key: cord-1013161-58tgkv39
authors: Oh, Chang-ki; Nakamura, Tomohiro; Beutler, Nathan; Zhang, Xu; Piña-Crespo, Juan; Talantova, Maria; Ghatak, Swagata; Trudler, Dorit; Carnevale, Lauren N.; McKercher, Scott R.; Bakowski, Malina A.; Diedrich, Jolene K.; Roberts, Amanda J.; Woods, Ashley K.; Chi, Victor; Gupta, Anil K.; Rosenfeld, Mia A.; Kearns, Fiona L.; Casalino, Lorenzo; Shaabani, Namir; Liu, Hejun; Wilson, Ian A.; Amaro, Rommie E.; Burton, Dennis R.; Yates, John R.; Becker, Cyrus; Rogers, Thomas F.; Chatterjee, Arnab K.; Lipton, Stuart A.
title: Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection
date: 2022-04-05
journal: bioRxiv
DOI: 10.1101/2022.04.05.487060
sha: 39ebeb6296fff5b05425db8394e9858a76c5e150
doc_id: 1013161
cord_uid: 58tgkv39

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

1a-c). Interestingly, multiple cysteine residues have been shown to be of importance in ACE2 and TMPRSS2 activity, so S-nitrosylation might be expected to disrupt their activity 20,21 .

We focused on S-nitrosylation of ACE2 (forming SNO-ACE2), reasoning that this nitrosylation reaction might prevent binding of SARS-CoV-2 S protein to ACE2, thus inhibiting viral infection. To test this premise, we exposed HeLa cells stably expressing human ACE2 (HeLa-ACE2) to SNOC and assessed SNO-ACE2 formation by biotinswitch assay. To evaluate binding of the S protein to these HeLa-ACE2 cells, we then incubated the cells with purified recombinant SARS-CoV-2 Spike protein (S1+S2). Since NO dissipates very quickly from SNOC (<5 minutes at neutral pH), and Spike protein was added sequentially after this period, we could rule out the possibility of direct Snitrosylation of Spike protein by SNOC under these conditions 22, 23 . We found that the formation of SNO-ACE2 was stable for at least 12 h (Extended Data Fig. 1 ). The receptor binding domain (RBD) in the S1 subunit of the SARS-CoV-2 Spike glycoprotein binds to ACE2 expressed on the surface of host cells, while the C-terminal S2 membrane anchoring subunit functions to translocate virus into host cells 21, 24 . After preincubation of HeLa-ACE2 cells with SNOC, we found significantly decreased binding 5 of purified S protein to HeLa-ACE2-cells (Fig. 1d, e) , consistent with the notion that the cysteine residue(s) susceptible to S-nitrosylation in ACE2 affected S protein binding.

Human ACE2 protein contains eight cysteine residues, six of which participate in formation of three pairs of disulfide bonds, and the remaining two (Cys 261 and Cys 498 ) are present as free thiols (or thiolates) (Extended Data Fig. 2a ) 21 and thus potentially available for S-nitrosylation via reversible nucleophilic attack on a nitroso nitrogen to form an SNO-protein adduct 25 . Accordingly, we performed site-directed mutagenesis of these cysteine residues in ACE2 and found that C261A, C498A, or C261A/C498A mutation significantly inhibited SNOC-mediated S-nitrosylation on biotin-switch assays, consistent with the notion that these two cysteine residues are targets of S-nitrosylation (Extended Data Fig. 2b, c) . Moreover, mass spectrometry confirmed the presence of Snitrosylated ACE2 at Cys 261 and Cys 498 after exposure to SNOC (Extended Data Fig.   2d ).

