Systematic evaluation of methyl ester bioisosteres in the context of developing alkenyldiarylmethanes (ADAMs) as non-nucleoside reverse transcriptase inhibitors (NNRTIs) for anti-HIV-1 chemotherapy.

The alkenyldiarylmethanes (ADAMs) are a class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) targeting HIV-1. Four chemically and metabolically stabilized ADAMs incorporating N-methoxyimidoyl halide replacements of the methyl esters of the lead compound were previously reported. In this study, twenty-five new ADAMs were synthesized in order to investigate the biological consequences of installing nine different methyl ester bioisosteres at three different locations. Attempts to define a universal rank order of methyl ester bioisosteres and discover the 'best' one in terms of inhibitory activity versus HIV-1 reverse transcriptase (RT) led to the realization that the potencies are critically dependent on the surrounding structure at each location, and therefore the definition of universal rank order is impossible. This investigation produced several new non-nucleoside reverse transcriptase inhibitors in which all three of the three methyl esters of the lead compound were replaced by methyl ester bioisosteres, resulting in compounds that are more potent as HIV-1 RT inhibitors and antiviral agents than the lead compound itself and are expected to also be more metabolically stable than the lead compound.

In vitro effect of a non-immunosuppressive FKBP ligand, FK1706, on SARS-CoV-2 replication in combination with antivirals.

FKBP, a naturally occurring ubiquitous intracellular protein, has been proposed as a potential target for coronavirus replication. A non-immunosuppressive FKBP ligand, FK1706, was studied in vitro in a Vero cell model to assess potential activity alone and in combination with antivirals against SARS-CoV-2 replication. When combined with remdesivir, synergistic activity was seen (summary synergy score 24.7±9.56). FK1706 warrants in vivo testing as a potential new combination therapeutic for the treatment of COVID-19 infections.

Inhibition of human immunodeficiency virus type 1 by triciribine involves the accessory protein nef.

Triciribine (TCN) is a tricyclic nucleoside that inhibits human immunodeficiency virus type 1 (HIV-1) replication by a unique mechanism not involving the inhibition of enzymes directly involved in viral replication. This activity requires the phosphorylation of TCN to its 5' monophosphate by intracellular adenosine kinase. New testing with a panel of HIV and simian immunodeficiency virus isolates, including low-passage-number clinical isolates and selected subgroups of HIV-1, multidrug resistant HIV-1, and HIV-2, has demonstrated that TCN has broad antiretroviral activity. It was active in cell lines chronically infected with HIV-1 in which the provirus was integrated into chromosomal DNA, thereby indicating that TCN inhibits a late process in virus replication. The selection of TCN-resistant HIV-1 isolates resulted in up to a 750-fold increase in the level of resistance to the drug. DNA sequence analysis of highly resistant isolate HIV-1(H10) found five point mutations in the HIV-1 gene nef, resulting in five different amino acid changes. DNA sequencing of the other TCN-resistant isolates identified at least one and up to three of the same mutations observed in isolate HIV-1(H10). Transfer of the mutations from TCN-resistant isolate HIV-1(H10) to wild-type virus and subsequent viral growth experiments with increasing concentrations of TCN demonstrated resistance to the drug. We conclude that TCN is a late-phase inhibitor of HIV-1 replication and that mutations in nef are necessary and sufficient for TCN resistance.

Development of hexadecyloxypropyl tenofovir (CMX157) for treatment of infection caused by wild-type and nucleoside/nucleotide-resistant HIV.

CMX157 is a lipid (1-0-hexadecyloxypropyl) conjugate of the acyclic nucleotide analog tenofovir (TFV) with activity against both wild-type and antiretroviral drug-resistant HIV strains, including multidrug nucleoside/nucleotide analog-resistant viruses. CMX157 was consistently >300-fold more active than tenofovir against multiple viruses in several different cell systems. CMX157 was active against all major subtypes of HIV-1 and HIV-2 in fresh human peripheral blood mononuclear cells (PBMCs) and against all HIV-1 strains evaluated in monocyte-derived macrophages, with 50% effective concentrations (EC(50)s) ranging between 0.20 and 7.2 nM. The lower CMX157 EC(50)s can be attributed to better cellular uptake of CMX157, resulting in higher intracellular levels of the active antiviral anabolite, TFV-diphosphate (TFV-PP), inside target cells. CMX157 produced >30-fold higher levels of TFV-PP in human PBMCs exposed to physiologically relevant concentrations of the compounds than did TFV. Unlike conventional prodrugs, including TFV disoproxil fumarate (Viread), CMX157 remains intact in plasma, facilitating uptake by target cells and decreasing relative systemic exposure to TFV. There was no detectable antagonism with CMX157 in combination with any marketed antiretroviral drug, and it possessed an excellent in vitro cytotoxicity profile. CMX157 is a promising clinical candidate to treat wild-type and antiretroviral drug-resistant HIV, including strains that fail to respond to all currently available nucleoside/nucleotide reverse transcriptase inhibitors.

