The Front Cover illustrates the L26F variant of the M2 protein of influenza A virus which is resistant to the amantadine class of drugs. It shows the combination of two methods (MD simulations and MAS NMR) that reveal the binding of a potent inhibitor of the L26F M2 proton channel, RL208, or compound 61 in the corresponding research. The ligand RL208 is an isomer of amantadine and is described as a triple blocker of WT, L26F, V27A M2 variants. Cover design by Marianna Stampolaki and Kyriakos Georgiou; image Virus Bacterium Infection by PublicDomainPictures from Pixabay used in accordance with the Pixabay license. More information can be found in the Research Article by Μarianna Stampolaki, Anja Hoffmann, Loren B. Andreas, Michaela Schmidtke, Antonios Kolocouris et al.

We compared the anti-influenza potencies of 57 adamantyl amines and analogs against influenza A virus with serine-31 M2 proton channel, usually termed as WT M2 channel, which is amantadine sensitive. We also tested a subset of these compounds against viruses with the amantadine-resistant L26F, V27A, A30T, G34E M2 mutant channels. Four compounds inhibited WT M2 virus in vitro with mid-nanomolar potency, with 27 compounds showing sub-micromolar to low micromolar potency. Several compounds inhibited L26F M2 virus in vitro with sub-micromolar to low micromolar potency, but only three compounds blocked L26F M2-mediated proton current as determined by electrophysiology (EP). One compound was found to be a triple blocker of WT, L26F, V27A M2 channels by EP assays, but did not inhibit V27A M2 virus in vitro, and one compound inhibited WT, L26F, V27A M2 in vitro without blocking V27A M2 channel. One compound blocked only L26F M2 channel by EP, but did not inhibit virus replication. The triple blocker compound is as long as rimantadine, but could bind and block V27A M2 channel due to its larger girth as revealed by molecular dynamics simulations, while MAS NMR informed on the interaction of the compound with M2(18-60) WT or L26F or V27A.

Influenza A viruses can cause a serious future threat due to frequent mutations. Amantadine and rimantadine drugs inhibit influenza A M2 wild-type (WT; bearing in the protein M2 proton channel serine at position-31) viruses by binding and blocking M2 WT channel-mediated proton current. The resistant to these drugs influenza A viruses bearing the S31N mutant in the M2 proton channel can be inhibited by amantadine – aryl conjugates, in which amantadine and an aryl group are linked through a methylene, which block M2 S31N channel-mediated proton current. However, the M2 amantadine/rimantadine resistant viruses bearing one of the four mutations L26F, V27A, A30T, G34E in residues that line the M2 channel pore pose an additional concern for public health.Here, we designed 33 compounds based on the structure of three previously published and potent amantadine-aryl conjugates against M2 S31N virus, by replacing amantadine with 16 amantadine variants. The compounds were tested against M2 WT and the five M2 amantadine resistant viruses aiming at identifying inhibitors against multiple M2 mutant – amantadine resistant viruses.We identified 16 compounds that inhibited in vitro two influenza A viruses with M2 WT or L26F channels. Additionally, compounds 21 or 32 or 33, which are conjugates of the rimantadine variant with CMe2 (instead of CHMe in rimantadine) or the diamantylamine or the 4-(1-adamantyl)benzenamine with the 2-hydroxy-4-methoxyphenyl aryl group, were in vitro inhibitors against three influenza A viruses with M2 WT or L26F or S31N, while compound 21 inhibited also in vitro the M2 G34E virus and compound 32 inhibited also in vitro the M2 A30T virus. Also, using electrophysiology, we showed that compound 21 was an efficient blocker of the M2 WT and M2 L26F channels, compound 32 blocked efficiently the M2 WT channel and compound 33 blocked the M2 WT, L26F and V27A channels. The drug metabolism and pharmacokinetics studies showed that these compounds need further optimization.

