NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.
Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.
ML300 (CID 46861530) is being declared as a new first in class 3CLpro probe inhibitor. ML300 belongs to a new series of triazole-based SARS main proteinase 3CLpro inhibitors that follow our first dipeptide probe ML188. Based upon X-ray crystal data from a related inhibitor, ML300 is believed to interact within the 3CLpro enzyme active site via a novel binding mode distinct from both our first probe ML188 and other known peptidomimetic inhibitors. Relative to ML188 and based upon X-ray crystal data, the MW of the triazole series described here can be reduced resulting in truncation to a single backbone amide. Structure-activity relationships taking into account ligand efficiency and studies leading to the identification of ML300 will be described. ML300 should allow others in the field to access a novel non-covalent starting point for 3CLpro inhibitor design and optimization.
Assigned Assay Grant #: MH084162
Screening Center Name & PI: Scripps Research Institute Molecular Screening Center, Hugh Rosen
Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley
Assay Submitter & Institution: Andrew Mesecar, Purdue University
PubChem Summary Bioassay Identifier (AID): 1859
Probe Structure & Characteristics
| CID/ML# | Target Name | IC50/EC50(nM) [SID, AID] | Anti-target Name(s) | IC50/EC50 (μM) [SID, AID] | Fold Selective | Secondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]§ |
|---|---|---|---|---|---|---|
| CID 46861530/ML300 | 3CLpro | 1500 [SID 99289112,AID 488967, AID 488958] | none | IC50 = 4.11±0.24 | NA | NA |
1. Recommendations for Scientific Use of the Probe
This probe (ML300, CID 46861530) represents a second class of non-covalent small molecule inhibitors of the SARS main proteinase 3CLpro using a simple dipeptide backbone. This probe possesses excellent selectivity versus a large panel of GPCRs, ion channels and transporters. In addition, based upon X-ray crystallography data of 3CLpro bound inhibitor SID 24808289 from the ML300 series, this probe displays a novel mode of inhibition at the active site distinct from our previous probe ML188. As a result of the unique interaction and SAR observed against 3CLpro by ML300 within this triazole, a unique opportunity to identify potent inhibitors of 3CLpro and potentially related PL enzymes from other coronavirus strains exists. To date, the coronavirus 3CL/PL field has been limited by probes which are high MW peptidomimetics with weak inhibition of 3CLpro and/or probes containing reactive covalent modifier groups. ML300 does not contain any apparent reactive functional groups and contains only one backbone amide, an improvement over the initial triazole lead SID 24808289, which contains two backbone amides occupying four residue pockets of the enzyme. Further, cellular studies using ML300 will provide insight into their potential relative to existing tool compounds including ML188.
In addition to structural insights provided by ML188, we believe the SAR and structure details provided by the ML300 analog SID 24808289 will provide a means upon which to facilitate rapid structure-based inhibitor optimization. In addition, this probe will be of use in examining its activity against other emerging CoV strains expressing 3CLpro. This includes the recently identified SAR-like virus called HCoV-EMC, which has been shown to be lethal in the few cases reported to date.