Multivalent binding oligomers inhibit HIV Tat-TAR interaction critical for viral replication.

We describe the development of a new type of scaffold to target RNA structures. Multivalent binding oligomers (MBOs) are molecules in which multiple sidechains extend from a polyamine backbone such that favorable RNA binding occurs. We have used this strategy to develop MBO-based inhibitors to prevent the association of a protein-RNA complex, Tat-TAR, that is essential for HIV replication. In vitro binding assays combined with model cell-based assays demonstrate that the optimal MBOs inhibit Tat-TAR binding at low micromolar concentrations. Antiviral studies are also consistent with the in vitro and cell-based assays. MBOs provide a framework for the development of future RNA-targeting molecules.

The structure-activity profile of mercaptobenzamides' anti-HIV activity suggests that thermodynamics of metabolism is more important than binding affinity to the target.

Mercaptobenzamide thioesters and thioethers are chemically simple HIV-1 maturation inhibitors with a unique mechanism of action, low toxicity, and a high barrier to viral resistance. A structure-activity relationship (SAR) profile based on 39 mercaptobenzamide prodrug analogs exposed divergent activity/toxicity roles for the internal and terminal amides. To probe the relationship between antiviral activity and toxicity, we generated an improved computational model for the binding of mercaptobenzamide thioesters (SAMTs) to the HIV-1 NCp7 C-terminal zinc finger, revealing the presence of a second low-energy binding orientation, hitherto undisclosed. Finally, using NMR-derived thiol-thioester exchange equilibrium constants, we propose that thermodynamics plays a role in determining the antiviral activity observed in the SAR profile.

Inhibition of tubulin polymerization by select alkenyldiarylmethanes.

During studies on the alkenyldiarylmethane (ADAM) class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), analogues were discovered that exhibit low micromolar and submicromolar cytotoxicities. Since the ADAMs are structurally related to the tubulin polymerization inhibitor CC-5079, a set of 14 ADAMs were tested for inhibition of tubulin polymerization in an attempt to identify the biological target responsible for their cytotoxicity. The results indicate that, overall, the ADAMs are poor inhibitors of tubulin polymerization. However, the two most cytotoxic compounds, 15 and 16, are in fact active as inhibitors of tubulin assembly with IC(50) values of 3.7+/-0.3 and 2.8+/-0.2 microM, respectively, and they both inhibit the binding of colchicine to tubulin. Both compounds were investigated for anticancer activity in the National Cancer Institute's panel of 60 human cancer cell lines, and both compounds consistently displayed submicromolar cytotoxicities with mean-graph midpoint (MGM) values of 0.31+/-0.08 and 0.47+/-0.09 microM, respectively.

Investigation of the alkenyldiarylmethane non-nucleoside reverse transcriptase inhibitors as potential cAMP phosphodiesterase-4B2 inhibitors.

The alkenyldiarylmethanes (ADAMs) are currently being investigated as non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) of potential value in the treatment of HIV infection and AIDS. During the course of these studies, a number of ADAM analogues have been identified that protect HIV-infected cells from the cytopathic effects of the virus by an unknown, HIV-1 RT-independent mechanism. Since the phosphodiesterase 4 family is required for HIV infection, the effect of various ADAMs on the activity of PDE4B2 was investigated in an effort to determine if the ADAMs could possibly be targeting phosphodiesterases. Six compounds representative of the ADAM class were tested for inhibition of cAMP hydrolysis by PDE4B2 enzymatic activity. Four ADAMs were found to be weak inhibitors of PDE4B2 and two of them were inactive. The experimental results are consistent with an antiviral mechanism that does not include inhibition of PDE4 isoforms.

Synthesis and biological evaluation of alkenyldiarylmethane HIV-1 non-nucleoside reverse transcriptase inhibitors that possess increased hydrolytic stability.