Future pandemic influenza necessitates the development of new drugs against the current circulating, amantadine and rimantadine drugs resistant, influenza A M2 S31N viruses. The possibility of an antigenic shift to M2 S31 necessitates ranking the biological activities of amantadine variants. Several amantadine variants have been tested by different laboratories, but various M2 wild type influenza A strains have been used with different sensitivity against amantadine and the unambiguous comparison between potencies is not straightforward. Here, we compared the anti-influenza activities of 57 synthetic amantadine variants against influenza A WSN/33 viruses with amantadine-sensitive M2 WT, with a range of over three digits providing a reference set of potencies for structure-activity relationships, and amantadine-resistant M2 S31N proteins (and observed no potent compounds). 17 compounds were selected and tested against M2 L26F, V27A, A30T, G34E viruses. We tested few reference compounds using electrophysiology and explored point mutations which both showed that M2 is the target of potent antiviral potency against the M2 WT, L26F, V27A viruses. Major findings are: (a) Several amantadine variants from Kolocouris group block only M2 WT and M2 L26F-mediated proton current and the corresponding viruses replication. (b) A compound from Vazquez’s group is a triple blocker of M2 WT, L26F, V27A channels and viruses replication. (c) A compound from Vazquez’s group blocks only M2 L26 channel and virus replication. (d) Several compounds from Kolocouris group have potent activity against several influenza A M2 WT and three M2 S31N viruses, eg. the pandemic A/H1N1/California/07/2009 (H1N1pdm09) or A/H1N1/PuertoRico/08/1934 without blocking M2 S31N. The compounds and their cocktails while not to be more toxic than amantadine might be useful for re-purposing of amantadine class of drugs in the case (i) of the prevalence of M2 L26F and or M2 V27A strains (ii) of an antigenic shift of the virus to M2 WT and (iii) because they inhibited a broad panel of M2 WT and M2 S31N viruses including the H1N1pdm09). (d) We showed that the mechanism of antiviral activity against A/California/07/2009 or A/PR/08/1934 and possibly also M2 WT viruses compared to WSN/33 viruses is not due to inhibition of an early stage of virus infection or a late stage of M2 channel function during endocytosis or inhibition of HA binding to host cells or a different pH for HA fusion or a lysosomotropic effect.

We report on using the synthetic aminoadamantane-CH2-aryl derivatives 1-6 as sensitive probes for blocking M2 S31N and influenza A virus (IAV) M2 wild-type (WT) channels as well as virus replication in cell culture. The binding kinetics measured using electrophysiology (EP) for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl headgroup realized in 2 and 3 and the girth and length of the adamantane adduct realized in 4 and 5. Study of 1-6 shows that, according to molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations, all bind in the M2 S31N channel with the adamantyl group positioned between V27 and G34 and the aryl group projecting out of the channel with the phenyl (or isoxazole in 6) embedded in the V27 cluster. In this outward binding configuration, an elongation of the ligand by only one methylene in rimantadine 2 or using diamantane or triamantane instead of adamantane in 4 and 5, respectively, causes incomplete entry and facilitates exit, abolishing effective block compared to the amantadine derivatives 1 and 6. In the active M2 S31N blockers 1 and 6, the phenyl and isoxazolyl head groups achieve a deeper binding position and high kon/low koff and high kon/high koff rate constants, compared to inactive 2-5, which have much lower kon and higher koff. Compounds 1-5 block the M2 WT channel by binding in the longer area from V27-H37, in the inward orientation, with high kon and low koff rate constants. Infection of cell cultures by influenza virus containing M2 WT or M2 S31N is inhibited by 1-5 or 1-4 and 6, respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 2-4 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection.

In an attempt to synthesize potent blockers of the influenza A M2 S31N proton channel with modifications of amantadine, we used MD simulations and MM-PBSA calculations to project binding modes of compounds 2-5, which are analogues of 1, a dual blocker. Blocking both the S31N mutant and the wild type (WT) M2, 1 is composed of amantadine linked to an aryl head group, (4-methoxy-2-hydroxy)-benzyl. Compound 6, used as control, has an 3-(thiophenyl)isoxazolyl aryl head group, and selectively blocks M2 S31N (but not WT) in an aryl head group “out” (i.e. N-ward) binding orientation. We then tested 1-6 as anti-virals in cell culture and for M2 binding efficacy with electrophysiology (EP). The new molecules 2-5 have a linker between the adamantane and amino group which can be as small as a CMe2 in rimantadine derivative 2, or longer like phenyl in 3. Alternatively, we explored the impact of expanding the diameter of adamantane with diamantyl or triamantyl in 4 and 5, respectively. Antiviral effects against A/WSN/33 and its M2 WT revertant (M2 N31S) were seen for all six compounds except for 5 vs. the native (S31N) virus and (as predicted from previous studies) 6 vs. the WT revertant. Compounds 1-5, projected to bind in a polar head group “in” (C-ward) orientation, strongly block proton currents through M2 WT expressed in voltage-clamped oocytes with fast association rate constants (kon), and slow dissociation rate constants (koff). Surprisingly, 2-5, projected to bind in a polar head group out orientation, do not effectively block M2 S31N-mediated proton currents in EP. The results from MD and MM-PBSA calculations suggested that compounds 2-5 can be fully effective at blocking the M2 channel when present. The low degree of blocking in M2 S31N is due to their kinetics of binding observed in EP, i.e. two orders of magnitude reduction in kon compared to 6, and a fast off rate constant similar to that of 6, which is consistent with steered-MDsimulations. The low kon values can be interpreted from MD simulations, which suggest distortions to V27 cluster of the M2 S31N caused by the longer (even by one methylene) hydrophobic segment from adamantane to aryl head group, appropriate to fit from G34 to V27. The deformations in the N-terminus may be sufficiently energetic for 2-5 (compared to 6) to cause the observed low kon.