S-Nitrosylation of ACE2 destabilizes dimer formation and thus Spike protein binding. Notably, these S-nitrosylation sites are located near the collectrin-like domain (CLD) region rather than the Spike protein-binding domain region of ACE2 (Fig. 1f ). This suggests that S-nitrosylation may affect the conformation of ACE2 protein at some distance from the S-nitrosylated cysteine residue(s) to diminish binding of ACE2 to trimeric S protein 21,24,26 . Accordingly, explicitly solvated, all-atom molecular dynamics simulations of the S-nitrosylated-ACE2/RBD complex in plasma membrane show that the distance between each S-nitrosylated ACE2 protomer's center of mass is overall much longer and more broadly distributed than in simulations of wild-type (WT) ACE2 dimer (Fig. 1f, g) 26 . This behavior indicates a certain extent of destabilization of the 6 dimer interface imparted by S-nitrosylation, particularly of C 498 . Specifically, at the beginning of the simulations, the S-nitrosylated-ACE2/RBD model displays a hydrogen bond between Q175A and Q139B, which is then interchanged with D136B (Fig. 1h) . This is the only interaction between the peptidase domains (PD) of the two protomers, as also reported for the initial cryo-EM structure 27 . Importantly, over the course of our simulations, this interaction was progressively lost (Fig. 1i) , leading to partial disruption of the PD dimeric interface and transient detachment of the two protomers. Therefore, we hypothesize that the addition of S-nitrosylation at the side chain of C 498 , which is located in the vicinity of Q 175 , could be sufficient to induce rearrangement in the packing of secondary structural elements of this region, leading in turn to the disruption of the only point of contact between the two PDs of ACE2. The loss of this contact may potentially trigger a further destabilization at the level of the dimeric interface between the neck domains. Alteration of ACE2 dimer stability has the potential to interfere with the SARS-CoV-2 Spike binding 28 , thus abrogating infection.

Screening aminoadamantane nitrate compounds against SARS-CoV-2 infection.

Next, we examined the effect of aminoadamantane nitrate compounds on SARS-CoV-2 entry into cells, causing infection. Aminoadamantanes have been reported to directly bind to the viroporin ion channel formed by the SARS-CoV-2 envelope (E) protein 10,29,30 . Therefore, we screened our series of aminoadamantane and nitro-aminoadamantane compounds 6-8 as potential therapeutic drugs against SARS-CoV-2 -these latter drugs might be expected to bind to the viral channel, thus targeting S-nitrosylation to ACE2 to inhibit its interaction with Spike protein and thus viral entry. Specifically, we tested in a masked fashion the efficacy against live SARS-CoV-2 in HeLa-ACE2 cells of 7 aminoadamantanes (memantine, blindly coded as NMT1, and amantadine/NMT4) and aminoadamantane nitrate compounds (NMT2, NMT3 and NMT5-NMT9) (Fig. 2 , full data set shown in Extended Data Table 1) . As positive controls, we used remdesivir, apilimod, and puromycin (Extended Data Table 1 ) 31,32 . In determining the therapeutic potential of these compounds, we considered the selectivity index (SI) that compares a compound's half-maximal non-specific cytotoxicity (CC50) in the absence of infection to its half-maximal effective antiviral concentration (EC50) (CC50/EC50) (Extended Data Table 1 ). The SI can be considered an in vitro indicator of therapeutic index and ideally would approach 10. The aminoadamantane compounds alone (amantadine and memantine) offered no efficacy, and thus were not studied further. In contrast, several of the aminoadamantane nitrate compounds offered some degree of protection from infection. However, NMT6 and NMT8 may have done this simply by killing the host cells irrespective of infection, as evidenced by its off-target killing of uninfected cells in the live/dead assay (Fig. 2 , Extended Data Table 1 ). Among the 7 aminoadamantane nitrate compounds tested, NMT5 displayed the best combination of EC50 and CC50 (SI = 9.2) with an EC50 for protection against SARS-CoV-2 of 5.28 μM (Fig. 2 , Extended Data Table 1 ); this concentration of compound is well within the micromolar amounts attainable in human tissues at well-tolerated doses, as tested in two animal species 6-9,33 .

Additionally, NMT3 (also known as NitroSynapsin), which was already being developed for CNS indications 6-9 displayed some degree of protection against SARS-CoV-2 with an EC50 of 87.7 μM, although this value may be artificially high due to the short half-life of NMT3 in aqueous solution under in vitro conditions 6, 9, 34 . Hence, these two compounds were advanced for further study. We next asked if NMT3 and NMT5 could S-nitrosylate ACE2. We found that NMT5 > NMT3 effectively S-nitrosylated ACE2 both 8 in vitro in HeLa-ACE2 cells and in vivo in Syrian hamsters, as assessed by the biotinswitch assay (Fig. 3a, b, Extended Data Fig. 3) . Notably, a statistically significant increase in the level of S-nitrosylated ACE2 was observed in the SARS-CoV-2 target tissues of lung and kidney at 48 h after oral administration of a single dose of drug at 10 mg/kg (Extended Data Fig. 3d-i) . Consistent with the structure-activity relationship (SAR) indicating that SNO-ACE2 was associated with the anti-viral effect of NMT5 and NMT3, the other aminoadamantane nitrates (including NMT6 and NMT8) did not Snitrosylate ACE2 at low micromolar concentrations (Extended Data Fig. 4 ).

