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PMC:7291971 / 9099-32284 JSONTXT 11 Projects

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Id Subject Object Predicate Lexical cue
T60 0-25 Sentence denotes 2 Results and discussion
T61 27-72 Sentence denotes 2.1 Rational design of bisubstrate compounds
T62 73-327 Sentence denotes In order to mimic the transition state of the cap RNA 2′-O-methylation, the design of the SAM mimetics relies on the coupling of the adenosine moiety of the SAM cofactor to another adenosine modified at 2′-O-position with an ethyl group to form the link.
T63 328-439 Sentence denotes Thus, we first synthesized the dinucleoside 1 with a 2′-O-ethyl amino link between both adenosines (Scheme 2 ).
T64 440-651 Sentence denotes The major advantage of this N-containing linker is the possibility to functionalize the secondary amine with a large variety of groups which may lead to additional binding with specific sites of RNA 2′-O-MTases.
T65 652-876 Sentence denotes According to the schematic representation of the transition state of the 2′-O-methylation (Scheme 1), an accurate mimic was represented by compound 2 with the α-amino acid chain of the SAM branched to the nitrogen atom [16].
T66 877-1005 Sentence denotes We further modified this side chain under α-amino-ester form or α-amino-amide form to result in compounds 3 and 4, respectively.
T67 1006-1133 Sentence denotes Then instead of polar modifications, we introduced hydrophobic substituents on the secondary amine in dinucleosides 5, 6 and 7.
T68 1134-1265 Sentence denotes Finally we chose to obtain compound 9 with a nitrobenzenesulfonamide moiety as a structural particular element of the dinucleoside.
T69 1266-1428 Sentence denotes Indeed, as a global observation in medicinal chemistry the N-arylsulfonamide motif is regularly found in antitumor agents as in some antiviral inhibitors [19,20].
T70 1429-1652 Sentence denotes Further, we explored the combination of the nitro group with another substituent (MeO, CF3, Cl) at diverse positions in the phenyl ring resulting in the compounds 10–13 and we also removed the nitro group in 14 (Scheme 3 ).
T71 1653-1869 Sentence denotes In addition, we chose to replace the sulfone by a methylene group in 15–16 (Scheme 4 ) to assess the sulfone contribution to the inhibitory activity obtained with dinucleosides containing the N-arylsulfonamide motif.
T72 1870-2656 Sentence denotes Scheme 2 Synthesis of dinucleosides 1–9. (a) K2CO3, KI, DMF, 50 °C, 24 h, 74%; (b) PhSH, K2CO3, DMF, 25 °C, 2 h, 76%; (c) TFA/H2O 8/2, 25 °C, 3 h, 76% for 1 and 34% for 9; (d) (i) 21, AcOH, CH2Cl2, 25 °C, 2 h, (ii) NaBH(OAc)3, 25 °C, 2 h, 93%; (e) (i) TFA/H2O 8/2, 25 °C, 3 h, (ii) 2 M aqueous solution LiOH, 25 °C, 0.5 h, 32%; (f) TFA/H2O 8/2, 25 °C, 3 h, 35%; (g) (i) TFA/H2O 8/2, 25 °C, 3 h, (ii) 7 M NH3/MeOH, 30 °C, 24 h, 38%; (h) 1-bromobutane, DIEA, NMP, microwave 110 °C, 4 h, 47% for 23; 1-bromo-3-phenylpropane, DIEA, NMP, microwave 110 °C, 4 h, 53% for 24, methyl 4-bromobutyrate, DIEA, NMP, microwave 110 °C, 3.5 h, 58% for 25; (i) (i) TFA/H2O 8/2, 25 °C, 6 h, 72% for 5; 3 h, 28% for 6; 5.5 h, 60% for 7; 5.5 h for 8. (ii) 2 M aqueous solution LiOH, 0 °C, 0.5 h, 36% for 8.
T73 2657-2829 Sentence denotes Scheme 3 Synthesis of dinucleosides 10–13. (a) Ns-Cl, NEt3, DMF, 25 °C, 3.5 h, 40–72%; (b) 17, KI, K2CO3, DMF, 50 °C, 24 h, 43–52%; (c) TFA/H2O 8/2, 25 °C, 3 h–5 h, 13–20%.
