Biophysical and structural study of La Crosse virus endonuclease inhibition for the development of new antiviral options.

The large Bunyavirales order includes several families of viruses with a segmented ambisense (-) RNA genome and a cytoplasmic life cycle that starts by synthesizing viral mRNA. The initiation of transcription, which is common to all members, relies on an endonuclease activity that is responsible for cap-snatching. In La Crosse virus, an orthobunyavirus, it has previously been shown that the cap-snatching endonuclease resides in the N-terminal domain of the L protein. Orthobunyaviruses are transmitted by arthropods and cause diseases in cattle. However, California encephalitis virus, La Crosse virus and Jamestown Canyon virus are North American species that can cause encephalitis in humans. No vaccines or antiviral drugs are available. In this study, three known Influenza virus endonuclease inhibitors (DPBA, L-742,001 and baloxavir) were repurposed on the La Crosse virus endonuclease. Their inhibition was evaluated by fluorescence resonance energy transfer and their mode of binding was then assessed by differential scanning fluorimetry and microscale thermophoresis. Finally, two crystallographic structures were obtained in complex with L-742,001 and baloxavir, providing access to the structural determinants of inhibition and offering key information for the further development of Bunyavirales endonuclease inhibitors.

An exonuclease-resistant chain-terminating nucleotide analogue targeting the SARS-CoV-2 replicase complex.

Nucleotide analogues (NA) are currently employed for treatment of several viral diseases, including COVID-19. NA prodrugs are intracellularly activated to the 5'-triphosphate form. They are incorporated into the viral RNA by the viral polymerase (SARS-CoV-2 nsp12), terminating or corrupting RNA synthesis. For Coronaviruses, natural resistance to NAs is provided by a viral 3'-to-5' exonuclease heterodimer nsp14/nsp10, which can remove terminal analogues. Here, we show that the replacement of the α-phosphate of Bemnifosbuvir 5'-triphosphate form (AT-9010) by an α-thiophosphate renders it resistant to excision. The resulting α-thiotriphosphate, AT-9052, exists as two epimers (RP/SP). Through co-crystallization and activity assays, we show that the Sp isomer is preferentially used as a substrate by nucleotide diphosphate kinase (NDPK), and by SARS-CoV-2 nsp12, where its incorporation causes immediate chain-termination. The same -Sp isomer, once incorporated by nsp12, is also totally resistant to the excision by nsp10/nsp14 complex. However, unlike AT-9010, AT-9052-RP/SP no longer inhibits the N-terminal nucleotidylation domain of nsp12. We conclude that AT-9052-Sp exhibits a unique mechanism of action against SARS-CoV-2. Moreover, the thio modification provides a general approach to rescue existing NAs whose activity is hampered by coronavirus proofreading capacity.

Macro1 domain residue F156: A hallmark of SARS-CoV-2 de-MARylation specificity.

SARS-CoV-2 is a large, enveloped and positive sense single stranded RNA virus. Its genome codes for 16 non-structural proteins. The largest protein of this complex is nsp3, that contains a well conserved Macro1 domain. Viral Macro domains were shown to bind to mono-ADP-ribose (MAR) and poly-ADP-ribose (PAR) in their free form or conjugated to protein substrates. They carry ADP-ribose hydrolase activities implicated in the regulation of innate immunity. SARS-CoV-2 and SARS-CoV show widely different induction and handling of the host interferon response. Herein, we have conducted a mutational study on the key amino-acid residue F156 in SARS-CoV-2, pinpointed by bioinformatic and structural studies, and its cognate residue N157 in SARS-CoV. Our data suggest that the exchange of these residues slightly modifies ADP-ribose binding, but drastically impacts de-MARylation activity. Alanine substitutions at this position hampers PAR binding, abolishes MAR hydrolysis of SARS-CoV-2, and reduces by 70% this activity in the case of SARS-CoV.

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5.

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2'-methyl-2'-fluoro guanosine 5'-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2'-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2'-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 ≈ 0.50 µM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

Inhibition of Arenaviridae nucleoprotein exonuclease by bisphosphonate.