Non-nucleoside inhibitors of HIV reverse transcriptase (NNRTIs), albeit not the mainstays of HIV/AIDS treatment, have become increasingly important in highly active antiretroviral therapy (HAART) due to their unique mechanism of action. Several years ago our group identified the alkenyldiarylmethanes (ADAMs) as a potent and novel class of NNRTIs; however, the most active compounds were found to be metabolically unstable. Subsequent work has led to the synthesis of 33 analogues, with improved metabolic profiles, through the replacement of labile esters with various heterocycles, nitriles, and thioesters. As a result, a number of hydrolytically stable NNRTIs were identified with anti-HIV activity in the nanomolar concentration range. Furthermore, an improved pharmacophore model has been developed based on the new ADAM series, in which a salicylic acid-derived aryl ring is oriented cis to the side chain and the aryl ring that is trans to the side chain contains a hydrogen bond acceptor site within the plane of the ring.

Synthesis and anti-HIV activity of new metabolically stable alkenyldiarylmethane non-nucleoside reverse transcriptase inhibitors incorporating N-methoxy imidoyl halide and 1,2,4-oxadiazole systems.

The alkenyldiarylmethanes (ADAMs) are a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are capable of inhibiting HIV-1 reverse transcriptase (RT) through an allosteric mechanism. However, the potential usefulness of the ADAMs is limited by the presence of metabolically labile methyl ester moieties that are hydrolyzed by nonspecific esterases present in blood plasma, resulting in the formation of the inactive carboxylic acid metabolites. Therefore, to discover metabolically stable ADAMs, the design and synthesis of a new class of ADAMs with N-methoxy imidoyl halide and 1,2,4-oxadiazole systems were attempted. The resulting new ADAM 6 displayed enhanced metabolic stability in rat plasma (t1/2 = 61 h) along with the ability to inhibit HIV-1 reverse transcriptase and the cytopathic effect of HIV-1RF and HIV-1IIIB at submicromolar concentrations.

The structure-activity relationships of 2,4(1H,3H)-pyrimidinedione derivatives as potent HIV type 1 and type 2 inhibitors.

Since the discovery of the 2,4 (1H,3H)-pyrimidinediones as potent non-nucleoside inhibitors of the HIV-1 reverse transcriptase (RT) this class of compounds has yielded a number of N-1 acyclic substituted pyrimidinediones with substantial antiviral activity, which is highly dependent upon their molecular fit into the binding pocket common to this inhibitory class. We have specifically examined the structure activity relationships of compounds with chemical modification made by substituting homocyclic rather than acyclic moieties at N-1 of the pyrimidinedione. Seventy-four compounds were synthesized and evaluated for antiviral activity against HIV-1 and HIV-2. The homocyclic modifications resulted in compounds with significant activity against both HIV-1 and HIV-2, suggesting these compounds represent a new class of non-nucleoside RT inhibitors. The structure-activity relationship (SAR) evaluations indicated that cyclopropyl, phenyl and 1- or 3-cyclopenten-1-yl substitutions at the N-1 of the pyrimidinedione, the addition of a methyl linker between the cyclic moiety and the N-1 and the addition of a benzoyl group at the C-6 of the pyrimidinedione had the greatest contribution to antiviral activity. Five pyrimidinedione analogues with therapeutic indexes (TIs) > 450,000 and a specific analogue (1-cyclopropylmethyl-5-isopropyl-6-(3,5-dimethylbenzoyl)-2,4(1H,3H)-pyrimidinedione), which exhibited a TI of > 2,000,000, were identified. None of the analogues were cytotoxic to target cells at the highest in vitro test concentration, which is the upper limit of compound solubility of the analogues in aqueous solution. Thus, we have identified a series of pyrimidinediones with substantially improved antiviral efficacy and range of action and with significantly reduced cellular cytotoxicity.

Probing Mercaptobenzamides as HIV Inactivators via Nucleocapsid Protein†7.

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein 7 (NCp7), a zinc finger protein, plays critical roles in viral replication and maturation and is an attractive target for drug development. However, the development of drug-like molecules that inhibit NCp7 has been a significant challenge. In this study, a series of novel 2-mercaptobenzamide prodrugs were investigated for anti-HIV activity in the context of NCp7 inactivation. The molecules were synthesized from the corresponding thiosalicylic acids, and they are all crystalline solids and stable at room temperature. Derivatives with a range of amide side chains and aromatic substituents were synthesized and screened for anti-HIV activity. Wide ranges of antiviral activity were observed, with IC50 values ranging from 1 to 100 µm depending on subtle changes to the substituents on the aromatic ring and side chain. Results from these structure-activity relationships were fit to a probable mode of intracellular activation and interaction with NCp7 to explain variations in antiviral activity. Our strategy to make a series of mercaptobenzamide prodrugs represents a general new direction to make libraries that can be screened for anti-HIV activity.