2. Materials and Methods
2.1. Assays
| AID | Name |
|---|---|
| AID 1706 | QFRET-based primary biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro) |
| AID 1859 | Summary of probe development efforts to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro) |
| AID 1879 | QFRET-based confirmation biochemical high throughput screening assay for inhibitors of the SARS coronavirus 3C-like Protease (3CLPro) |
| AID 1890 | QFRET-based dose response biochemical high throughput screening assay to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro) |
| AID 1944 | Luminescence-based counterscreen for inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): dose response biochemical high throughput screening assay to identify inhibitors of the papain-like protease |
| AID 435015 | Late stage counterscreen results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro); luminescence-based cell-based assay to identify cytotoxic compounds in Vero E6 cells. |
| AID 488958 | Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical dose-response assay for inhibitors of 3CLPro. |
| AID 488877 | Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): luminescence-based dose-response cell-based assay for restoration of viability of SARS-CoV-infected Vero cells. |
| AID 488967 | Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical assay for inhibitors of 3CLPro. |
| AID 488984 | Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical assay for inhibitors of 3CLPro: Set 2 |
| AID 493245 | Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical assay for inhibitors of 3CLPro: Set 3 |
| AID 488999 | Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical dose-response assay for inhibitors of 3CLPro; Set 2 |
| AID 588772 | Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical assay for inhibitors of 3CLPro: Set 4 |
| AID 588771 | Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical dose-response assay for inhibitors of 3CLPro; Set 3. |
| AID 588786 | Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical dose-response assay for inhibitors of 3CLPro; Set 4. |
| AID 602487 | Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical assay for inhibitors of 3CLPro: Set 5 |
| AID 602486 | Late stage assay provider results from the probe development effort to identify inhibitors of the SARS coronavirus 3C-like Protease (3CLPro): fluorescence-based biochemical dose-response assay for inhibitors of 3CLPro; Set 5 |
2.2. Probe Chemical Characterization
Probe compound ML300 was prepared according to the scheme in Figure 1 and provided the following characterization data: LC-MS (>98%) m/z = 432.1 [M+H]; 1H NMR (400 MHz, CDCl3) δ 8.05 (1H, s), 8.02 (1H, d, J = 8.4 Hz), 7.61 (2H, d, J = 8.6 Hz), 7.49 (2H, m), 7.36 (1H, p, J = 4.2 Hz), 7.23 (1H, m), 7.02 (3H, m), 6.95 (1H, d, J = 4.92 Hz), 5.15 (2H, s), 4.84 (2H, s), 1.55 (1H, m), 1.09 (2H, m), 0.85 (2H, m); 13C NMR (100 MHz, CDCl3) δ 172.6, 164.9, 145.7, 139.3, 136.7, 134.9, 133.7, 128.7, 128.1, 127.8, 126.2, 124.4, 124.1, 120.9, 119.7, 109.8, 49.7, 48.4, 15.5, 8.2; HRMS (ESI) m/z 432.1497 ([M+H]+, 100%) calcd for C23H22N5O2S, 432.1494.
Figure 1
Synthetic procedure and spectral data for ML300 (CID 46861530).
Solubility. Solubility in PBS at pH 7.4 was determined to be 29 μM or 13 μg/mL. ML300 shows good solubility up >100 μM DMSO and up to 40 mM DMSO which is currently used for original stocks of novel compounds shipped to the assay provider for testing. In addition, solubility in organic solvents for ML300 versus ML188 appears to be improved.
Stability. Stability was determined for ML300 in PBS buffer at room temperature with both 5 and 10% DMSO. ML300 was found to be completely stable (>99% intact) after 48h.
Compounds added to the SMR collection (MLS#s): MLS004084528 (ML300, CID 46861530, 20.4 mg); MLS004084529 (CID 46861523, 8.8 mg); MLS004084530 (CID 45382022, 5.5 mg); MLS004084531 (CID 45382030, 7.4 mg); MLS004084532 (CID 46861528, 16.1 mg); MLS004084533 (CID 46861529, 17.6 mg).