Arenaviruses are emerging enveloped negative-sense RNA viruses that cause neurological and hemorrhagic diseases in humans. Currently, no FDA-approved vaccine or therapeutic agent is available except for ribavirin, which must be administered early during infection for optimum efficacy. A hallmark of arenavirus infection is rapid and efficient immune suppression mediated by the exonuclease domain encoded by the nucleoprotein. This exonuclease is therefore an attractive target for the design of novel antiviral drugs since exonuclease inhibitors might not only have a direct effect on the enzyme but could also boost viral clearance through stimulation of the innate immune system of the host cell. Here, in silico screening and an enzymatic assay were used to identify a novel, specific but weak inhibitor of the arenavirus exonuclease, with IC50 values of 65.9 and 68.6 µM for Mopeia virus and Lymphocytic choriomeningitis virus, respectively. This finding was further characterized using crystallographic and docking approaches. This study serves as a proof of concept and may have assigned a new therapeutic purpose for the bisphosphonate family, therefore paving the way for the development of inhibitors against Arenaviridae.

Identification of potent inhibitors of arenavirus and SARS-CoV-2 exoribonucleases by fluorescence polarization assay.

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design. Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells.

A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase.

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.

Observation of arenavirus nucleoprotein heptamer assembly.

Arenaviruses are enveloped viruses containing a segmented, negative, and ambisense single-stranded RNA genome wrapped with a nucleoprotein (NP). The NP is the most abundant viral protein in infected cells and plays a critical role in both replication/transcription and virion assembly. The NP associates with RNA to form a ribonucleoprotein (RNP) complex, and this implies self-assembly while the exact structure of this polymer is not yet known. Here, we report a measurement of the full-length Mopeia virus NP by negative stain transmission electron microscopy. We observed RNP complex particles with diameter 15 ± 1 nm as well as symmetric circular heptamers of the same diameter, consistent with previous observations.

Brothers in Arms: Structure, Assembly and Function of Arenaviridae Nucleoprotein.

Arenaviridae is a family of viruses harbouring important emerging pathogens belonging to the Bunyavirales order. Like in other segmented negative strand RNA viruses, the nucleoprotein (NP) is a major actor of the viral life cycle being both (i) the necessary co-factor of the polymerase present in the L protein, and (ii) the last line of defence of the viral genome (vRNA) by physically hiding its presence in the cytoplasm. The NP is also one of the major players interfering with the immune system. Several structural studies of NP have shown that it features two domains: a globular RNA binding domain (NP-core) in its N-terminal and an exonuclease domain (ExoN) in its C-terminal. Further studies have observed that significant conformational changes are necessary for RNA encapsidation. In this review we revisited the most recent structural and functional data available on Arenaviridae NP, compared to other Bunyavirales nucleoproteins and explored the structural and functional implications. We review the variety of structural motif extensions involved in NP-NP binding mode. We also evaluate the major functional implications of NP interactome and the role of ExoN, thus making the NP a target of choice for future vaccine and antiviral therapy.

Remdesivir and SARS-CoV-2: Structural requirements at both nsp12 RdRp and nsp14 Exonuclease active-sites.

The rapid global emergence of SARS-CoV-2 has been the cause of significant health concern, highlighting the immediate need for antivirals. Viral RNA-dependent RNA polymerases (RdRp) play essential roles in viral RNA synthesis, and thus remains the target of choice for the prophylactic or curative treatment of several viral diseases, due to high sequence and structural conservation. To date, the most promising broad-spectrum class of viral RdRp inhibitors are nucleoside analogues (NAs), with over 25 approved for the treatment of several medically important viral diseases. However, Coronaviruses stand out as a particularly challenging case for NA drug design due to the presence of an exonuclease (ExoN) domain capable of excising incorporated NAs and thus providing resistance to many of these available antivirals. Here we use the available structures of the SARS-CoV RdRp and ExoN proteins, as well as Lassa virus N exonuclease to derive models of catalytically competent SARS-CoV-2 enzymes. We then map a promising NA candidate, GS-441524 (the active metabolite of Remdesivir) to the nucleoside active site of both proteins, identifying the residues important for nucleotide recognition, discrimination, and excision. Interestingly, GS-441524 addresses both enzyme active sites in a manner consistent with significant incorporation, delayed chain termination, and altered excision due to the ribose 1'-CN group, which may account for the increased antiviral effect compared to other available analogues. Additionally, we propose structural and function implications of two previously identified RdRp resistance mutations in relation to resistance against Remdesivir. This study highlights the importance of considering the balance between incorporation and excision properties of NAs between the RdRp and ExoN.

Discovery, SAR study and ADME properties of methyl 4-amino-3-cyano-1-(2-benzyloxyphenyl)-1H-pyrazole-5-carboxylate as an HIV-1 replication inhibitor.

