl ketone (1 equiv.) was dissolved in methanol at 0 °C and added powdered potassium hydroxide (6 equiv.) followed by Iodobenzene diacetate (1.1 equiv.). The mixture was warmed to room temperature, stirred for 3 h, and then evaporated to dryness under reduced pressure. The reaction was worked up by diluting with ethyl acetate and washed with water. The organic phase was collected and dried over anhydrous Na2SO4. Filtered and thesolvent was removed under reduced pressure to give the crude residue. The residue was dissolved in a mixture of methanol and aqueous hydrochloric acid (2M) and stirred overnight at rt, after that time evaporated the reaction mixture to dryness under reduced pressure The reaction was worked up by diluting with ethyl acetate and washed with water. The organic phase was collected and dried over anhydrous Na2SO4. Filtered and the solvent was removed under reduced pressure to give the crude residue. Purification by silica gel chromatography (ethyl acetate/ hexanes) provided the pure product.
General procedure-5 (Suzuki reaction); 15 the arylbromide (1 equiv.), boronic acid (1.3 equiv.), K2CO3 (1.5 equiv.) and Pd(dppf)Cl2 . DCM complex (0.1 equiv.) were placed in a vial equipped with a stir bar. The vial was sealed with a septum screw-cap, and then it was evacuated and filled with nitrogen (three cycles). 1, 4-dioxane and water (5:1 ratio) was added, and the resulting homogeneous reaction mixture was stirred vigorously at 110 °C for 1h. After which time TLC and LCMS indicated complete consumption of starting material. The reaction was worked up by diluting with ethyl acetate and washed with water and brine. The organic phase was collected and dried over anhydrous Na2SO4. Filtered and the solvent was removed under reduced pressure to give the crude residue. Purification by silica gel chromatography (ethyl acetate/ hexanes) provided the pure product.
General procedure-6 (aldehyde reduction); into a round bottom flask equipped with magnetic stir bar and septum under nitrogen, the aldehyde compound (1 equiv.) was dissolved in dry THF, solid NaBH4 (1.5 equiv.) was added at 0 °C. After addition the reaction was allowed to stir at rt for 3 h, after which time TLC and LCMS indicated complete consumption of starting material. The reaction was worked up by diluting with EtOAc and washed with water and brine. The organic phase was collected and dried over anhydrous Na2SO4. Filtered and the solvent was removed under reduced pressure to give the crude residue. Purification by silica gel chromatography (ethyl acetate/ hexanes) provided the pure product.
General procedure-7 (reductive amination); 12 into a round bottom flask equipped with magnetic stir bar and septum under nitrogen, the aldehyde compound (1 equiv.) was dissolved in DCM, treated with methylamine (2M solution in THF, 1.5 equiv.) followed by AcOH (0.2 equiv.) at rt. The solution was stirred at rt for 20 minutes and then treated with sodium triacetoxyborohydride (STAB, 1.5 equiv.). The reaction was stirred at rt for 16 h. LCMS confirmed complete consumption of starting material. The reaction was worked up by diluting with DCM and washed with aq. sodium bicarbonate. The organic phase was collected and dried over anhydrous Na2SO4. Filtered and the solvent was removed under reduced pressure to give the crude residue. Purification by silica gel chromatography (ethyl acetate/ hexanes) provided the pure product.
General procedure-8 (Wittig); 16 into a round bottom flask equipped with magnetic stir bar and septum under nitrogen, the aldehyde compound (1 equiv.) was dissolved in dry DCM, (ethoxycarbonylmethylene)triphenylphosphorane (1.1 equiv.) was added at rt. The reaction was allowed to stir for 16 h, after which time TLC and LCMS indicated complete consumption of starting material. The reaction was worked up by diluting with DCM and washed with water and brine. The organic phase was collected and dried over anhydrous Na2SO4. Filtered and the solvent was removed under reduced pressure to give the crude residue. Purification by silica gel chromatography (ethyl acetate/ hexanes) provided the pure product.