Therapy or prophylaxis of herpes simplex virus type 2 (HSV-2) infections with the nucleoside analog aciclovir (ACV) can lead to the emergence of drug-resistant HSV-2 strains, particularly in immunocompromised patients. In this context, multiple amino acid (aa) changes can accumulate in the ACV-converting viral thymidine kinase (TK) which hampers sequence-based diagnostics significantly. In this study, the so far unknown or still doubted relevance of several individual aa changes for drug resistance in HSV-2 was clarified. For this purpose, ten recombinant fluorescent HSV-2 strains differing in the respective aa within their TK were constructed using the bacterial artificial chromosome (BAC) pHSV2(MS)Lox. Similar TK expression levels and similar replication behavior patterns were demonstrated for the mutants as compared to the unmodified BAC-derived HSV-2 strain. Subsequently, the resulting strains were tested for their susceptibility to ACV as well as penciclovir (PCV) in parallel to a modified cytopathic effect (CPE) inhibition assay and by determining the relative fluorescence intensity (quantified using units, RFU) as a measure for the viral replication capacity. While aa changes Y53N and R221H conferred ACV resistance with cross-resistance to PCV, the aa changes G25A, G39E, T131M, Y133F, G150D, A157T, R248W, and L342W maintained a susceptible phenotype against both antivirals. The CPE inhibition assay and the measurement of relative fluorescence intensity yielded comparable results for the phenotypic testing of recombinant viruses. The latter test showed some technical advantages. In conclusion, the significance of single aa changes in HSV-2 TK on ACV/PCV resistance was clarified by the construction and phenotypic testing of recombinant viral strains. This was facilitated by the fluorescence based method.

Recently, the binding kinetics of a ligand-target interaction, such as the residence time of a small molecule on its protein target, are seen as increasingly important for drug efficacy. Here, we investigate these concepts to explain binding and proton blockage of rimantadine variants bearing progressively larger alkyl groups to influenza A virus M2 wild type (WT) and M2 S31N protein proton channel. We showed that resistance of M2 S31N to rimantadine analogues compared to M2 WT resulted from their higher koff rates compared to the kon rates according to electrophysiology (EP) measurements. This is due to the fact that, in M2 S31N, the loss of the V27 pocket for the adamantyl cage resulted in low residence time inside the M2 pore. Both rimantadine enantiomers have similar channel blockage and binding kon and koff against M2 WT. To compare the potency between the rimantadine variants against M2, we applied approaches using different mimicry of M2, i.e., isothermal titration calorimetry and molecular dynamics simulation, EP, and antiviral assays. It was also shown that a small change in an amino acid at site 28 of M2 WT, which does not line the pore, seriously affects M2 WT blockage kinetics.