Inspired by the antiviral activity of known pyrazole-based HIV inhibitors, we screened our in-house library of pyrazole-based compounds to evaluate their in cellulo activity against HIV-1 replication. Two hits with very similar structures appeared from single and multiple-round infection assays to be non-toxic and active in a dose-dependent manner. Chemical expansion of their series allowed an in-depth and consistent structure-activity-relationship study (SAR) to be built. Further ADME evaluation led to the selection of 4-amino-3-cyano-1-(2-benzyloxyphenyl)-1H-pyrazole-5-carboxylate with an advantageous pharmacokinetic profile. Finally, examination of its mode of action revealed that this compound does not belong to the three main classes of anti-HIV drugs, a feature of prime interest in the context of viral resistance.

Identification of a new natural gastric lipase inhibitor from star anise.

The identification and isolation of bioactive compounds are of great interest in the drug delivery field, despite being a difficult task. We describe here an innovative strategy for the identification of a new gastric lipase inhibitor from star anise for the treatment of obesity. After plant screening assays for gastric lipase inhibition, star anise was selected and investigated by bioactivity guided fractionation. MALDI-TOF mass spectrometry and peptide mass fingerprinting allowed the detection of an inhibitor covalently bound to the catalytic serine of gastric lipase. A mass-directed screening approach using UPLC-HRMS and accurate mass determination searching identified the flavonoid myricitrin-5-methyl ether (M5ME) as a lipase inhibitor. The inhibitory activity was rationalized based on molecular docking, showing that M5ME is susceptible to nucleophilic attack by gastric lipase. Overall, our data suggest that M5ME may be considered as a potential candidate for future application as a gastric lipase inhibitor for the treatment of obesity.

Metal chelators for the inhibition of the lymphocytic choriomeningitis virus endonuclease domain.

Arenaviridae is a viral family whose members are associated with rodent-transmitted infections to humans responsible of severe diseases. The current lack of a vaccine and limited therapeutic options make the development of efficacious drugs of high priority. The cap-snatching mechanism of transcription of Arenavirus performed by the endonuclease domain of the L-protein is unique and essential, so we developed a drug design program targeting the endonuclease activity of the prototypic Lymphocytic ChorioMeningitis Virus. Since the endonuclease activity is metal ion dependent, we designed a library of compounds bearing chelating motifs (diketo acids, polyphenols, and N-hydroxyisoquinoline-1,3-diones) able to block the catalytic center through the chelation of the critical metal ions, resulting in a functional impairment. We pre-screened 59 compounds by Differential Scanning Fluorimetry. Then, we characterized the binding affinity by Microscale Thermophoresis and evaluated selected compounds in in vitro and in cellula assays. We found several potent binders and inhibitors of the endonuclease activity. This study validates the proof of concept that the endonuclease domain of Arenavirus can be used as a target for anti-arena-viral drug discovery and that both diketo acids and N-hydroxyisoquinoline-1,3-diones can be considered further as potential metal-chelating pharmacophores.

Crystal structures of Lymphocytic choriomeningitis virus endonuclease domain complexed with diketo-acid ligands.

The Arenaviridae family, together with the Bunyaviridae and Orthomyxoviridae families, is one of the three negative-stranded RNA viral families that encode an endonuclease in their genome. The endonuclease domain is at the N-terminus of the L protein, a multifunctional protein that includes the RNA-dependent RNA polymerase. The synthesis of mRNA in arenaviruses is a process that is primed by capped nucleotides that are 'stolen' from the cellular mRNA by the endonuclease domain in cooperation with other domains of the L protein. This molecular mechanism has been demonstrated previously for the endonuclease of the prototype Lymphocytic choriomeningitis virus (LCMV). However, the mode of action of this enzyme is not fully understood as the original structure did not contain catalytic metal ions. The pivotal role played by the cap-snatching process in the life cycle of the virus and the highly conserved nature of the endonuclease domain make it a target of choice for the development of novel antiviral therapies. Here, the binding affinities of two diketo-acid (DKA) compounds (DPBA and L-742,001) for the endonuclease domain of LCMV were evaluated using biophysical methods. X-ray structures of the LCMV endonuclease domain with catalytic ions in complex with these two compounds were determined, and their efficacies were assessed in an in vitro endonuclease-activity assay. Based on these data and computational simulation, two new DKAs were synthesized. The LCMV endonuclease domain exhibits a good affinity for these DKAs, making them a good starting point for the design of arenavirus endonuclease inhibitors. In addition to providing the first example of an X-ray structure of an arenavirus endonuclease incorporating a ligand, this study provides a proof of concept that the design of optimized inhibitors against the arenavirus endonuclease is possible.

