LitCovid-sentences  

PMC:7571312 / 21128-34225 JSONTXT 11 Projects

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Id Subject Object Predicate Lexical cue
T134 0-42 Sentence denotes Reversible Inhibitors of SARS CoV-1 3CLpro
T135 43-492 Sentence denotes Peptidyl HMK inhibitors and their corresponding ethers have been designed as potent reversible cysteine protease inhibitors of cathepsin K34 and cruzain.43 High-resolution crystal structures of two such inhibitors complexed with cruzain highlighted the presence of a strong hydrogen bond between the catalytic His and the α-hydroxyl moiety of the inhibitor but interestingly no covalent bond between the active-site Cys and the ketone pharmacophore.
T136 493-836 Sentence denotes Utilizing the crystal structure of 28 in complex with SARS CoV-1 3CLpro, molecular modeling of an HMK-based design indicated the potential for establishing a covalent bond between the sulfur of the active-site cysteine (Cys145) and the HMK carbonyl as well as hydrogen bond interactions between catalytic His41 and the terminal hydroxy moiety.
T137 837-1009 Sentence denotes Additionally, the structure of 28 suggested the possibility to increase the lipophilic filling of the deep hydrophobic S2 pocket as a strategy to increase binding affinity.
T138 1010-1157 Sentence denotes To evaluate these structure-based observations, a series of reversible HMK inhibitors containing P2 diversity were prepared as depicted in Table 3.
T139 1158-1225 Sentence denotes Table 3 SARS 3CLpro Inhibition Data for P2-Modified HMK Inhibitors
T140 1226-1394 Sentence denotes a&b See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.
T141 1395-1429 Sentence denotes c This value is a Ki measurement.
T142 1430-1478 Sentence denotes d These values are from a single determination.
T143 1479-1630 Sentence denotes As suggested by molecular modeling, the S2 site appears to accommodate a variety of linear, branched, and cyclic alkyl moieties (entries 36 and 39–41).
T144 1631-1774 Sentence denotes Interestingly, the P2 Phe derivative 42 displayed less potent inhibition of the SARS CoV-1 3CLpro than the corresponding saturated analogue 41.
T145 1775-1920 Sentence denotes Also noteworthy is the large attenuation in enzyme inhibition seen with P2-N-methyl-Leu inhibitor 38 (IC50 = 83 nM) as compared to 4 (Ki = 4 nM).
T146 1921-2101 Sentence denotes This loss of potency is consistent with the inhibitor ligand interactions observed with 2 and 4 that show the P2 NH involved in a hydrogen bond with the side-chain amide of Gln189.
T147 2102-2298 Sentence denotes Additionally, the methyl substitution in 38 would be expected to alter the conformation of the 4-methoxy indole cap and perturb the observed ligand–enzyme hydrogen bond network present in 2 and 4.
T148 2299-2443 Sentence denotes The results for 4 and the other examples in Table 3 reveal a high antiviral EC50/enzyme IC50 ratio, which may arise from poor cell permeability.
T149 2444-2581 Sentence denotes Indeed, these HMK inhibitors exhibit very low permeability and high levels of efflux beyond the sensitivity of the Caco-2 in vitro assay.
T150 2582-2751 Sentence denotes However, the impact of high efflux on antiviral potencies from Vero 76 cells (derived from monkey kidney) versus disease-relevant human lung epithelial cells is unknown.
T151 2752-2948 Sentence denotes To better understand the observed high antiviral EC50/enzyme IC50 ratio for 4, we evaluated the role of efflux in the Vero 76 cell line by the in vitro experimental design discussed further below.
T152 2949-3121 Sentence denotes Concurrently, a strategy to design molecules reducing efflux by active transporters, such as P-glycoprotein, was pursued to decrease high antiviral EC50/enzyme IC50 ratios.
T153 3122-3391 Sentence denotes An analysis of the physicochemical properties of 4 suggested that increasing logP, reducing polar surface area (PSA), and reducing the number of hydrogen bond donors/acceptors were design strategies with the potential to improve cellular permeability and reduce efflux.
T154 3392-3568 Sentence denotes An examination of the crystal structure of 2 in complex with SARS CoV-1 3CLpro suggested a limited functional role for the 4-methoxy substituent contained in the P3 indole cap.