Drug candidate NMT5 can S-nitrosylate ACE2 and inhibit entry of SARS-CoV-2 variants in pseudovirus assays. Since we had found that S-nitrosylation of ACE2 inhibited the binding of SARS-CoV-2 Spike protein, we next asked if NMT3-or NMT5mediated SNO-ACE2 formation could prevent viral entry into host cells. To test this premise, we employed a replication-deficient Maloney murine leukemia virus (MLV)based SARS-CoV-2 Spike protein pseudotype virus, initially using the most prevalent strain of Spike protein (D614) as of early 2020 35 . We examined whether NMT3 and NMT5 could suppress infection with this SARS-CoV-2 pseudovirus. We found that NMT5 inhibited SARS-CoV-2 pseudoviral entry in a dose-dependent manner, with 5 µM inhibiting 53%, 10 µM 76%, and 20 µM 92% (Fig. 3c ). NMT3 showed more limited ability to suppress pseudovirus entry, ~24% at 10 µM. The fact that S-nitrosylation of ACE2 manifested inhibition in the pseudovirus assay (as shown in Fig. 3c) at the approximately the same EC50 of 5 µM as found in the live virus infection assay (Fig. 2) strongly implies that SNO-ACE2 formation is indeed the predominant mechanism by which NMT5 prevents viral infection. As a control, the NMT5 metabolite lacking the nitro 9 group did not suppress SARS-CoV-2 infection in the pseudovirus entry assay (Fig. 3c transmissibility or severity as well as altered antigenicity 36 . We found that NMT5 was also effective in reducing infectivity of these SARS-CoV-2 variants, including the delta and omicron variants, by up to 95% (Fig. 3d-f ). These results are consistent with the notion that NMT5 >> NMT3-mediated S-nitrosylation of ACE2 can inhibit SARS-CoV-2 entry into host cells.

NMT5 predominantly S-nitrosylates ACE2 on cysteine residue 498 and inhibits Spike protein binding to ACE2. Next, we sought to determine if NMT5 could modify ACE2 at both of the cysteine residues (Cys 261 and Cys 498 ) that we demonstrated to be susceptible to S-nitrosylation by SNOC. Analysis by cysteine mutation revealed that NMT5 preferentially S-nitrosylated Cys 498 over Cys 261 (Fig. 3g, h) . Interestingly, the crystal structure of ACE2 shows that an acid/base motif (comprised of Glu 495 and Asp 499 ), which under some conditions may facilitate S-nitrosylation, is present near Cys 498 , while only a partial motif (represented by Asp 609 ) is found near Cys 261 (Fig. 3i) Fig. 6 ), demonstrating relative selectivity of NMT5 for ACE2 at the cell surface. While we cannot rule out the possibility that proteins associated with virus intracellular trafficking are S-nitrosylated, this would be less likely as aminoadamantane nitrate compounds are known to act on extracellular rather than intracellular targets 5-9 .

To further investigate the effect of NMT5 on SARS-CoV-2 Spike protein binding to ACE2, we performed co-immunoprecipitation (co-IP) experiments of these two proteins in the presence and absence of NMT5 using anti-ACE2 antibody for IP. As expected, the two proteins co-IP'd, as evidenced on immunoblots. NMT5 (5 µM) significantly diminished this co-IP, consistent with the notion that the drug inhibited the binding of Spike protein to ACE2 (Fig. 3j, k) . As controls, the Spike protein was not co-IP'd with cysteine mutant ACE2(C498A) or with double mutant ACE2(C261A/C498A), although mutant ACE2(C261A) was still co-IP'd. These data suggest that S-nitrosylation predominantly of C 498 of ACE2 is important for Spike protein binding to ACE2.