Viral neuraminidases are an established drug target to combat influenza. Severe complications observed in influenza patients are primarily caused by secondary infections with e.g., Streptococcus pneumoniae. These bacteria engage in a lethal synergism with influenza A viruses (IAVs) and also express neuraminidases. Therefore, inhibitors with dual activity on viral and bacterial neuraminidases are expected to be advantageous for the treatment of influenza infections. Here we report on the discovery and characterization of diazenylaryl sulfonic acids as dual inhibitors of viral and Streptococcus pneumoniae neuraminidase. The initial hit came from a virtual screening campaign for inhibitors of viral neuraminidases. For the most active compound, 7-[2-[4-[2-[4-[2-(2-hydroxy-3,6-disulfo-1-naphthalenyl)diazenyl]-2-methylphenyl]diazenyl]-2-methylphenyl]diazenyl]-1,3-naphthalenedisulfonic acid (NSC65847; 1), the Ki-values measured in a fluorescence-based assay were lower than 1.5 μM for both viral and pneumococcal neuraminidases. The compound also inhibited N1 virus variants containing neuraminidase inhibitor resistance-conferring substitutions. Via enzyme kinetics and nonlinear regression modeling, 1 was suggested to impair the viral neuraminidases and pneumococcal neuraminidase with a mixed-type inhibition mode. Given its antiviral and antipneumococcal activity, 1 was identified as a starting point for the development of novel, dual-acting anti-infectives.

Recent findings from solid state NMR (ssNMR) studies suggested that the (R)-enantiomer of rimantadine binds to the full M2 protein with higher affinity than the (S)-enantiomer. Intrigued by these findings, we applied functional assays, such as antiviral assay and electrophysiology (EP), to evaluate the binding affinity of rimantadine enantiomers to the M2 protein channel. Unexpectedly, no significant difference was found between the two enantiomers. Our experimental data based on the full M2 protein function were further supported by alchemical free energy. calculations and isothermal titration calorimetry (ITC) allowing an evaluation of the binding affinity of rimantadine enantiomers to the M2TM pore. Both enantiomers have similar channel blockage, affinity, and antiviral potency.

Background: Genotypic resistance testing of varicella-zoster virus (VZV) strains to antivirals is of high relevance in immunocompromised patients with VZV reactivations unresponsive to therapy. However, the knowledge on mutations associated with natural gene polymorphism or resistance is limited. Objectives: To examine the genotype of the thymidine kinase (TK) and DNA polymerase(pol) of unselected clinical VZV isolates collected between 1984 and 2014 and to verify the phenotype related to novel amino acid (aa) substitutions. Study design: The TK and DNA pol genes of 169 VZV isolates were analyzed by amplification and sequencing. Sequences were compared to that of the reference strain Dumas. The phenotype to acyclovir and other antivirals was examined in isolates with novel aa substitutions using modified plaque reduction assay. Results: In the TK of four strains, four different aa substitutions were detected, apart from the known change S288L that was present in all strains compared to Dumas. All four substitutions have hitherto not been described in the literature and were phenotypically classified as natural gene polymorphisms although two out of them (S51L, K186R) were localized in conserved gene centers. The DNA pol of 34 isolates exhibited 19 different substitutions, 14 out of them were novel, and two (R753K, V777I) were within conserved gene regions. Again, these changes were characterized as natural gene polymorphisms. Conclusions: Non-synonymous mutations in VZV TK or DNA pol conferring natural gene polymorphism are rare events. Nevertheless, the phenotypic characterization of 18 novel polymorphisms can help to provide a better identification of resistance mutations.

While aminoadamantanes are well-established inhibitors of the influenza A M2 proton channel, the mechanisms by which they are rendered ineffective against M2S31N are unclear. Solid state NMR, isothermal titration calorimetry, electrophysiology, antiviral assays, and molecular dynamics simulations suggest stronger binding interactions for aminoadamantanes to M2WT compared to negligible or weak binding to M2S31N. This is due to reshaping of the M2 pore when N31 is present, which, in contrast to wild-type (WT), leads (A) to the loss of the V27 pocket for the adamantyl cage and to a predominant orientation of the ligand's ammonium group toward the N-terminus and (B) to the lack of a helical kink upon ligand binding. The kink, which reduces the tilt of the C-terminal helical domain relative to the bilayer normal, includes the W41 primary gate for proton conductance and may prevent the gate from opening, representing an alternative view for how these drugs prevent proton conductance.