2.3. Probe Preparation
N-(4-(2-(1H-benzo[d][1,2,3]triazol-1-yl)-N-(thiophen-3- ylmethyl)acetamido)phenyl)cyclopropanecarboxamide (ML300, CID 46861530)
Step 1: Preparation of tert-butyl (4-((thiophen-3-ylmethyl)amino)phenyl)carbamate. 3-thiophenecarboxylic acid (0.383 mL, 4.4 mmol, 1.0 equiv) and N-Boc-p-phenylenediamine (1.12 g, 5.4 mmol, 1.22 equiv) were dissolved in dichloroethane (44 mL, 0.1 M) in a 100 mL round bottom flask. Sodium triacetoxyborohydride (1.43 g, 6.7 mmol, 1.52 equiv) was added and the reaction was stirred at room temperature for 1 h. The reaction was determined to be complete by LC-MS, quenched with NH4Cl (sat’d, aqueous), and extracted with EtOAc (3 × 40 mL). The combined organic layers were dried over MgSO4, concentrated, and purified by silica gel column chromatography, eluting with EtOAc/hexanes. The title compound eluted at 35% EtOAc/hexanes and was isolated as a light yellow solid in quantitative yield (1.34 g). LC-MS (>98%) m/z = 305.2 [M+H].
Step 2: Preparation of tert-butyl (4-(2-(1H-benzo[d][1,2,3]triazol-1-yl)-N-(thiophen-3-ylmethyl)acetamido)phenyl)carbamate.Tert-butyl (4-((thiophen-3-ylmethyl)amino)phenyl)carbamate (1.1 g, 3.6 mmol, 1.0 equiv) and HATU (1.71 g, 4.5 mmol, 1.25 equiv) were combined in DMF (30 mL, 0.12 M) in a 100 mL round bottom flask. N,N-diisopropylethylamine (1.56 mL, 9 mmol, 2.5 equiv) was added and the reaction was stirred at room temperature for 10 min. 1-Benzotriazolylacetic acid (736 mg, 4.16, 1.15 equiv) was added and the reaction was stirred at room temperature for 16 h after which no further formation of the product was observed by LC-MS. The mixture was diluted with EtOAc (100 mL) and the organic layer was washed with water (3 × 25 mL). The organic layer was dried over MgSO4, concentrated under reduced pressure, and purified by silica gel column chromatography eluting with EtOAc/hexanes. The title compound eluted at 50% EtOAc/hexanes and was isolated as an off-white solid in 56% yield (931 mg). LC-MS (>98%) m/z = 464.2 [M+H].
Step 3: Preparation of N-(4-(2-(1H-benzo[d][1,2,3]triazol-1-yl)-N-(thiophen-3-ylmethyl)acetamido)phenyl)cyclopropanecarboxamide.Tert-butyl (4-(2-(1H-benzo[d][1,2,3]triazol-1-yl)-N-(thiophen-3-ylmethyl)acetamido)phenyl)carbamate (920 mg, 1.98 mmol, 1.0 equiv) was dissolved in CH2Cl2 (3 mL, 0.66 M) in a 20 mL scintillation vial. Trifluoroacetic acid (TFA, 2 mL, 26.1 mmol, 13 equiv) was added, and after stirring at room temperature for 1 h the starting material was determined to be consumed by LC-MS. The solvents were evaporated under reduced pressure and the resulting de-Boc protected aniline was used without purification. LC-MS (>98%) m/z = 364.1 [M+H].
The aniline (1.98 mmol, 1.0 equiv) was re-dissolved in CH2Cl2 (20 mL, 0.1 M) and Et3N (2.48 mL, 17.82 mmol, 9 equiv) was added. (Excess Et3N was used to neutralize any residual TFA, and the reaction mixture was determined to be basic by checking with pH paper.) Cyclopropanecarbonyl chloride (0.23 mL, 2.48, 1.25 equiv) was added and the reaction mixture was stirred for 1 h, after which the starting material was determined to be consumed by LC-MS. The reaction was quenched with NH4Cl (sat’d aqueous) and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were dried over MgSO4, concentrated, and purified by silica gel column chromatography eluting with EtOAc\hexanes. The title compound eluted at 75% EtOAc/hexanes to yield a tan solid in 39% yield (330 mg). LC-MS (>98%) m/z = 432.1 [M+H]; 1H NMR (400 MHz, CDCl3) δ 8.05 (1H, s), 8.02 (1H, d, J = 8.4 Hz), 7.61 (2H, d, J = 8.6 Hz), 7.49 (2H, m), 7.36 (1H, p, J = 4.2 Hz), 7.23 (1H, m), 7.02 (3H, m), 6.95 (1H, d, J = 4.92 Hz), 5.15 ( 2H, s), 4.84 (2H, s), 1.55 (1H, m), 1.09 (2H, m), 0.85 (2H, m); 13C NMR (100 MHz, CDCl3) δ 172.6, 164.9, 145.7, 139.3, 136.7, 134.9, 133.7, 128.7, 128.1, 127.8, 126.2, 124.4, 124.1, 120.9, 119.7, 109.8, 49.7, 48.4, 15.5, 8.2; HRMS (ESI) m/z 432.1497 ([M+H]+, 100%) calcd for C23H22N5O2S, 432.1494.
