Influenza viruses are respiratory pathogens that are responsible for both seasonal influenza epidemics and occasional influenza pandemics. The narrow therapeutic window of oseltamivir, coupled with the emergence of drug resistance, calls for the next-generation of antivirals. With our continuous interest in developing AM2-S31N inhibitors as oral influenza antivirals, we report here the progress of optimizing the in vitro pharmacokinetic (PK) properties of AM2-S31N inhibitors. Several AM2-S31N inhibitors, including compound 10b, were discovered to have potent channel blockage, single to submicromolar antiviral activity, and favorable in vitro PK properties. The antiviral efficacy of compound 10b was also synergistic with oseltamivir carboxylate. Interestingly, binding kinetic studies (K, K, and K) revealed several AM2-S31N inhibitors that have similar K values but significantly different K and K values. Overall, this study identified a potent lead compound (10b) with improved in vitro PK properties that is suitable for the in vivo mouse model studies.

Equilibrative nucleoside transporters (ENT) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/Cas9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [ 3 H]uridine uptake in the presence of the ENT specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse transcriptase inhibitors and thirty-seven nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC 50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine if there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, while nevirapine and lexibulin were ENT1 and ENT2 non-transported inhibitors. Bayesian models generated using Assay Central ® machine learning software yielded reasonably high internal validation performance (ROC > 0.7). ENT1 IC 50-based models were generated from ChEMBL; subvalidations using this training dataset correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC 50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 This article has not been copyedited and formatted. The final version may differ from this version.

Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [ 3 H] uridine uptake by NBMPR was biphasic, with IC 50 values of 11.3 nM for ENT1 and 9.6 mM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1-and ENT2-mediated [ 3 H]uridine uptake revealed no difference in J max (16.53 and 30.40 pmol cm 22 min 21 ) and an eightfold difference in K t (13.6 and 108.9 mM). The resulting fivefold difference in intrinsic clearance (J max /K t ) for ENT1-and ENT2 transport accounted for observed inhibition of [ 3 H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC 50 values of ∼200 mM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [ 3 H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENTThis study characterizes an in vitro model using S-[(4-nitrophenyl) methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1-and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.

Majority of current circulating influenza A viruses carry the S31N mutation in their M2 genes, rendering AM2-S31N as a high profile antiviral drug target. With our continuous interest in developing AM2-S31N channel blockers as novel antivirals targeting both oseltamivir-sensitive and-resistant influenza A viruses, we report herein the structure− property relationship studies of AM2-S31N inhibitors. The goal was to identify lead compounds with improved microsomal stability and membrane permeability. Two lead compounds, 10d and 10e, were found to have high mouse and human liver microsomal stability (T 1/2 > 145 min) and membrane permeability (>200 nm/s). Both compounds also inhibit both currently circulating oseltamivir-sensitive and-resistant human influenza A viruses (H1N1 and H3N2) with EC 50 values ranging from 0.4 to 2.8 μM and a selectivity index of >100. We also showed for the first time that AM2-S31N channel blockers such as 10e inhibited influenza virus replication at both low and high multiply of infection (10 2 −10 6 pfu/mL) and the inhibition was not cell-type dependent. Overall, these studies have identified two promising lead candidates for further development as antiviral drugs against drug-resistant influenza A viruses.

The blood-testis barrier (BTB) formed by adjacent Sertoli cells (SCs) limits the entry of many chemicals into seminiferous tubules. Differences in rodent and human substrate-transporter selectivity or kinetics can misrepresent conclusions drawn using rodent in vitro models. Therefore, human in vitro models are preferable when studying transporter dynamics at the BTB. This study describes a hTERT-immortalized human SC line (hT-SerC) with significantly increased replication capacity and minor phenotypic alterations compared to primary human SCs. Notably, hT-SerCs retained similar morphology and minimal changes to mRNA expression of several common SC genes, including AR and FSHR. The mRNA expression of most xenobiotic transporters was within the 2-fold difference threshold in RT-qPCR analysis with some exceptions (OAT3, OCT3, OCTN1, OATP3A1, OATP4A1, ENT1, and ENT2). Functional analysis of the equilibrative nucleoside transporters (ENTs) revealed that primary human SCs and hT-SerCs predominantly express ENT1 with minimal ENT2 expression at the plasma membrane. ENT1-mediated uptake of [3H] uridine was linear over 10 min and inhibited by NBMPR with an IC50 value of 1.35 ± 0.37 nM. These results demonstrate that hT-SerCs can functionally model elements of transport across the human BTB, potentially leading to identification of other transport pathways for xenobiotics, and will guide drug discovery efforts in developing effective BTB-permeable compounds.

The emergence of multidrug-resistant influenza viruses poses a persistent threat to public health. The current prophylaxis and therapeutic interventions for influenza virus infection have limited efficacy due to the continuous antigenic drift and antigenic shift of influenza viruses. As part of our ongoing effort to develop the next generation of influenza antivirals with broad-spectrum antiviral activity and a high genetic barrier to drug resistance, in this study we report the discovery of dapivirine, an FDA-approved HIV nonnucleoside reverse transcriptase inhibitor, as a broad-spectrum antiviral against multiple strains of influenza A and B viruses with low micromolar efficacy. Mechanistic studies revealed that dapivirine inhibits the nuclear entry of viral ribonucleoproteins at the early stage of viral replication. As a result, viral RNA and protein synthesis were inhibited. Furthermore, dapivirine has a high in vitro genetic barrier to drug resistance, and its antiviral activity is synergistic with oseltamivir carboxylate. In summary, the in vitro antiviral results of dapivirine suggest it is a promising candidate for the development of the next generation of dual influenza and HIV antivirals.

Influenza virus infection causes substantial morbidity and mortality worldwide. The limited efficacy of oseltamivir in delayed treatment, coupled with the increasing incidences of oseltamivirresistant strains, calls for next-generation of antiviral drugs. In this study, we discovered NMS-873, an allosteric and specific p97 inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. NMS-873 shows potent antiviral activity with lownanomolar EC 50 s against multiple human influenza A and B viruses, including adamantine-, oseltamivir-, or double resistant strains. Our data further showed that silencing of p97 via siRNA or inhibiting p97 by NMS-873 both inhibited virus replication and retained viral ribonucleoproteins (vRNPs) in the nucleus, confirming p97 is the drug target. Mechanistic studies have shown that the nuclear retention of vRNP with NMS-873 treatment is a combined result of two effects: the reduced viral M1 protein level (indirect effect), and the disruption of p97-NP interactions (direct effect). Taken together, our results suggest that p97 could be a novel antiviral target and its inhibitor, NMS-873, is a promising antiviral drug candidate.

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