Expanding the Antiviral Spectrum of 3-Fluoro-2-(phosphonomethoxy)propyl Acyclic Nucleoside Phosphonates: Diamyl Aspartate Amidate Prodrugs.

Acyclic nucleosides containing a 3-fluoro-2-(phosphonomethoxy)propyl (FPMP) side chain are known to be moderately potent antihuman immunodeficiency virus (HIV) agents, while being completely devoid of antiviral activity against a wide range of DNA viruses. The derivatization of the phosphonic acid functionality of FPMPs with a diamyl aspartate phenoxyamidate group led to a novel generation of compounds that not only demonstrate drastically improved antiretroviral potency but also are characterized by an expanded spectrum of activity that also covers hepatitis B and herpes viruses. The best compound, the (S)-FPMPA amidate prodrug, exerts anti-HIV-1 activity in TZM-bl and peripheral blood mononuclear cells at low nanomolar concentrations and displays excellent potency against hepatitis B virus (HBV) and varicella-zoster virus (VZV). This prodrug is stable in acid and human plasma media, but it is efficiently processed in human liver microsomes with a half-life of 2 min. The (R) isomeric guanine derivative emerged as a selectively active anti-HIV and anti-HBV inhibitor, while being nontoxic to human hepatoblastoma cells. Notably, the pyrimidine containing prodrug (S)-Asp-FPMPC is the only congener within this series to demonstrate micromolar antihuman cytomegalovirus (HCMV) potency.

Replacement of the metabolically labile methyl esters in the alkenyldiarylmethane series of non-nucleoside reverse transcriptase inhibitors with isoxazolone, isoxazole, oxazolone, or cyano substituents.

The alkenyldiarylmethanes (ADAMs) are a unique class of non-nucleoside reverse transcriptase inhibitors that have potential value in the treatment of HIV/AIDS. However, the potential usefulness of the ADAMs is limited by the presence of metabolically labile methyl ester moieties. A series of novel ADAMs were therefore designed and synthesized in order to replace the metabolically labile methyl ester moieties of the existing ADAM lead compounds with hydrolytically stable, fused isoxazolone, isoxazole, oxazolone, or cyano substituents on the aromatic rings. The methyl ester and methoxy substituents on both of the aromatic rings in the parent compound 1 were successfully replaced with metabolically stable moieties with retention of anti-HIV activity and a general decrease in cytotoxicity.

Synthesis and Antiviral Evaluation of Octadecyloxyethyl Benzyl 9-[(2-Phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG), a Potent Inhibitor of Transient HPV DNA Amplification.

Human papillomavirus (HPV) high-risk genotypes such as HPV-16 and HPV-18 cause the majority of anogenital tract carcinomas, including cervical cancer, the second most common malignancy in women worldwide. Currently there are no approved antiviral agents that reduce or eliminate HPV and reverse virus-associated pathology. We synthesized and evaluated several alkoxyalkyl acyclic nucleoside phosphonate diesters and identified octadecyloxyethyl benzyl 9-[(2-phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG) as an active compound which strongly inhibited transient amplification of HPV-11, -16, and -18 origin-containing plasmid DNA in transfected cells at concentrations well below its cytotoxic concentrations. ODE-Bn-PMEG demonstrated increased uptake in human foreskin fibroblast cells and was readily converted in vitro to the active antiviral metabolite, PMEG diphosphate. The P-chiral enantiomers of ODE-Bn-PMEG were obtained and appeared to have equivalent antiviral activities against HPV. ODE-Bn-PMEG is a promising candidate for the local treatment of HPV-16 and HPV-18 and other high-risk types, an important unmet medical need.

An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV.

Synthesis, anti-HIV activity, and metabolic stability of new alkenyldiarylmethane HIV-1 non-nucleoside reverse transcriptase inhibitors.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) are part of the combination therapy currently used to treat HIV infection. Based on analogy with known HIV-1 NNRT inhibitors, 18 novel alkenyldiarylmethanes (ADAMs) containing 5-chloro-2-methoxyphenyl, 3-cyanophenyl, or 3-fluoro-5-trifluoromethylphenyl groups were synthesized and evaluated as HIV inhibitors. Their stabilities in rat plasma have also been investigated. Although introducing 5-chloro-2-methoxyphenyl or 3-fluoro-5-trifluoromethylphenyl groups into alkenyldiarylmethanes does not maintain the antiviral potency, the structural modification of alkenyldiarylmethanes with a 3-cyanophenyl substituent can be made without a large decrease in activity. The oxazolidinonyl group was introduced into the alkenyldiarylmethane framework and found to confer enhanced metabolic stability in rat plasma.