Moreover, NMT5 inhibited co-IP of the Spike protein and ACE2(C261A), while having no effect on mutant ACE2(C498A) or ACE2(C261A/C498A) binding (Fig. 3j, k) . Taken together, these results are consistent with the notion that NMT5 inhibits SARS-CoV-2 Spike protein from binding to ACE2 and thus virus entry into the cell via S-nitrosylation of ACE2.

NMT5 targets S-nitrosylation to ACE2 via blockade of the E-protein viroporin channel. Intriguingly, we found that the presence of the envelope (E) protein of SARS-CoV-2 served to target S-nitrosylation by NMT5 to nearby ACE2 receptor proteins ( Fig.   4a , b). To investigate this action further, we assessed the ability of the aminoadamantane compound, memantine, and the lead aminoadamantane nitrate candidate, NMT5, to block ion channel activity of the E protein 11 using the patch-clamp technique. To test direct interaction with the viroporin channel, we transiently transfected HEK293T cells with a construct encoding the E protein and assessed voltage-dependent currents (vs. uninfected cells) in the presence and absence of drug ( Fig. 4c-f ). Under our conditions, we found that the presence of the E protein resulted in a robust voltage-dependent current carried by K + that was inhibited in by memantine and with greater potency by NMT5. Notably, the low micromolar concentrations needed to see these effects are within attainable levels in mammalian plasma and tissues, as shown in pharmacokinetic (PK) studies, and have proven to be safe in animal toxicity studies 6-9,33 .

19. In preparation for in vivo drug candidate efficacy testing in a COVID-19 small animal model, we next performed 48-h PK studies after a single oral dose of NMT3 or NMT5 at 10 mg/kg in ~150 gm Syrian hamsters. We found a half-life in plasma for NMT3 of 7.9 h and for NMT5 of 10.6 h ( Fig. 5a , b, Extended Data Table 2 ). The mean Cmax for NMT5 was 0.2 µM and ~0.4 µM for NMT3; NMT3 also displayed a hydroxylated metabolite (full detailed PK dataset shown in Extended Data Tables 3, 4). The fact that NMT5 was found to be more stable than NMT3 by mass spectrometry analysis ( Fig. 5a , b, Extended Data Table 2 ) was also consistent with prior findings 6-9,33 . Moreover, these drugs are concentrated in tissues up to ~30-fold over plasma levels. Utilizing a Bayesian-like adaptive clinical trial design, we determined the maximal tolerated dose (MTD) of NMT3 and NMT5 in vivo based on dose-ranging toxicity and efficacy studies in 52 Syrian hamsters. To assess treatment efficacy in the Syrian hamster model of COVID-19 at the MTD for NMT5, we administered by oral gavage 200 mg/kg in two equally divided doses separated by 12 h, with the initial dose timed right after challenge with the virus and the second dose 12 h later 35, 42 . Based on the PK results, at this dose, drug levels in tissue should approach or exceed the EC50 found in our in vitro screens to significantly decrease viral infectivity. We found in the Syrian hamster model that this regimen of NMT5, but not NMT3, knocked down live viral titers of SARS-CoV-2 postinfection by ~100 fold, as measured by plaque assay (Fig. 5c ). In the absence of depletion by antibodies 35,43 , virus can persist in lung tissue for several days even in the absence of infection, and thus contribute to plaque assay titers, any significant decrement is encouraging as a potential treatment. More importantly in this model is the histological examination of the lungs for large hemorrhages related to actual SARS-13 CoV-2 infection, reflecting direct blood vessel damage as also seen in human lungs with fatal COVID-19 44 . In this regard, on histological examination, NMT5 virtually eliminated large COVID-19-related hemorrhages in the lungs of infected hamsters compared to vehicle when examined up to 5 days after infection (Fig. 5d ). This translational model revealed a striking absence of large SARS-CoV-2-induced hemorrhages in the lungs of NMT5-treated hamsters vs. controls, with all controls displaying such hemorrhages while no NMT-5 treated animals did so (n = 12, P < 0.01 by Fisher Exact Test) 45 . While some inflammatory changes were noted in the NMT5-treated lung tissue compared to uninfected controls, it was far less than in the infected/untreated tissue ( 

