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Allosteric inhibition of aminopeptidase N functions related to tumor growth and virus infection

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ABSTRACT

Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.

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Catalytic site-binding drugs inhibit CoV cell infection.(a) Inhibition of TGEV infection by various pAPN-binding drugs. Relative survival of ST cells infected with TGEV at different m.o.i., treated with 50 μM ABS1-4, actinonin (Ac) or DMSO (Methods). See Supplementary Fig. S3b. Mean ± SEM (n = 3). (b) Inhibition of TGEV cell entry by ABS4. Virus RNA was quantified by qRT-PCR 6 h post-infection, in samples without inhibitor (−), with ABS4 as indicated (μM) or with TGEV RBD-Fc (40 μg/ml) as positive control (RBD). Background signal determined with uninfected BHK cells (BHK). Mean ± SEM (n = 3). (c) Inhibition of TGEV cell infection with increasing ABS4 concentrations. ST cell survival was determined as in a. Mean ± SEM (n = 4).
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f5: Catalytic site-binding drugs inhibit CoV cell infection.(a) Inhibition of TGEV infection by various pAPN-binding drugs. Relative survival of ST cells infected with TGEV at different m.o.i., treated with 50 μM ABS1-4, actinonin (Ac) or DMSO (Methods). See Supplementary Fig. S3b. Mean ± SEM (n = 3). (b) Inhibition of TGEV cell entry by ABS4. Virus RNA was quantified by qRT-PCR 6 h post-infection, in samples without inhibitor (−), with ABS4 as indicated (μM) or with TGEV RBD-Fc (40 μg/ml) as positive control (RBD). Background signal determined with uninfected BHK cells (BHK). Mean ± SEM (n = 3). (c) Inhibition of TGEV cell infection with increasing ABS4 concentrations. ST cell survival was determined as in a. Mean ± SEM (n = 4).

Mentions: APN catalytic activity is not necessary for CoV cell entry and infection31. We nonetheless found that active site-binding molecules hindered CoV S protein binding and might inhibit virus infection. Studies with low affinity binding drugs such as bestatin show no reduction in TGEV infection31. Virus particles have high receptor-binding avidity, and these drugs might not have sufficient affinity to maintain most APN molecules closed. The selective compounds ABS1-4 have high affinity for APN and, at 1–10 μM concentrations, inhibit capillary tube formation in cell cultures, with no cytotoxicity27. In our cultures, we observed no toxicity at ABS concentrations <100 μM (not shown). We therefore analyzed the TGEV-mediated cytopathic effect for each of the four ABS molecules and actinonin at a 50 μM concentration and monitored inhibition of virus infection with ABS4 (2 log) and ABS2 (1 log) (Fig. 5a); at the same concentration, the lower-affinity ABS1 and ABS3 compounds or actinonin did not inhibit. ABS4 has a bromo substituent that is predicted to interact with the phenylalanine that plugs the substrate in the closed conformation32; this interaction likely helped maintain the closed ectodomain and efficiently prevented virus binding.


Allosteric inhibition of aminopeptidase N functions related to tumor growth and virus infection
Catalytic site-binding drugs inhibit CoV cell infection.(a) Inhibition of TGEV infection by various pAPN-binding drugs. Relative survival of ST cells infected with TGEV at different m.o.i., treated with 50 μM ABS1-4, actinonin (Ac) or DMSO (Methods). See Supplementary Fig. S3b. Mean ± SEM (n = 3). (b) Inhibition of TGEV cell entry by ABS4. Virus RNA was quantified by qRT-PCR 6 h post-infection, in samples without inhibitor (−), with ABS4 as indicated (μM) or with TGEV RBD-Fc (40 μg/ml) as positive control (RBD). Background signal determined with uninfected BHK cells (BHK). Mean ± SEM (n = 3). (c) Inhibition of TGEV cell infection with increasing ABS4 concentrations. ST cell survival was determined as in a. Mean ± SEM (n = 4).
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getmorefigures.php?uid=PMC5385526&req=5

f5: Catalytic site-binding drugs inhibit CoV cell infection.(a) Inhibition of TGEV infection by various pAPN-binding drugs. Relative survival of ST cells infected with TGEV at different m.o.i., treated with 50 μM ABS1-4, actinonin (Ac) or DMSO (Methods). See Supplementary Fig. S3b. Mean ± SEM (n = 3). (b) Inhibition of TGEV cell entry by ABS4. Virus RNA was quantified by qRT-PCR 6 h post-infection, in samples without inhibitor (−), with ABS4 as indicated (μM) or with TGEV RBD-Fc (40 μg/ml) as positive control (RBD). Background signal determined with uninfected BHK cells (BHK). Mean ± SEM (n = 3). (c) Inhibition of TGEV cell infection with increasing ABS4 concentrations. ST cell survival was determined as in a. Mean ± SEM (n = 4).
Mentions: APN catalytic activity is not necessary for CoV cell entry and infection31. We nonetheless found that active site-binding molecules hindered CoV S protein binding and might inhibit virus infection. Studies with low affinity binding drugs such as bestatin show no reduction in TGEV infection31. Virus particles have high receptor-binding avidity, and these drugs might not have sufficient affinity to maintain most APN molecules closed. The selective compounds ABS1-4 have high affinity for APN and, at 1–10 μM concentrations, inhibit capillary tube formation in cell cultures, with no cytotoxicity27. In our cultures, we observed no toxicity at ABS concentrations <100 μM (not shown). We therefore analyzed the TGEV-mediated cytopathic effect for each of the four ABS molecules and actinonin at a 50 μM concentration and monitored inhibition of virus infection with ABS4 (2 log) and ABS2 (1 log) (Fig. 5a); at the same concentration, the lower-affinity ABS1 and ABS3 compounds or actinonin did not inhibit. ABS4 has a bromo substituent that is predicted to interact with the phenylalanine that plugs the substrate in the closed conformation32; this interaction likely helped maintain the closed ectodomain and efficiently prevented virus binding.

View Article: PubMed Central - PubMed

ABSTRACT

Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.

No MeSH data available.


Related in: MedlinePlus