T155 3569-3705 Sentence denotes Additionally, the terminal α-hydroxyl moiety located at P1′ appeared to provide a potential opportunity to remove a hydrogen bond donor.
T156 3706-3865 Sentence denotes Although it was anticipated that the α-hydroxyl moiety would form a hydrogen bond to the catalytic His41, the directionality of this interaction was uncertain.
T157 3866-4098 Sentence denotes As noted earlier, His41 presents a hydrogen bond donor to the interior of the protease, leaving an acceptor to participate in the proton transfer cycling from neutral to cationic in the putative catalytic mechanism44 of proteolysis.
T158 4099-4271 Sentence denotes If His41 is protonated in the inhibitor-bound complex, a lone pair from the α-hydroxyl moiety would function as a hydrogen bond acceptor from the proton on the His residue.
T159 4272-4435 Sentence denotes On the other hand, if His41 is deprotonated, the hydrogen of the α-hydroxyl moiety would act as a hydrogen bond donor to the nitrogen lone pair of the His residue.
T160 4436-4626 Sentence denotes Consistent with the strategy to decrease PSA and hydrogen bond donors, a series of inhibitors were prepared that contained alterations to the P3 cap and substitution of the α-hydroxyl group.
T161 4627-4737 Sentence denotes Table 4 summarizes this effort where two of the optimal P2 residues (Leu and β-tert-butyl-Ala) were conserved.
T162 4738-4995 Sentence denotes Removal of the methoxy group from the indole generally led to slightly weaker potency in both the enzymatic and antiviral assays across pairs (43 vs 36, 44 vs 37, 50 vs 48 and 49 vs 4) and therefore no improvement in the antiviral/protease inhibition ratio.
T163 4996-5107 Sentence denotes The impact of more significant changes to the P3 cap is illustrated by tetrahydrofuranyl derivatives 46 and 47.
T164 5108-5282 Sentence denotes These derivatives lack the lipophilic aryl ring and NH hydrogen bond donor present in the indole P3 cap and exhibit a pronounced reduction in enzymatic and antiviral potency.
T165 5283-5446 Sentence denotes A noteworthy reduction in biochemical and antiviral potency was observed in comparing each hydroxymethylketone derivative with its corresponding ether counterpart.
T166 5447-5620 Sentence denotes These results suggest that modest structural and physicochemical alterations of 4 fail to significantly decrease the high EC50/IC50 ratios for this class of peptidomimetics.
T167 5621-5928 Sentence denotes However, in vitro analysis of 4 revealed this derivative to possess high levels of metabolic stability in human liver microsomes (t1/2 = 107 min).45 Further evaluation of this breakthrough derivative in several aqueous-based formulations suitable for IV administration indicated high levels of solubility.46
T168 5929-6043 Sentence denotes Table 4 SARS CoV-1 3CLpro Inhibition Data for P3- and P2-Modified Hydroxymethyl and Alkoxymethylketone Inhibitors
T169 6044-6212 Sentence denotes a&b See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.
T170 6213-6247 Sentence denotes c This value is a Ki measurement.
T171 6248-6296 Sentence denotes d These values are from a single determination.
T172 6297-6612 Sentence denotes Cocrystal structures of 4 bound to the 3CLpro of both SARS CoV-1 and CoV-2 were solved at 1.47 and 1.26 Å resolutions, respectively (PDB codes 6XHL and 6XHM).47 As expected, the ligand binding sites of the main protease from SARS CoV-1 and SARS CoV-2 are conserved in sequence and are nearly identical structurally.
T173 6613-6739 Sentence denotes The schematic diagram in Figure 5 is representative of the covalent adduct between 4 and the 3CLpro from both CoV-1 and CoV-2.
T174 6740-6916 Sentence denotes The warhead HMK carbonyl carbon of 4 forms a covalent bond to the 3CLpro active-site cysteine (Cys145) sulfur generating a tetrahedral carbinol complex (1.8 Å C–S bond length).
T175 6917-7054 Sentence denotes This carbinol hydroxyl forms hydrogen bonds with the backbone NH of Cys145 and with the amide NH of Gly143 via a bridging water molecule.