T74 2830-3205 Sentence denotes Scheme 4 Synthesis of dinucleosides 14–16. (a) 4-chlorobenzenesulfonyl chloride, NEt3, CH2Cl2, 0 °C, 3 h, 90%. (b) (i) 4-chloro-3-nitrobenzaldehyde, AcOH, DCE, 40 °C, 20 min; (ii) NaBH(OAc)3, 40 °C, 16 h, 87%. (c) (i) 4-chlorobenzaldehyde, AcOH, DCE, 40 °C, 20 min. (ii) NaBH(OAc)3, 40 °C, 16 h, 72%. (d) TFA/H2O 8/2, 25 °C, 3 h, 48% for 14; 6 h, 25% for 15; 6 h, 37% for 16.
T75 3207-3221 Sentence denotes 2.2 Chemistry
T76 3222-3462 Sentence denotes The retrosynthetic analysis of the dinucleoside 1 structure suggested the coupling reaction between the tosyl derivative 17 previously described by us (Scheme 2) [14], and the readily accessible 5′-amino-2′,3′-isopropylidene adenosine [21].
T77 3463-3678 Sentence denotes The direct N-alkylation at room temperature did not afford the secondary amine in satisfactory yields and when increasing the temperature to enhance the reactivity of the primary amine, we noticed degradation of 17.
T78 3679-3875 Sentence denotes To circumvent the lack of reactivity of primary amines, a synthetic method using nitrobenzenesulfonamides (Ns-amides) as both a protecting and activating group has been developed by Fukuyama [22].
T79 3876-3989 Sentence denotes The main advantage of this nosyl strategy is that both alkylation and deprotection proceed under mild conditions.
T80 3990-4175 Sentence denotes Recently, the 2-nitrobenzenesulfonamide has been used successfully to synthetize transition state analogs of DNA methylation based on the coupling of cytosine analogs to adenosine [15].
T81 4176-4300 Sentence denotes In the same way, we envisaged the coupling between 17 and the 5′-nosyl adenosine 18 to obtain the dinucleoside 1 (Scheme 2).
T82 4301-4570 Sentence denotes The building block 18 was prepared in 74% yield by reacting 4-nitrobenzenesulfonyl chloride [22] that has a similar reactivity to 2-nitrobenzenesulfonyl chloride as used in Ref. [15], with 5′-amino-2′,3′-isopropylidene adenosine prepared upon published procedures [23].
T83 4571-4729 Sentence denotes N-alkylation of Ns-amide 18 with 17 in the presence of K2CO3 in DMF at room temperature did not afford the expected dinucleoside 19, even at high temperature.
T84 4730-4882 Sentence denotes Nevertheless, according to the literature [24], the addition of a catalytic amount of KI to the reaction mixture was beneficial to give 19 in 74% yield.
T85 4883-5005 Sentence denotes Facile deprotection of 19 by treatment with a nucleophilic thiolate produced the desired secondary amine 20 in high yield.
T86 5006-5116 Sentence denotes Removal of sugar protecting groups has been accomplished in acidic medium to give dinucleoside 1 in 76% yield.
T87 5117-5282 Sentence denotes Likewise, the acidic treatment was applied to the intermediate Ns-amide 19 to afford the 4-nitrobenzenesulfonamide-containing dinucleoside 9 in 34% yield (Scheme 2).
T88 5283-5473 Sentence denotes Compounds 2, 3 and 4 were obtained from key compound 20 via reductive amination of the aldehyde 21 that was prepared in three steps from l-aspartic acid following a published procedure [25].
T89 5474-5577 Sentence denotes Reductive amination was conducted in the presence of sodium triacetoxyborohydride and acetic acid [26].
T90 5578-5657 Sentence denotes The resulting fully protected dinucleoside 22 was isolated in high yield (93%).
T91 5658-5763 Sentence denotes Then, sugar hydroxyls and amine were deprotected by TFA treatment and afforded methyl ester derivative 3.