3. Results
3.1. Dose Response Curves for Probe
Figure 2M-M Inhibition Curve for ML300
3.2. Cellular Activity
Cellar activity measurements are still in progress using against SARS-CoV Urbani infected Vero E6 cells using ML300. Prior studies using ML188 suggest cellular activity above 10 micromolar is likely for ML300. Although it remains to be seen, based on the EC50/IC50 ratio observed for ML188 (<10) as a minimum, we expect improvements in potency below micromolar level will be required to achieve good cellular activity for the triazole series below 10 μM.
3.3. Profiling Assays
The probe molecule (ML188) was tested at Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM), and was found to have one hit against Melatonin (MT1) at 75% inhibition.
4. Discussion
4.1. Comparison to Existing Art and How the New Probe is an Improvement
Based on recent literature searches, a lack of legitimate non-covalent 3CLpro inhibitors continues to exist. The field has been limited mostly to covalently bound inhibitors.(1–5 and references therein) Among non-covalent inhibitors to date, these are primarily limited to large refined peptidomimetics with poor inhibitory activity (4) or so-called small molecules, that in fact maintain a covalent modifier group or reactive elements that would limit their potential for development as an antiviral. A summary of second generation small molecule 3CLpro inhibitors, some containing reactive warhead groups and are known to have a covalent mode of action, are included in Figure 3.
Figure 3
Second generation small molecule 3CLpro inhibitors. Some contain reactive warhead groups and are known to have a covalent mode of action
This probe molecule (ML300) is a non-covalent inhibitor of the SARS-CoV 3CLpro enzyme. It is selective against the related PLpro enzyme. This inhibitor can be synthetically accessed in a straight forward four-step sequence and no chiral chromatography is required. Current inhibitors within the covalent modifier class require multiple steps to access a final compound. The 3CLpro-ML188 and SID-289 X-ray crystal structures were critical in guiding the optimization process and encouraged the exploration of the P3-truncated effort within the benzotriazole series. With this additional probe 3CLpro inhibitor declared, a renewed interest to develop selective small molecule inhibitors against 3CLpro and other coronavirus proteinase targets will follow as the field continues to search for novel antivirals against SARS and SARS-like infections.
5. References
- 1.
- Anand K, Ziebuhr J, Wadhwani R, Mesters JR, Hilgenfeld R. Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs. Science. 2003;300:1763–1767. [PubMed: 12746549]
- 2.
- Ghosh AK, Xi K, Johnson ME, Baker SC, Mesecar AD. Progress in Anti-SARS Coronavirus Chemistry, Biology and Chemotherapy. Ann. Rep. Med. Chem. 2006;41:183–196. [PMC free article: PMC2718771] [PubMed: 19649165]
- 3.
- Ghosh AK, Gong G, Grum-Tokars V, Mulhearn DC, Baker SC, Coughlin M, Prabhakar BS, Sleeman K, Johnson ME, Mesecar AD. Design, synthesis and antiviral efficacy of a series of potent choropyridyl ester-derived SARS-CoV 3CLpro inhibitors. Bioorg Med Chem Lett. 2008;18:5684–5688. [PMC free article: PMC2745596] [PubMed: 18796354]
- 4.