In summary, development of an oral drug to combat acute SARS-Cov-2 infection remains a high priority to treat the COVID-19 pandemic, particularly for the unvaccinated segment of the world population. Our findings provide proof that the cellular receptor of SARS-CoV-2, ACE2, can be S-nitrosylated to inhibit binding of SARS-CoV-2 Spike protein, thus inhibiting viral entry, infectivity, and cytotoxicity. Taking advantage of this finding, we developed a novel aminoadamantane nitrate compound, NMT5, that provides inhibition of SARS-CoV-2 activity by protein S-nitrosylation with a 14 nitro group that is targeted to ACE2 by aminoadamantane-mediated viroporin channel blockade of the E protein 2,3,10-12 . The discovery that ACE2 could be S-nitrosylated was quite unexpected, as most authorities had postulated that the beneficial effects of NO on COVID-19 patients was due to a direct effect on the virus itself. These mechanistic insights should facilitate development of aminoadamantane nitrate drugs for acute antiviral therapy for human COVID-19. A key concept of this novel approach to ameliorating infection by SARS-CoV-2 is that these nitro-aminoadamantane compounds should also prevent new variants of the Spike protein from binding to ACE2 because ACE2 itself is blocked. In this manner, the aminoadamantane nitrate approach to COVID-19 drug therapy complements vaccine and antibody therapies, which are dependent on Spike protein antigenic sites and thus may eventually be susceptible to evasion by further Spike protein mutation. Critically, the binding of NMT5 to the viroporin channel also confers the ability to block spread of SARS-CoV-2 from one host to another. Mechanistically, NMT5 binds to the E protein viroporin channel on SARS-CoV-2 and then transfers NO to ACE2 on the host cell to prevent infection (Fig. 4a, b) .

However, if a patient is already infected and takes NMT5, the newly produced viral particles will bind NMT5 via their E protein viroporin channels and hence viral infectivity will be limited when a new host is exposed to this virus because the new host's ACE2 target protein will be S-nitrosylated by the drug attached to the viral particles as the virus approaches ACE2 on the new host. (Newton, MA), and have been described previously 6-9,33 . The aminoadamantane compounds memantine and amantadine (blindly coded NMT1 and NMT4) were also obtained from EuMentis Therapeutics, Inc. All compounds were sent to Scripps

Research for testing in a masked fashion, and compound identities were not revealed until after experiments were completed and analyzed blindly.

Biotin-switch assays and immunoblotting. For analysis of S-nitrosylated proteins, we performed the biotin-switch assay as previously described [47] [48] [49] preparation then was buffer-exchanged to 1x PBS for the S-nitrosylation assay.

Triton X-100 in PBS. Equivalent protein quantities were immunoprecipitated with anti- Replicate data were analyzed using median condensing. The full dataset is supplied in Extended Data Table 1 .

Pseudoviral entry assay. To measure SARS-CoV-2 viral infectivity, we performed pseudoviral entry assays as previously described 35 Reporting summary. Further information on research design is available in the Nature

Research Reporting Summary linked to this paper.

All data are available in the main text or the supplementary materials. All plasmids generated in this study are available from S.A.L. under a material transfer agreement with The Scripps Research Institute. cells. Cells were exposed to 100 μM SNOC or, as a control, 'old' SNOC (from which NO had been dissipated). After 20 minutes, cell lysates were subjected to biotin-switch assay to assess S-nitrosylated (SNO-) and input (total) proteins detected by immunoblotting with cognate antibody. The ascorbate minus (Asc-) sample served as a negative control. b, c, Ratio of SNO-ACE2/input ACE2 protein and SNO-TMPRSS2/input TMPRSS2 protein. Data are mean + s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA with Tukey's multiple comparisons. n = 3 biological replicates. d,

HeLa and HeLa-ACE2 cells were pre-exposed to 100 μM SNOC or old SNOC. After 30 minutes, 10 μg/ml of purified recombinant SARS-CoV-2 Spike (S1+S2) protein was 