How to Control HTLV-1-Associated Diseases: Preventing de Novo Cellular Infection Using Antiviral Therapy.

Five to ten million individuals are infected by Human T-cell Leukemia Virus type 1 (HTLV-1). HTLV-1 is transmitted through prolonged breast-feeding, by sexual contacts and by transmission of infected T lymphocytes through blood transfusion. One to ten percent of infected carriers will develop a severe HTLV-1-associated disease: Adult-T-cell leukemia/lymphoma (ATLL), or a neurological disorder named Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (TSP/HAM). In vivo, HTLV-1 is mostly detected in CD4+ T-cells, and to a lesser extent in CD8+ T cells and dendritic cells. There is a strong correlation between HTLV-1 proviral load (PVL) and clinical status of infected individuals. Thus, reducing PVL could be part of a strategy to prevent or treat HTLV-1-associated diseases among carriers. Treatment of ATLL patients using conventional chemotherapy has very limited benefit. Some chronic and acute ATLL patients are, however, efficiently treated with a combination of interferon α and zidovudine (IFN-α/AZT), to which arsenic trioxide is added in some cases. On the other hand, no efficient treatment for TSP/HAM patients has been described yet. It is therefore crucial to develop therapies that could either prevent the occurrence of HTLV-1-associated diseases or at least block the evolution of the disease in the early stages. In vivo, reverse transcriptase (RT) activity is low in infected cells, which is correlated with a clonal mode of viral replication. This renders infected cells resistant to nucleoside RT inhibitors such as AZT. However, histone deacetylase inhibitors (HDACi) associated to AZT efficiently induces viral expression and prevent de novo cellular infection. In asymptomatic STLV-1 infected non-human primates, HDACi/AZT combination allows a strong decrease in the PVL. Unfortunately, rebound in the PVL occurs when the treatment is stopped, highlighting the need for better antiviral compounds. Here, we review previously used strategies targeting HTLV-1 replication. We also tested a series of HIV-1 RT inhibitors in an in vitro anti-HTLV-1 screen, and report that bis-POM-PMEA (adefovir dipivoxil) and bis-POC-PMPA (tenofovir disoproxil) are much more efficient compared to AZT to decrease HTLV-1 cell-to-cell transmission in vitro. Our results suggest that revisiting already established antiviral drugs is an interesting approach to discover new anti-HTLV-1 drugs.

Synthesis and substrate properties towards HIV-1 reverse transcriptase of new diphosphate analogues of 9-[(2-phosphonomethoxy)ethyl]adenine.

Background The replacement of ß,γ-pyrophosphate by ß,γ-phosphonate moieties within the triphosphate chain of 5'-triphosphate nucleoside analogues was previously studied for various antiviral nucleoside analogues such as AZT and 2',3'-dideoxynucleosides. Thus, it has been shown that these chemical modifications could preserve, in some cases, the terminating substrate properties of the triphosphate analogue for HIV-RT. Herein, we aimed to study such 5'-triphosphate mimics based on the scaffold of the well-known antiviral agent 9-[(2-phosphonomethoxy)ethyl]adenine (PMEA, Adefovir). Methods Synthesis involved coupling of a morpholidate derivative of PMEA with appropriate pyrophosphoryl analogues. The relative efficiencies of incorporation of the studied diphosphate phosphonates were measured using subtype B WT HIV-1 RT in an in vitro susceptibility assay, in comparison to the parent nucleotide analogue (PMEApp). Results Searching for nucleoside 5'-triphosphate mimics, we have synthesized and studied a series of diphosphate analogues of PMEA bearing non hydrolysable bonds between the and phosphorus atoms. We also examined their relative inhibitory capacity towards HIV-1 reverse transcriptase in comparison to the parent nucleotide analogue (PMEApp). Only one of them appeared as a weak inhibitor (IC50 = 403.0 ± 75.5 µM) and proved to be less effective than PMEApp (IC50 = 6.4 ± 0.8 µM). Conclusion PMEA diphosphoryl derivatives were designed as potential substrates and/or inhibitors of various viral polymerases. These modifications dramatically affect their ability to inhibit HIV-RT.

Novel 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,3,4-oxadiazole and 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole derivatives as dengue virus inhibitors targeting NS5 polymerase.