Efforts to develop novel neuraminidase inhibitors (NAIs) for the treatment of influenza are ongoing. Novel NAIs should in particular be also effective against seasonal and/or pandemic N1 that carry a H274Y or N294S substitution (N2 numbering), which are most commonly linked to oseltamivir resistance. Here we report a platform for profiling the efficacy of novel NAIs in the N1 genetic background of influenza A virus. Employing reverse genetics, a set of influenza virus variants containing an amino acid substitution associated with oseltamivir resistance in N1 isolates (H274Y, N294S, Y155H or Q136L) was generated. In parallel, so far unreported mutations of I427 (I427Q and I427M) were investigated. These possibly interfere with the side chain orientation of R371 and alter the binding affinity of most relevant NAIs. The profiling platform was validated with both oseltamivir and zanamivir and exemplarily applied to three analogs with differing decorations at positions 4 and 5. Besides confirming the inhibition profile of zanamivir and oseltamivir, the distinct effect of I427Q/M on the activity of both NAIs was shown. For 5-amidino and 5-guanidino analogs of oseltamivir a significantly stronger inhibition of virus variants carrying a NA-H274Y was confirmed, and additionally shown for NA-N294S and NA-Y155H substitutions as compared to the parent compound. Hence, the herein presented profiling platform is a valid tool for defining the inhibition profile of novel NAIs in the N1 background.

Influenza virus neuraminidase (NA) is the primary target for influenza therapeutics. Severe complications are often related to secondary pneumonia caused by Streptococcus pneumoniae (pneumococci), which also express NAs. Recently, a NA-mediated lethal synergism between influenza A viruses and pneumococci was described. Therefore, dual inhibitors of both viral and bacterial NAs are expected to be advantageous for the treatment of influenza. We investigated the traditional Chinese herbal drug sāng bái pí (mulberry root bark) as source for anti-infectives. Two prenylated flavonoid derivatives, sanggenon G (4) and sanggenol A (5) inhibited influenza A viral and pneumococcal NAs and, in contrast to the approved NA inhibitor oseltamivir, also planktonic growth and biofilm formation of pneumococci. Evaluation of 27 congeners of 5 revealed a correlation between the degree of prenylation and bioactivity. Abyssinone-V 4′-methyl ether (27) inhibited pneumococcal NA with IC50 = 2.18 μM, pneumococcal growth with MIC = 5.63 μM and biofilm formation with MBIC = 4.21 μM, without harming lung epithelial cells. Compounds 5 and 27 also disrupt the synergism between influenza A virus and pneumococcal NA in vitro, hence functioning as dual-acting anti-infectives. The results warrant further studies on whether the observed disruption of this synergism is transferable to in vivo systems.

ABSTRACT Aims: In this study, we analyze the challenges involved in detecting novel neuraminidase inhibitors (NAIs) and offer strategies to overcome them with complementary bioassays. Materials & Methods: We investigated the inhibitory activities of NAIs (oseltamivir, zanamivir, DANA, katsumadain A and remazol) as well as non-NAIs (amantadine, nucleozin and rifampicin) on influenzaviral and bacterial (Streptococcus pneumoniae, Clostridium perfringens and Vibrio cholerae) neuraminidases (NAs) with chemiluminescence (CL)- and fluorescence (FL)-based assays. Furthermore, hemagglutination-based NA inhibition assays were established. Results: Our study shows three types of signal interference affecting the readout of biochemical assays: self-FL (katsumadain A and remazol), FL quenching (rifampicin) and CL quenching (rifampicin, remazol, nucleozin and katsumadain A). These challenges were overcome by hemagglutination-based assays. Conclusion: The latter allow a robust performance in discriminating NAIs and non-NAIs.

The neuraminidase (NA) inhibitor zanamivir (1) is potently active against a broad panel of influenza A and B strains, including mutant viruses, but suffers from pharmacokinetic (PK) shortcomings. Here, distinct prodrug approaches are described that aimed at overcoming zanamivir's lack of oral bioavailability. Lowering the high basicity of the 4-guanidino group in zanamivir and of a bioisosteric 4-acetamidine analog (5) by N-hydroxylation was deemed to be a plausible tactic. The carboxylic acid and glycerol side chain were also masked with different ester groups. The bioisosteric amidine 5 turned out to be potently active against a panel of H1N1 (IC50 = 2-10 nM) and H3N2 (IC50 = 5-10 nM) influenza A viruses (NA inhibition assay). In vitro PK studies showed that all prodrugs were highly soluble, exhibited low protein binding, and were bioactivated by N-reduction to the respective guanidines and amidines. The most promising prodrug candidates, amidoxime ester 7 and N-hydroxyguanidine ester 8, were subjected to in vivo bioavailability studies. Unfortunately, both prodrugs were not orally bioavailable to a convincing degree (F ≤ 3.7%, rats). This finding questions the general feasibility of improving the oral bioavailability of 1 by lipophilicity-increasing prodrug strategies, and suggests that intrinsic structural features represent key hurdles.