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HOPE T32 Training Grant T32AI007384 (to L.N.C.), California Institute for Regenerative Medicine (CIRM) grant DISC2 COVID19-11811, COVID-19 awards from Fast Grants (to S.A.L.), and grants from the Bill & Melinda Gates Foundation #OPP1107194 (to Calibr) and INV-004923 (to I.A.W). The molecular dynamics simulations were supported by NIH R01 GM132826, NSF RAPID (MCB-2032054), an award from the RCSA Research Corp. and a UC San Diego Moore's Cancer Center 2020 SARS-CoV-2 seed grant (to R.E.A.), and the Interfaces Graduate Training Program

formulated the detailed research plans, interpreted experimental results, and wrote the first draft of the manuscript. C.O. performed a majority of molecular and biochemical experiments, and X.Z. carried out biochemical experiments with mutant ACE2

ns: not significant by ANOVA with Tukey's multiple comparisons. n = 4 biological replicates. c, HeLa-ACE2 cells were incubated with SARS-CoV-2 Spike (D614) or VSV-G (control) pseudovirus particles in the presence and absence of MEM (memantine), NMT3, or NMT5. After 48 h, viral transduction efficiency was monitored by luciferase activity

After 48 h, viral transduction efficiency was monitored by luciferase activity. Data are mean + s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed Student's t test for single comparison (d) or ANOVA with Tukey's for multiple comparisons (e, f). ns: not significant, n = 3 biological replicates. g, HEK293T cells were transfected with plasmids encoding human WT ACE2 or nonnitrosylatable mutant ACE2 (C262A, C498A, or C261A/C498A). One day later, cells were treated with 10 μM NMT5, and 1-h later subjected to biotin-switch assay for detection of S-nitrosylated proteins by immunoblotting with anti-ACE2 and anti-TMPRSS2 antibodies. The absence of ascorbate (Asc-) served as a negative control. h, Ratio of SNO-ACE2/input ACE2

Glu 495 and Asp 499 , acidic amino-acid residues, surround Cys 498 (right panel). j, HEK293T cells were transfected with plasmids encoding human WT ACE2 or non-nitrosylatable mutant ACE2 (C262A, C498A, or C261A/C498A). One day later, cells were exposed to 1 μg/ml of purified recombinant SARS-CoV-2

after 1 h, cells were lysed and subjected to co-IP with anti-ACE2 antibody

Spike protein were detected by immunoblotting with anti-ACE2 and anti-Spike protein antibodies. k, Ratio of IP-ACE2/IP-Spike protein. Data are mean + s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant by ANOVA with Fisher's LSD multiple comparisons

After 1 day, cells were harvested and plated onto HeLa-ACE2 cells in the presence or absence of 5 µM NMT5. After 30 min, cell lysates were subjected to biotin-switch assay to monitor protein S-nitrosylation of ACE2, detected by immunoblotting. b, Ratio of SNO-ACE2/total input ACE2 protein. Data are mean + s.e.m., *P < 0.05, **P < 0.01 by ANOVA with Tukey's multiple comparisons

and transiently transfected (n = 15) HEK293T cells before and after application of memantine or NMT5 during patch-clamp recording. Whole-cell +90 mV in increments of 10 mV. f, Current-voltage (I-V) curves from steadystate current density (pA/pF) versus holding potential (mV) for memantine (MEM

Cell lysates were then subjected to biotin-switch assay to assess protein S-nitrosylation, which was detected by immunoblotting with anti-ACE2 antibody. b, Ratio of SNO-ACE2/input ACE2 protein

Extended Data Fig. 2 | Identification of cysteine residues in ACE2 that are Snitrosylated. a, List of human ACE2 peptides (± 7 amino acid residues flanking a cysteine residue); gray: peptides involved in disulfide bond formation; black: peptides containing a free cysteine thiol (red) that could potentially be S-nitrosylated. b, HEK293T cells were transiently transfected with plasmids containing human WT ACE2 or cysteine mutant ACE2 (C261A, C498A, or C261/498A)

After 20 minutes, cells were subjected to biotinswitch assay. Absence of ascorbate (Asc -) served as a negative control. c, Ratio of SNO-ACE2/input ACE2

Extended Data Fig. 3 | NMT5 S-nitrosylates ACE2 in vitro and in vivo. a, Detection of SNO-ACE2 in vitro

After 1 h, cells were subjected to the biotin-switch assay in the presence or absence of ascorbate. SNO-ACE2 and input ACE2 were detected by immunoblotting with anti-ACE2 antibody. b, c, Ratio of SNO-ACE2/input ACE2. Data are mean ± s.e.m., **P < 0.01 by two-tailed Student's t test. ns: not significant, n = 5 biological replicates. d-i, Detection of SNO-ACE2 in vivo