T92 5764-5926 Sentence denotes Subsequent basic treatment with LiOH converted the methyl ester in carboxylic acid and dinucleoside 2 with α-amino acid chain similar to that of SAM was obtained.
T93 5927-6110 Sentence denotes Finally, the SAM analogue 4 with an amide function instead of a carboxylic acid in α-amino acid chain was prepared from 22 upon a final treatment with 7 M methanolic ammonia solution.
T94 6111-6368 Sentence denotes Dinucleosides 5, 6 and 7 were rather synthesized through N-alkylation of 20 with 1-bromobutane, 1-bromo-3-phenylpropane or methyl-4-bromobutyrate, respectively, in N-methylmorpholine in the presence of diisopropylethylamine (DIEA) at 110 °C under microwave.
T95 6369-6480 Sentence denotes These specific conditions were optimized for a successful synthesis of 23, 24 and 25 with an average 50% yield.
T96 6481-6592 Sentence denotes This moderate yield results from double N-alkylations (observed in mass spectrometry) and incomplete reactions.
T97 6593-6678 Sentence denotes Increasing temperature of the reactions neither did drive the reaction to completion.
T98 6679-6790 Sentence denotes Next, 23, 24 and 25 were deprotected upon TFA treatment to obtain N-alkyl derivatives 5, 6 and 7, respectively.
T99 6791-6958 Sentence denotes Subsequently, an additional basic treatment with 2 M LiOH was applied to 7 to convert the methyl ester moiety into the carboxylic acid-ended alkyl chain of compound 8.
T100 6959-7159 Sentence denotes The same synthetic route used for the preparation of nosyl-containing dinucleoside 9 was followed to synthesize compounds 10–13 with diverse Ns-amide moieties as analogs of the nosyl group (Scheme 3).
T101 7160-7429 Sentence denotes The reaction of 5′-amino-2′,3′-isopropylidene adenosine [23] with four diversely substituted (OMe, CF3, Cl) and commercially available nitrobenzenesulfonyl chloride reagents afforded the corresponding N-nosyl adenosines 26–29 with 40–72% yield [[27], [28], [29], [30]].
T102 7430-7573 Sentence denotes Their subsequent coupling with 17 in the presence of K2CO3 and KI gave the corresponding dinucleosides 30–33 in moderate yields from 43 to 52%.
T103 7574-7724 Sentence denotes A final TFA treatment provided the respective N-nosyl adenine dinucleosides 10–13 which were purified by reversed-phase chromatography (Yield 13–20%).
T104 7725-7850 Sentence denotes Scheme 4 depicts the synthesis of the adenine dinucleosides 14–16 from the intermediate NH-linked dinucleoside 20 (Scheme 2).
T105 7851-7979 Sentence denotes The reaction of 4-chlorobenzenesulfonyl chloride with 20 in the presence of NEt3 [31,32] followed by a TFA treatment yielded 14.
T106 7980-8208 Sentence denotes The treatment of 20 with 4-chloro-3-nitrobenzaldehyde or 4-chlorobenzaldehyde and sodium triacetoxyborohydride, followed by the removal of protective groups in acidic conditions resulted in dinucleosides 15 and 16, respectively.
T107 8209-8325 Sentence denotes It is noteworthy that this reductive amination conducted at 40 °C increased the yields to 71% and 81%, respectively.
T108 8327-8369 Sentence denotes 2.3 RNA methyltransferase activity assays
T109 8370-8467 Sentence denotes Compounds 1–16 were tested for their ability to inhibit the methylation of the RNA cap structure.
T110 8468-8744 Sentence denotes The inhibition induced by each compound (50 μM) was determined by a radioactive MTase assay (filter binding assay) which consists in measuring the [3H] radiolabeled methyl transferred from the methyl donor SAM onto RNA substrate (GpppAC4) synthetized by using T7 primase [33].
T111 8745-8965 Sentence denotes Compounds 1–16 designed as mimics of the transition state of RNA 2′-O-methylation were screened against several viral RNA 2′-O-MTases from SARS-CoV (nsp10/nsp16 complex), Zika, West-Nile, Dengue, Vaccinia (VP39) viruses.