T176 7055-7176 Sentence denotes Another key active-site interaction is the hydrogen bond between the primary alcohol moiety of 4 and the catalytic His41.
T177 7177-7313 Sentence denotes Similar interactions between catalytic His residues and the OH moiety of HMK in other protease–inhibitor complexes have been reported.43
T178 7314-7406 Sentence denotes Figure 5 Cocrystal structure of the covalent adduct of 4 bound to SARS CoV-2 3CLpro (6XHM).
T179 7407-7478 Sentence denotes The Connolly surface for the inhibitor binding pocket is shown in gray.
T180 7479-7572 Sentence denotes The bonds are represented as dashed lines, with the bond length between heavy atoms depicted.
T181 7573-7745 Sentence denotes The schematic rendering of the active site with dashed lines represented as hydrogen bonds with key residues and curved lines to show S1 and S2 binding pockets is depicted.
T182 7746-7988 Sentence denotes In S1, the lactam carbonyl of 4 forms a strong hydrogen bond with the side chain of His163, while the lactam NH is within the hydrogen-bonding distance but poorly aligned with the side-chain oxygen of Glu166 and the backbone oxygen of Phe140.
T183 7989-8064 Sentence denotes These three residues partially define the bottom and edge of the S1 pocket.
T184 8065-8222 Sentence denotes Additionally, the backbone carbonyl and NH of Glu166 form β-sheet-like hydrogen-bonding interactions with the NH and C2-carbonyl of the indole fragment of 4.
T185 8223-8365 Sentence denotes The NH of the P2 Leu of inhibitor 4 and the side chain of Gln189 form a hydrogen bond, while the carbonyl of P2 Leu is exposed to the solvent.
T186 8366-8452 Sentence denotes The inhibitor P1 NH forms a strong hydrogen bond with the backbone carbonyl of His164.
T187 8453-8613 Sentence denotes The inhibitor’s lipophilic leucine residue is bound to the SARS 3CLpro in the hydrophobic S2 pocket formed by residues Asp187, Arg188, Gln189, Met49, and His41.
T188 8614-8810 Sentence denotes The indole portion of 4 does not protrude into the S3 pocket but rather rests across a partially collapsed S3, making extended van der Waals interactions across the 189–191 residue backbone atoms.
T189 8811-9228 Sentence denotes The closed S3 is the only significant conformational difference compared to a recently reported structure of SARS CoV-2 3CLpro inhibited by α-ketoamides.8 The energetics of this extended interaction, the associated protein conformation stabilities, or warhead difference may contribute to the 3-order-of-magnitude increased SARS CoV-2 3CLpro potency of HMK 4 (see below) compared to that of the referenced ketoamides.
T190 9229-9326 Sentence denotes Hydroxymethylketone 4 was evaluated against a panel of other viral and human proteases (Table 5).
T191 9327-9564 Sentence denotes In general, 4 is highly selective for 3CLpro inhibition, displaying IC50 values of >10 μM against many of the other proteases and possessing modest levels of inhibition of cathepsin B (IC50 = 1.3 μM) and rhinovirus 3Cpro (IC50 = 1.7 μM).
T192 9565-9697 Sentence denotes High levels of similarity between the catalytic sites of 3CLpro proteases suggest the potential of 4 as a pan-coronavirus inhibitor.
T193 9698-9919 Sentence denotes Testing revealed that 4 demonstrates potent inhibition of the related hCoV 229E protease (IC50 = 0.004 μM) but more importantly, given the current global pandemic, potently inhibits SARS CoV-2 3CLpro with a Ki of 0.27 nM.