Kidney and lung tissues were subjected to biotin-switch assay in the presence or absence of ascorbate. Note that in some samples, low levels of SNO-ACE2 were observed in control tissue, suggesting endogenous S-nitrosylation of ACE2 may occur at low levels. Graphs show ratio of SNO-ACE2/input ACE2

After 1 h, cell lysates were subjected to biotin-switch assay for protein S-nitrosylation, detected by immunoblotting with anti-ACE2 antibody. The ascorbate minus (Asc-) sample served as a negative control. b, Ratio of SNO-ACE2/input ACE2 protein

Extended Data Fig. 5 | Critical role of nitro group of NMT5 suppressing SARS-CoV-2 infection on pseudovirus entry assay. a, Chemical structure of NMT5

HeLa-ACE2 cells were incubated in the presence and absence of 5 µM NMT5-Met with SARS-CoV-2 Spike (D614) pseudovirus particles

Purified recombinant SARS-CoV-2 Spike (S1+S2) protein and ACE2 protein were exposed to 100 μM SNOC; 30 min later, samples were subjected to biotin-switch assay in the presence or absence of ascorbate (Asc) to assess protein S-nitrosylation. b, Lack of E protein S-nitrosylation by NMT5. HA-tagged E protein plasmid was transiently transfected into HEK293 cells. One day after transfection, cells were exposed to 10 μM NMT5. After 1 hour, the cells were harvested and subjected to biotin-switch assay in the presence or absence of ascorbate (Asc) to assess protein S-nitrosylation

Dose-response analysis of in vitro infection assay and uninfected host cell cytotoxicity data for control (apilimod, remdesivir, puromycin) and test compounds, including dose-response curves and curve fit parameters. For the infection assay two assay metrics (% infected cells and total cells per well) are reported

We thank David Nemazee (Scripps Research) for providing HeLa-ACE2 cells and plasmids for pseudovirus. This work was supported in part by NIH grants RF1

The authors declare that S.A.L. is an inventor on patents for the use of memantine and 

Extended data is available for this paper at https://doi.org/xxxxx/xxxxx.

Supplementary information The online version contains supplementary material available at https://doi.org/xxxxxx.Correspondence and requests for materials should be addressed to Stuart A. Lipton.Peer review information Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work.Reprints and permissions information is available at http://www.nature.com/reprints. 61 Extended Data Table 2 | Summary values of PK data for NMT5 and NMT3. Plasma concentrations measured by LC-MS/MS of NMT5 or NMT3 in Syrian hamsters collected at timepoints ranging from 0.5 hours to 48 hours after a single 10 mg/kg dose administered by oral gavage. Pharmacokinetic (PK) analysis of NMT5 (n = 3) and NMT3 (n = 6) in hamsters was assessed using non-compartmental analysis (NCA) of extrapolated plasma concentrations over time using Phoenix WinNonLin software.

Full dataset for plasma concentrations of NMT5 and metabolite quantified by LC-MS/MS after a single 10 mg/kg dose administered by oral gavage to Syrian hamsters.Extrapolation of plasma concentrations was determined from an NMT5-spiked standard curve with a linear range of 0.3 ng/ml -1250 ng/ml, and a standard curve with a linear 62 range of 0.3 ng/ml -5000 ng/ml for the metabolite. Determination of PK parameters was conducted by NCA analysis of serially collected samples using Phoenix WinNonLin software. This table is located in a separate EXCEL file with multiple tabs accessible by clicking on the bottom of the page.

Full dataset for plasma concentrations of NMT3 and metabolite quantified by LC-MS/MS after a single 10 mg/kg dose administered by oral gavage to Syrian hamsters.Extrapolation of plasma concentrations was determined from an NMT3-spiked standard curve with a linear range of 4.88 ng/ml -10,000 ng/ml. The metabolite for NMT3 was quantified by extrapolation from a standard curve with a linear concentration range of 2.44 ng/ml -10,000 ng/ml. Determination of limited PK parameters was conducted by NCA analysis of sparsely collected samples using Phoenix WinNonLin software. This table is located in a separate EXCEL file with multiple tabs accessible by clicking on the bottom of the page.