Using a functional high-throughput screening (HTS) and subsequent SAR studies, we have discovered a novel series of non-nucleoside dengue viral polymerase inhibitors. We report the elaboration of SAR around hit compound 1 as well as the synthesis and antiviral evaluation of 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole and 5-phenyl-2-[2-(2-thienyl)ethenyl]-1,3,4-oxadiazole analogues derived from a rapid and easily accessible chemical pathway. A large number of compounds prepared by this method were shown to possess in vitro activity against the polymerase of dengue virus. The most potent inhibitors were tested against Dengue virus clinical isolates on infected cells model and exhibit submicromolar activity on the four dengue virus serotypes.

Synthesis and structural characterization of monomeric and dimeric peptide nucleic acids prepared by using microwave-promoted multicomponent reactions.

A solution phase synthesis of peptide nucleic acid monomers and dimers was developed by using microwave-promoted Ugi multicomponent reactions. A mixture of a functionalized amine, a carboxymethyl nucleobase, paraformaldehyde and an isocyanide as building blocks generates PNA monomers which are then partially deprotected and used in a second Ugi 4CC reaction, leading to PNA dimers. Conformational rotamers were identified by using NMR and MD simulations.

Enzymatic synthesis of acyclic nucleoside thiophosphonate diphosphates: effect of the α-phosphorus configuration on HIV-1 RT activity.

The acyclic nucleosides thiophosphonates (9-[2-(thiophosphonomethoxy)ethyl]adenine (S-PMEA) and (R)-9-[2-(thiophosphonomethoxy)propyl]adenine (S-PMPA), exhibit antiviral activity against HIV-1, -2 and HBV. Their diphosphate forms S-PMEApp and S-PMPApp, synthesized as stereoisomeric mixture, are potent inhibitors of wild-type (WT) HIV-1 RT. Understanding HIV-1 RT stereoselectivity, however, awaits resolution of the diphosphate forms into defined stereoisomers. To this aim, thiophosphonate monophosphates S-PMEAp and S-PMPAp were synthesized and used in a stereocontrolled enzyme-catalyzed phosphoryl transfer reaction involving either nucleoside diphosphate kinase (NDPK) or creatine kinase (CK) to obtain thiophosphonate diphosphates as separated isomers. We then quantified substrate preference of recombinant WT HIV-1 RT toward pure stereoisomers using in vitro steady-state kinetic analyses. The crystal structure of a complex between Dictyostelium NDPK and S-PMPApp at 2.32Å allowed to determine the absolute configuration at the α-phosphorus atom in relation to the stereo-preference of studied enzymes. The RP isomer of S-PMPApp and S-PMEApp are the preferred substrate over SP for both NDPK and HIV-1 RT.

Ester prodrugs of acyclic nucleoside thiophosphonates compared to phosphonates: synthesis, antiviral activity and decomposition study.

9-[2-(Thiophosphonomethoxy)ethyl]adenine [S-PMEA, 8] and (R)-9-[2-(Thiophosphonomethoxy)propyl]adenine [S-PMPA, 9] are acyclic nucleoside thiophosphonates we described recently that display the same antiviral spectrum (DNA viruses) as approved and potent phosphonates PMEA and (R)-PMPA. Here, we describe the synthesis, antiviral activities in infected cell cultures and decomposition study of bis(pivaloyloxymethoxy)-S-PMEA [Bis-POM-S-PMEA, 13] and bis(isopropyloxymethylcarbonyl)-S-PMPA [Bis-POC-S-PMPA, 14] as orally bioavailable prodrugs of the S-PMEA 8 and S-PMPA 9, in comparison to the equivalent "non-thio" derivatives [Bis-POM-PMEA, 11] and [Bis-POC-PMPA, 12]. Compounds 11, 12, 13 and 14 were evaluated for their in vitro antiviral activity against HIV-1-, HIV-2-, HBV- and a broad panel of DNA viruses, and found to exhibit moderate to potent antiviral activity. In order to determine the decomposition pathway of the prodrugs 11, 12, 13 and 14 into parent compounds PMEA, PMPA, 8 and 9, kinetic data and decomposition pathways in several media are presented. As expected, bis-POM-S-PMEA 13 and bis-POC-S-PMPA 14 behaved as prodrugs of S-PMEA 8 and S-PMPA 9. However, thiophosphonates 8 and 9 were released very smoothly in cell extracts, in contrast to the release of PMEA and PMPA from "non-thio" prodrugs 11 and 12.