Multiple Computational Approaches for Predicting Drug Interactions with Human Equilibrative Nucleoside Transporter 1

Abstract

Equilibrative nucleoside transporters (ENTs) participate in the pharmacokinetics and disposition of nucleoside analog drugs. Understanding drug-interactions with the ENTs may inform and facilitate the development of new drugs, including chemotherapeutics and antivirals that require access to sanctuary sites such as the male genital tract. This study created 3-D pharmacophores for ENT1 and ENT2 substrates and inhibitors, using K t and IC 50 data curated from the literature. Substrate pharmacophores for ENT1 and ENT2 are distinct, with partial overlap of hydrogen bond donors, while the inhibitor pharmacophores predominantly feature hydrogen bond acceptors. Mizoribine and ribavirin mapped to the ENT1 substrate pharmacophore and proved to be substrates of the ENTs. The presence of the ENT-specific inhibitor, NBMPR, decreased mizoribine accumulation in ENT1 and ENT2 cells (ENT1, ~70% decrease, p = 0.0046; ENT2, ~50% decrease p = 0.0012). NBMPR also decreased ribavirin accumulation in ENT1 and ENT2 cells (ENT1: ~50% decrease p = 0.0498; ENT2: ~30% decrease p = 0.0125). Darunavir mapped to the ENT1 inhibitor pharmacophore and NBMPR did not significantly influence darunavir accumulation in either ENT1 or ENT2 cells (ENT1: p = 0.28; ENT2: p = 0.53), indicating that darunavir's interaction with the ENTs is limited to inhibition. These computational and in vitro models can inform compound selection in the drug discovery and development process, thereby reducing time and expense of identification and optimization of ENT interacting compounds. Significance StatementThis study developed computational models of human equilibrative nucleoside transporters (ENTs) to predict drug interactions and validated these models with two compounds in vitro.

Defending Antiviral Cationic Amphiphilic Drugs That May Cause Drug-Induced Phospholipidosis

“…46 We can also target transporters in order to reach viral sanctuary sites such as the testes, for example, using human equilibrative nucleoside transporters (ENT) whose structure-inhibitor and structure-substrate relationships we are just beginning to elucidate. 47 Therefore, perhaps before advocating for the abrogation of FDA-approved drugs or novel compounds that are CADs demonstrating phospholipidosis from SARS-CoV-2 in vitro screens (and ultimately from in vivo testing), we should consider the potential for missing promising clinical candidates which represent the needle in the proverbial haystack (Figure 1). As an example, out of the compounds in Table 1 that demonstrated in vitro and in vivo activity against SARS-CoV-2 we would suggest several may meet the logP and pK a criteria as likely CADs; however, we are not aware of any published reports of phospholipidosis for these compounds.…”

“…It is unlikely that these would impact SARS-CoV-2 viral entry; however, it should also be pointed out that some transporters can be leveraged by viruses for entry, such as the bile acid transporter sodium taurocholate cotransporting polypeptide (NTCP) . We can also target transporters in order to reach viral sanctuary sites such as the testes, for example, using human equilibrative nucleoside transporters (ENT) whose structure-inhibitor and structure-substrate relationships we are just beginning to elucidate …”

Transporter Inhibition Profile for the Antivirals Tilorone, Quinacrine and Pyronaridine

“…Therefore, there is considerable interest in identifying compounds that cross the blood-testis barrier (BTB), as this compartment can act as a viral reservoir . The equilibrative nucleoside transporters (ENTs) are present at the BTB, where they can facilitate antiviral drug disposition to eliminate a sanctuary site for viruses detectable in semen. , Predicting interactions with these transporters can be used to aid the development of novel compounds that can cross the BTB using the ENT1-ENT2 transepithelial transport pathway. ,, These include novel antivirals and chemotherapeutics that are substrates for these widely expressed transporters. Most recently, we applied computational and experimental approaches to investigate interactions of the antiviral drugs remdesivir, tilorone, pyronaridine, quinacrine, hydroxychloroquine, molnupiravir and its active metabolite EIDD-1931 with ENT1 and ENT2 .…”

“… 49 , 50 Predicting interactions with these transporters can be used to aid the development of novel compounds that can cross the BTB using the ENT1-ENT2 transepithelial transport pathway. 25 , 51 , 52 These include novel antivirals and chemotherapeutics that are substrates for these widely expressed transporters. Most recently, we applied computational and experimental approaches to investigate interactions of the antiviral drugs remdesivir, tilorone, pyronaridine, quinacrine, hydroxychloroquine, molnupiravir and its active metabolite EIDD-1931 with ENT1 and ENT2.…”

Inosine: A bioactive metabolite with multimodal actions in human diseases

“…Inosine can be metabolized into hypoxanthine, xanthine, and uric acid ( Sorensen, 1970 ; Zoref-Shani et al, 1988 ; Doyle et al, 2018 ; Garcia-Gil et al, 2021 ). Cell membrane transport of inosine is mediated by equilibrative and concentrative nucleoside transporters ( Körber et al, 1975 ; Belt et al, 1993 ; Cass et al, 1999 ; Miller et al, 2021 ). Inosine functions are mediated in receptor-dependent or–independent manners.…”