T112 8966-9166 Sentence denotes At the same time, the compounds were tested against human RNA N7-MTase (hRNMT) and selected viral N7-MTases such as SARS-CoV nsp14 and Vaccinia D1-D12 complex to evaluate their specificity (Table 1 ).
T113 9167-9264 Sentence denotes Table 1 Screening for inhibitory activity of sinefungin and compounds 1–16 at 50 μM on N7-MTases.
T114 9265-9313 Sentence denotes Compounds Percentage of inhibition at 50 μM (%)a
T115 9314-9355 Sentence denotes SARS-CoV nsp14 Vaccinia virusD1-D12 hRNMT
T116 9356-9399 Sentence denotes Sinefungin 98.3 ± 0.2 99.8 ± 0.1 99.8 ± 0.2
T117 9400-9434 Sentence denotes 1 31.0 ± 6.8 20.3 ± 0.8 35.2 ± 4.9
T118 9435-9469 Sentence denotes 2 72.0 ± 1.2 85.8 ± 2.5 77.4 ± 1.2
T119 9470-9504 Sentence denotes 3 30.6 ± 9.3 32.1 ± 2.4 33.2 ± 4.3
T120 9505-9540 Sentence denotes 4 13.1 ± 13.3 53.2 ± 2.6 12.2 ± 2.1
T121 9541-9561 Sentence denotes 5 n.i n.i 27.5 ± 6.6
T122 9562-9597 Sentence denotes 6 38.4 ± 11.7 11.6 ± 7.1 23.1 ± 9.7
T123 9598-9626 Sentence denotes 7 n.i 69.2 ± 1.9 32.8 ± 16.1
T124 9627-9647 Sentence denotes 8 43.0 ± 4.0 n.i n.i
T125 9648-9682 Sentence denotes 9 88.6 ± 1.3 49.8 ± 3.2 66.0 ± 6.1
T126 9683-9717 Sentence denotes 10 96.6 ± 0.9 4.6 ± 0.3 31.8 ± 3.3
T127 9718-9752 Sentence denotes 11 47.6 ± 2.8 5.3 ± 4.3 44.2 ± 8.5
T128 9753-9788 Sentence denotes 12 94.6 ± 1.1 10.1 ± 5.5 23.3 ± 3.6
T129 9789-9823 Sentence denotes 13 97.2 ± 2.7 2.8 ± 0.8 33.9 ± 3.3
T130 9824-9859 Sentence denotes 14 96.2 ± 1.5 19.7 ± 3.8 20.2 ± 9.4
T131 9860-9887 Sentence denotes 15 94.0 ± 1.1 4.3 ± 3.9 n.i
T132 9888-9923 Sentence denotes 16 75.9 ± 2.5 4.5 ± 15.1 14.7 ± 1.3
T133 9924-9978 Sentence denotes aValues are the mean of three independent experiments.
T134 9979-10040 Sentence denotes The MTase activity was measured using a filter binding assay.
T135 10041-10232 Sentence denotes Assays were carried out in reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM MgCl2, 2 μM SAM and 0.1 μM 3H-SAM] in the presence of 0.7 μM GpppAC4 synthetic RNA and incubated at 30 °C.
T136 10233-10339 Sentence denotes SARS-CoV nsp14 (50 nM), vaccinia virus capping enzyme (D1-D12) (41 U), human RNA N7 MTase (hRNMT) (50 nM).
T137 10340-10427 Sentence denotes Compounds were previously dissolved in 100% DMSO. n.i: no inhibition detected at 50 μM.
T138 10428-10555 Sentence denotes Unexpectedly, all the bisubstrate compounds were barely active against the 2′-O MTases of flaviviruses or coronavirus SARS-CoV.
T139 10556-10625 Sentence denotes In contrast, most of the compounds displayed inhibition of N7-MTases.
T140 10626-10805 Sentence denotes Dinucleoside 2 bearing the amino acid chain of the SAM showed some significant inhibition of both viral N7-MTases with a better activity on Vaccinia D1-D12 than on SARS-CoV nsp14.