- Wu C.-Y, Jan J.-T, Ma S.-H, Kuo C.-J, Juan H.-F, Cheng EY.-S, Hsu H.-H, Huang H.-C, Wu D, Brik A, Liang F.-S, Liu R.-S, Fang J.-M, Chen S.-T, Liang P.-H, Wong C.-H. Small molecules targeting severe acute respiratory syndrome human coronavirus. Proc Natl Acad Sci. 2004;101:10012. [PMC free article: PMC454157] [PubMed: 15226499]
- 5.
- Ghosh AK, Xi K, Ratia K, Santarsiero BD, Fu W, Harcourt BH, Rota PA, Baker SC, Johnson ME, Mesecar AD. Design and Synthesis of Peptidomimetic Severe Acute Respiratory Syndrome Chymotrypsin-like Protease Inhibitors. J Med Chem. 2005;48:6767–6770. [PubMed: 16250632]
- PMCPubMed Central citations
- PubChem BioAssay for Chemical ProbePubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter
- PubChem SubstanceRelated PubChem Substances
- PubMedLinks to PubMed
- Review Discovery of non-covalent inhibitors of the SARS main proteinase 3CLpro.[Probe Reports from the NIH Mol...]Review Discovery of non-covalent inhibitors of the SARS main proteinase 3CLpro.Jacobs J, Zhou S, Dawson E, Daniels JS, Hodder P, Tokars V, Mesecar A, Lindsley CW, Stauffer SR. Probe Reports from the NIH Molecular Libraries Program. 2010
- Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: identification of ML300 and noncovalent nanomolar inhibitors with an induced-fit binding.[Bioorg Med Chem Lett. 2013]Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: identification of ML300 and noncovalent nanomolar inhibitors with an induced-fit binding.Turlington M, Chun A, Tomar S, Eggler A, Grum-Tokars V, Jacobs J, Daniels JS, Dawson E, Saldanha A, Chase P, et al. Bioorg Med Chem Lett. 2013 Nov 15; 23(22):6172-7. Epub 2013 Sep 7.
- Discovery, synthesis, and structure-based optimization of a series of N-(tert-butyl)-2-(N-arylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease.[J Med Chem. 2013]Discovery, synthesis, and structure-based optimization of a series of N-(tert-butyl)-2-(N-arylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease.Jacobs J, Grum-Tokars V, Zhou Y, Turlington M, Saldanha SA, Chase P, Eggler A, Dawson ES, Baez-Santos YM, Tomar S, et al. J Med Chem. 2013 Jan 24; 56(2):534-46. Epub 2013 Jan 3.
- Broad-spectrum inhibitors against 3C-like proteases of feline coronaviruses and feline caliciviruses.[J Virol. 2015]Broad-spectrum inhibitors against 3C-like proteases of feline coronaviruses and feline caliciviruses.Kim Y, Shivanna V, Narayanan S, Prior AM, Weerasekara S, Hua DH, Kankanamalage AC, Groutas WC, Chang KO. J Virol. 2015 May; 89(9):4942-50. Epub 2015 Feb 18.
- Review Design of HIV protease inhibitors targeting protein backbone: an effective strategy for combating drug resistance.[Acc Chem Res. 2008]Review Design of HIV protease inhibitors targeting protein backbone: an effective strategy for combating drug resistance.Ghosh AK, Chapsal BD, Weber IT, Mitsuya H. Acc Chem Res. 2008 Jan; 41(1):78-86. Epub 2007 Aug 28.
- Non-covalent triazole-based inhibitors of the SARS main proteinase 3CLpro - Prob...Non-covalent triazole-based inhibitors of the SARS main proteinase 3CLpro - Probe Reports from the NIH Molecular Libraries Program
Your browsing activity is empty.
Activity recording is turned off.
See more...