T194 9920-9953 Sentence denotes Table 5 Protease Inhibition by 4
T195 9954-10005 Sentence denotes protease source % inhibition @ 10 μMa IC50 (μM)a
T196 10006-10044 Sentence denotes 3CLpro SARS CoV-1   0.004 ± 0.0003b
T197 10045-10082 Sentence denotes 3CLpro SARS CoV-2 0.00027 ± 0.0001b
T198 10083-10116 Sentence denotes cathepsin B human 93 1.3 ± 0.1
T199 10117-10143 Sentence denotes cathepsin D human 3 >10
T200 10144-10179 Sentence denotes leukocyte elastase human –31 >10
T201 10180-10207 Sentence denotes chymotrypsin human 8 >10
T202 10208-10233 Sentence denotes thrombin human –28 >10
T203 10234-10256 Sentence denotes pepsin human –2 >10
T204 10257-10283 Sentence denotes caspase 2 human –20 >10
T205 10284-10304 Sentence denotes HIV-1 HIV-1 0 >10
T206 10305-10322 Sentence denotes HCV HCV 16 >10
T207 10323-10343 Sentence denotes HRV HRV   1.7 ± 1
T208 10344-10379 Sentence denotes hCoV 229E human   0.004 ± 0.0004
T209 10380-10546 Sentence denotes a See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.
T210 10547-10611 Sentence denotes b For SARS CoV-1 and CoV-2 3CLpro, Ki values are reported here.
T211 10612-10694 Sentence denotes The antiviral specificity of 4 was evaluated against a panel of viruses (Table 6).
T212 10695-10894 Sentence denotes No antiviral activity was observed when 4 was tested against two human rhinovirus strains (HRV-14 and HRV-16), human immunodeficiency virus-1 (HIV-1), or human cytomegalovirus (HCMV) in cell culture.
T213 10895-10948 Sentence denotes This compound also does not inhibit the HCV replicon.
T214 10949-11127 Sentence denotes As anticipated by the potent hCoV 229E protease activity, 4 is a potent inhibitor of the human coronavirus hCoV 229E in MRC-5 cells (human lung-derived) with an EC50 of 0.090 μM.
T215 11128-11158 Sentence denotes Table 6 Viral Inhibition by 4
T216 11159-11188 Sentence denotes virus EC50 (μM)a CC50 (μM)a
T217 11189-11215 Sentence denotes SARS CoV-1 4.8 ± 2.2 452
T218 11216-11243 Sentence denotes hCoV 229E 0.09 ± 0.04 >40
T219 11244-11264 Sentence denotes HRV-14 >100 >1,260
T220 11265-11285 Sentence denotes HRV-16 >100 >1,260
T221 11286-11304 Sentence denotes HIV-1 RF >32 >32
T222 11305-11329 Sentence denotes HCV replicon >320 >320
T223 11330-11352 Sentence denotes HCMV RC256 >100 >100
T224 11353-11519 Sentence denotes a See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.
T225 11520-11732 Sentence denotes The hCoV 229E antiviral potency is more than 50-fold greater than that for SARS CoV-1 in the Vero 76 line, even though 4 possesses nearly equivalent 3CLpro biochemical potency for the hCoV 229E and CoV-1 viruses.
T226 11733-11992 Sentence denotes While undefined differences between hCoV 229E and SARS CoV-1 could account for these large differences in antiviral EC50/enzyme IC50 ratios, different active transporter expression levels between MRC-5 and Vero 76 cell lines may also be a contributing factor.
T227 11993-12268 Sentence denotes Consistent with the previously stated goal to characterize the impact of efflux on potency measured in the SARS CoV-1 antiviral assay, we co-dosed a known P-glycoprotein transport inhibitor at a fixed concentration (0.5 μM CP-100356)48 in a full dose–response of inhibitor 4.
T228 12269-12434 Sentence denotes This resulted in a pronounced potency shift of SARS CoV-1 antiviral activity (EC50 = 0.11 μM) that now more closely matched the antiviral potency seen for hCoV 229E.
T229 12435-12678 Sentence denotes As a control, the same co-dosing of efflux inhibitor led to no such shift in the hCoV 229E antiviral potency of 4, demonstrating that the human lung-derived MRC-5 cells show less P-glycoprotein-based transporter under similar assay conditions.
T230 12679-12949 Sentence denotes These results suggest that the high antiviral EC50/enzyme IC50 ratio observed in Vero 76 cells is an artifact of the high efflux potential of that assay cell line and may underestimate the antiviral potency in human lung cells, the relevant tissue for SARS and COVID-19.
T231 12950-13097 Sentence denotes Assays to directly measure the antiviral potency of 4 against SARS CoV-2 in clinically relevant human lung cells are currently under investigation.