T141 10806-10973 Sentence denotes However, compound 2 also displayed a potent inhibition of hRNMT in the same range as the viral MTases displaying a lack of specificity against human and viral enzymes.
T142 10974-11115 Sentence denotes The amino acid group of 2 seems essential for inhibition since compound 1 with a non-substituted NH linker weakly inhibited the three MTases.
T143 11116-11252 Sentence denotes The replacement of the amino acid group with an α-amino-ester at the extremity in compound 3 is detrimental for the inhibitory activity.
T144 11253-11440 Sentence denotes Interestingly, the dinucleoside 4 bearing an α-amino-amide specifically inhibited the viral protein Vaccinia D1-D12 complex whereas did not show any inhibition of SARS-CoV nsp14 or hRNMT.
T145 11441-11652 Sentence denotes Replacing the amino acid chain by a more hydrophobic butyl or phenylpropyl chain in dinucleosides 5 and 6, respectively, we aimed at favoring the Van der Waals interactions in hydrophobic pockets of the protein.
T146 11653-11729 Sentence denotes Only compound 6 showed a moderate but specific inhibition of SARS-CoV nsp14.
T147 11730-11987 Sentence denotes The removal of the NH2 of the amino acid chain of the broader spectrum inhibitor 2 with an ester-ended butyl chain in compound 7 or with an acid-ended butyl chain in 8 induced weaker but more specific inhibitions of Vaccinia D1-D12 MTase and SARS-CoV nsp14.
T148 11988-12092 Sentence denotes In the synthetic pathway of dinucleoside 1, the intermediate 19 bearing a 4-Ns-amide group was prepared.
T149 12093-12277 Sentence denotes In view of the valuable properties of such motif in some antiviral or anticancer drugs [19,20] it seemed interesting to us to obtain dinucleoside 9 by simple acidic deprotection of 19.
T150 12278-12454 Sentence denotes Of special interest, compound 9 showed a good and specific inhibition on SARS-CoV nsp14 confirming that the nosyl group contributes to the inhibitory activity with specificity.
T151 12455-12713 Sentence denotes Then, we modulated the initial nosyl moiety with the nitro group in “para” position by introducing diverse hydrophobic substituents (Cl, OMe, CF3) at different positions on the phenyl ring and/or by varying the position of the nitro group in compounds 10–13.
T152 12714-12799 Sentence denotes The addition of such substituents aimed at increasing the interactions with proteins.
T153 12800-12893 Sentence denotes Like 9, the four dinucleosides 10–13 maintained a high inhibitory activity on SARS-CoV nsp14.
T154 12894-13095 Sentence denotes The role of the nitro group on the phenyl ring was demonstrated by removing it in dinucleoside 14 bearing solely one chlorine atom in “para” position, thus the inhibitory effect was slightly decreased.
T155 13096-13173 Sentence denotes These data indicated the importance of the hydrophobic Cl in “para” position.
T156 13174-13430 Sentence denotes Finally, the resulting decreased inhibition when the sulfone moiety of 14 was replaced by a methylene group in compounds 15 and 16 stressed the importance of the N-arylsulfonylbenzene motif in the dinucleoside structure to maintain an effective inhibition.
T157 13432-13513 Sentence denotes 2.4 Dose-response testing of selected compounds against SARS-CoV nsp14 and hRNMT
T158 13514-13711 Sentence denotes To confirm the observed inhibition of some adenine dinucleosides against SARS-CoV nsp14 N7-MTase, we further tested 10 compounds in a dose-response assay (Table 2 , Supporting Information Fig. S2).
T159 13712-13835 Sentence denotes The selection of these compounds was based on their percentage inhibition higher than 50% against SARS-CoV nsp14 (Table 1).
T160 13836-13945 Sentence denotes After pre-incubation with increasing concentrations of dinucleosides, the MTase activity was measured by FBA.
T161 13946-14140 Sentence denotes The IC50 of compounds, deduced from Hill slope equation (Y = 100/(1+((X/IC50)^Hillslope) curve-fitting, ranged from 0.6 μM to 70.4 μM on SARS-CoV nsp14 and 10.9 μM to > 500 μM on hRNMT activity.
T162 14141-14395 Sentence denotes Among all the potential inhibitors, three compounds 2, 6 and 11 showed the lowest inhibitory activity on SARS-CoV nsp14 with IC50 > 40 μM, the others have IC50 values in the micromolar range except 13 that showed an IC50 value in the submicromolar range.
T163 14396-14513 Sentence denotes The potency of the derivative 13 to inhibit SARS-CoV nsp14 was comparable to the broad spectrum inhibitor sinefungin.
T164 14514-14718 Sentence denotes However, it is noteworthy that compound 13 specifically inhibits the N7-MTase of SARS-CoV with a high 413-fold of specificity in dose-response testing whereas sinefungin is also active against hRNMT [11].
T165 14719-15019 Sentence denotes Remarkably 13 bearing a chlorine atom in “para” position and a nitro group in “meta” position displayed the best inhibition which was 6.5–9.5 fold more potent than the derivatives 10 and 12, respectively that contain the nitro in “ortho” position and a lipophilic group CF3 or MeO in “para” position.
T166 15020-15179 Sentence denotes The comparison of IC50 values of 14 and 13 indicates that the addition of a nitro group in the phenyl ring increased the inhibitory activity by 2.5 fold in 13.
T167 15180-15344 Sentence denotes These results confirm that the chlorine atom in “para” position and the presence of a nitro group seem crucial for submicromolar SARS-CoV nsp14 inhibitory activity.
T168 15345-15451 Sentence denotes Table 2 Comparison of IC50 values of 10 most active compounds on SARS-CoV nsp14 and human RNMT activities.
T169 15452-15502 Sentence denotes Compounds SARS-CoV nsp14 IC50a (μM) hRNMTIC50 (μM)
T170 15503-15525 Sentence denotes Sinefunginb 0.36 <0.05
T171 15526-15549 Sentence denotes 2 40.6 ± 3.2 10.9 ± 1.0
T172 15550-15567 Sentence denotes 6 55.5 ± 5.1 >500
T173 15568-15584 Sentence denotes 9 2.6 ± 0.2 >500
T174 15585-15602 Sentence denotes 10 3.9 ± 0.4 >500
T175 15603-15629 Sentence denotes 11 70.4 ± 4.9 169.3 ± 30.4
T176 15630-15655 Sentence denotes 12 5.7 ± 0.5 275.9 ± 28.7
T177 15656-15681 Sentence denotes 13 0.6 ± 0.1 247.5 ± 14.9
T178 15682-15698 Sentence denotes 14 1.5 ± 0.1 n.d
T179 15699-15715 Sentence denotes 15 2.4 ± 0.2 n.i
T180 15716-15732 Sentence denotes 16 9.9 ± 0.9 n.d
T181 15733-15829 Sentence denotes a Concentration inhibiting MTase activity by 50%; mean value from three independent experiments.
T182 15830-15864 Sentence denotes b values from the literature [11].
T183 15865-16070 Sentence denotes The MTase activity for IC50 determinations was measured using a filter binding assay as described above. n.i: no inhibition detected at 50 μM; n.d: not determined due to inhibition lower than 50% at 50 μM.
T184 16072-16120 Sentence denotes 2.5 Thermal shift DSF assays for SARS-CoV nsp14
T185 16121-16321 Sentence denotes SARS-CoV nsp14-inhibitor interactions were further investigated by monitoring the thermal stability of the protein using differential scanning fluorimetry (DSF) (Supporting Information, Fig. S3) [34].
T186 16322-16534 Sentence denotes The change in thermal stability of SARS-CoV nsp14 was monitored in response to binding of the natural cofactor substrate SAM, sinefungin and the 9 most active and specific compounds 6 and 9–16 with IC50 < 100 μM.
T187 16535-16744 Sentence denotes As expected, SARS-CoV nsp14 displayed an increased melting temperature (T m) value with SAM (+6.5 °C) and sinefungin (+4.4 °C) whose structure only differs by a C–NH2 in place of S+-CH3 group in SAM (Fig. 2 ).
T188 16745-16960 Sentence denotes The binding experiments with the bisubstrate inhibitors showed that all dinucleosides stabilized the SARS-CoV nsp14 protein (T m > 40 °C) with a T m shift from +4.6 °C to +10.8 °C (Supporting Information, Table S1).
T189 16961-17095 Sentence denotes Eight of the 9 examined compounds increased the stability of SARS-CoV nsp14 more efficiently than the well-known inhibitor sinefungin.
T190 17096-17298 Sentence denotes More interestingly, T m values for SARS-CoV nsp14 were larger in the presence of five compounds 10 and 12–15 than with the natural enzyme substrate SAM, suggesting strong protein-inhibitor interactions.
T191 17299-17620 Sentence denotes Remarkably, the highest T m was observed with the most efficient inhibitor 13 (IC50 0.6 ± 0.1 μM) that stabilizes SARS-CoV nsp14 against thermal denaturation with a ΔT m +10.8 °C and exhibits notable binding affinity (apparent KD 1.3 ± 0.87 μM), as deduced from TSA performed with increasing concentration of compound 13.
T192 17621-17717 Sentence denotes This demonstrates a favorable interaction and highlights the inhibitor potential of compound 13.
T193 17718-17923 Sentence denotes The T m comparison of compounds 16, 15 and 13 showed a respective increase in SARS-CoV nsp14 stability, settling the importance of the sulfone group, the Cl and NO2 substituents in protein binding with 13.
T194 17924-18034 Sentence denotes Thus the N-(4-Cl-3-NO2-phenylsulfonamide) moiety is notably preferred for optimal SARS-CoV nsp14 interactions.
T195 18035-18240 Sentence denotes Another highlight is the thermal shift (+5.4 °C) for SARS-CoV nsp14 observed in the presence of compound 10 compared to 11, both only differ by the position of OCH3 and NO2 substituents in the phenyl ring.
T196 18241-18367 Sentence denotes A higher stability and inhibition of SARS-CoV nsp14 was observed when the OCH3 group is in “para” position in dinucleoside 10.
T197 18368-18501 Sentence denotes Fig. 2 Thermal shifts (ΔTm) of SARS-CoV nsp14 in the absence or presence of SAM, sinefungin and 9 dinucleoside inhibitors 6 and 9–16.
T198 18502-18609 Sentence denotes Thermal stability of SARS-CoV nsp14 upon ligand binding was monitored by differential scanning fluorimetry.
T199 18610-18810 Sentence denotes Assays were carried out in reaction mixture [20 mM HEPES (pH 7.5), 150 mM NaCl, 1x SYPRO orange dye] in the presence of 5 μM SARS-CoV nsp14 protein and 1 mM compound previously dissolved in 100% DMSO.
T200 18811-18932 Sentence denotes The bars and error bars correspond to the mean values from three independent measurements and their s.d.’s, respectively.
T201 18934-19014 Sentence denotes 2.6 Molecular docking studies of SARS-CoV nsp14 in complex with dinucleoside 13
T202 19015-19185 Sentence denotes To address the molecular bases of N7-MTase nsp14 inhibition by the dinucleosides, we performed computational docking studies of the best inhibitor 13 using Autodock Vina.
T203 19186-19285 Sentence denotes The docking was based on the SARS-CoV nsp14-nsp10 complex structure solved in presence of SAM [35].
T204 19286-19505 Sentence denotes SARS-CoV nsp14 is a bifunctional enzyme carrying RNA cap guanine N7-MTase at the C-terminal domain for mRNA capping (which is not influenced by nsp10) and 3′-5′-exoribonuclease at the N-terminal domain for proofreading.
T205 19506-19679 Sentence denotes The N7-MTase domain exhibits an original fold and the methyl receptor cap RNA (GpppA-RNA) and SAM bind in proximity in a highly constricted pocket to achieve methyltransfer.
T206 19680-19774 Sentence denotes The compound 13 was modeled in the SAM binding pocket of the SARS-CoV nsp14 structure (PDB ID:
T207 19775-19794 Sentence denotes 5C8T [35] & PDB ID:
T208 19795-19806 Sentence denotes 5NFY [36]).
T209 19807-19950 Sentence denotes At first sight, the overlay of the N-adenosine of 13 with the adenosine of SAM bounded structure is suitable (Supporting Information, Fig. S4).
T210 19951-20162 Sentence denotes More interestingly, the nitrobenzenesulfonamide core of 13 binds into a SARS-CoV nsp14 well known binding site formed by Trp385, Phe401, Tyr420, Phe426 and Phe506 (Fig. 3 , Supporting Information, Fig. S5) [35].
T211 20163-20263 Sentence denotes Naturally, the side chains of these amino acids enclose the nucleobase guanine of the cap structure.
T212 20264-20412 Sentence denotes In this cap-binding site, Phe426 was shown to have the largest influence on the N7-MTase activity, and F426A mutation reduced MTase activity by 50%.
T213 20413-20551 Sentence denotes With 13, the orientation of the Phe426 residue all around the nitrobenzenesulfonamide leads to the formation of π-π stacking interactions.
T214 20552-20685 Sentence denotes In addition, there are other hydrophobic interactions between the phenylsulfonamide moiety and aromatic residues of the binding site.
T215 20686-20810 Sentence denotes All these interactions may explain the strong inhibition observed with phenyl-containing compounds, notably compounds 13–15.
T216 20811-20976 Sentence denotes Moreover, Asn386 is located in proximity to the methylation site and forms two hydrogen bonds with the guanine moiety favoring its right orientation for methylation.
T217 20977-21098 Sentence denotes Here, fixed on the nitrobenzenesulfonamide core of 13, the chlorine atom forms a halogen bond with Asn386 (Fig. 4 ) [37].
T218 21099-21301 Sentence denotes The formation of a double hydrogen bond interaction was observed between the nitro group and Arg310 that normally interacts with the second phosphate group of the triphosphate bond in the cap structure.
T219 21302-21452 Sentence denotes The docking also suggests that the sulfone group of 13 avoids flexibility of the N-nosyl substituent, thus increasing its orientation into the pocket.
T220 21453-21627 Sentence denotes This constraint may explain the difference in activity (IC50) and stabilizing effect (T m) between compounds 13 and 15 that contains a methylene group instead of the sulfone.
T221 21628-21737 Sentence denotes Finally, the common element of all dinucleosides is an adenosine linked to a N-adenosine by the 2′O position.
T222 21738-21923 Sentence denotes Its contribution is well defined by the formation of intermolecular hydrogen bonds between the adenosine and Gly333 (3′OH), Ile338 (5′OH), Lys336 (N7) and His424 (N1) residues (Fig. 4).
T223 21924-22053 Sentence denotes All the major interactions maintain 13 in a suitable orientation in the binding site in place of the natural substrate GpppA-RNA.
T224 22054-22210 Sentence denotes The docking model of 13 is consistent with our inhibition experimental data and high thermal stability of SARS-CoV nsp14 in the presence of these compounds.
T225 22211-22287 Sentence denotes Fig. 3 Modeling results in the SAM binding pocket of SARS-CoV nsp14 (PDB ID:
T226 22288-22357 Sentence denotes 5C8T, resolution 3.2 Å). (A) Amino acids surrounding dinucleoside 13.
T227 22358-22465 Sentence denotes Hydrogen and halogen bonds are indicated by red dashes. π-π stacking interaction is indicated by red curve.
T228 22466-22621 Sentence denotes Distances are given in Å. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
T229 22622-22698 Sentence denotes Fig. 4 Modeling results in the SAM binding pocket of SARS-CoV nsp14 (PDB ID:
T230 22699-22864 Sentence denotes 5C8T, resolution 3.2 Å). (A) Contribution of the nitrobenzenesulfonamide core of 13. (B) Contribution of the 2′O linked adenosine of all dinucleosides, including 13.
T231 22865-23185 Sentence denotes Hydrogen bonds (yellow), halogen bond (green) and π-π stacking interaction (cyan) are represented. (Atoms not involved in protein-ligand interaction are not represented for clarity purpose). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)