Antiviral activity of hop constituents against a series of DNA and RNA viruses.

We investigated whether crude hop extracts and purified hop components representing every major chemical class of hop compound have antiviral activity. These hop constituents were tested for antiviral activity against bovine viral diarrhea virus (BVDV) as a surrogate model of hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza A virus (FLU-A), influenza B virus (FLU-B), rhinovirus (Rhino), respiratory syncytial virus (RSV), yellow fever virus (YFV), cytomegalovirus (CMV), hepatitis B virus (HBV), and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The extracts all failed to prevent the replication of HIV, FLU-A, FLU-B, RSV and YFV. A xanthohumol-enriched hop extract displayed a weak to moderate antiviral activity against BVDV (therapeutic index (TI)=6.0), HSV-2 (TI=>5.3), Rhino (TI=4.0) and HSV-1 (TI=>1.9) with IC(50) values in the low microg/ml range. Pure iso-alpha-acids demonstrated low to moderate antiviral activity against both BVDV (TI=9.1) and CMV (TI=4.2) with IC(50) values in the low microg/ml range. No antiviral activity was detected using beta-acids or a hop oil extract. Ultra-pure preparations (>99% pure) were used to show that xanthohumol accounted for the antiviral activity observed in the xanthohumol-enriched hop extract against BVDV, HSV-1 and HSV-2. Xanthohumol was found to be a more potent antiviral agent against these viruses than the isomer iso-xanthohumol. With Rhino, the opposite trend was observed with iso-xanthohumol showing superior antiviral activity to that observed with xanthohumol. Xanthohumol also showed antiviral activity against CMV, suggesting that it might have a generalized anti-herpesvirus antiviral activity. Again, superior antiviral activity was observed with the xanthohumol isomer against CMV. In summary, iso-alpha-acids and xanthohumol were shown to have a low-to-moderate antiviral activity against several viruses. These hop constituents might serve as interesting lead compounds from which more active anti-HCV, anti-Rhino and anti-herpesvirus antiviral agents could be synthesized.

Anti-HSV activity of serpin antithrombin III.

Natural serine protease inhibitors (serpins) elicit sensing of a microbial cell intruder and activate an intrinsic cellular immune response in HIV and HCV infected cells. Here, we demonstrate in vitro inhibition of HSV with serpin antithrombin III (ATIII) early during infection pointing towards inhibition of an entry event. We also found reduction of mortality from 90% to 40% in an abrasion mice model demonstrating a strong reduction of infection in vivo. Our data also indicated that this treatment might be suitable for drug-resistant viruses since high inhibition of an acyclovir-resistant HSV-1 strain was found. Thus, an ATIII tropical treatment might be used for immunocompromised patients where prolonged treatment leads to drug resistant HSV-1 strains. Understanding how ATIII regulates HSV-1 infections may reveal new avenues for therapeutic interventions.

The Continuing Evolution of HIV-1 Therapy: Identification and Development of Novel Antiretroviral Agents Targeting Viral and Cellular Targets.

During the past three decades, over thirty-five anti-HIV-1 therapies have been developed for use in humans and the progression from monotherapeutic treatment regimens to today's highly active combination antiretroviral therapies has had a dramatic impact on disease progression in HIV-1-infected individuals. In spite of the success of AIDS therapies and the existence of inhibitors of HIV-1 reverse transcriptase, protease, entry and fusion, and integrase, HIV-1 therapies still have a variety of problems which require continued development efforts to improve efficacy and reduce toxicity, while making drugs that can be used throughout both the developed and developing world, in pediatric populations, and in pregnant women. Highly active antiretroviral therapies (HAARTs) have significantly delayed the progression to AIDS, and in the developed world HIV-1-infected individuals might be expected to live normal life spans while on lifelong therapies. However, the difficult treatment regimens, the presence of class-specific drug toxicities, and the emergence of drug-resistant virus isolates highlight the fact that improvements in our therapeutic regimens and the identification of new and novel viral and cellular targets for therapy are still necessary. Antiretroviral therapeutic strategies and targets continue to be explored, and the development of increasingly potent molecules within existing classes of drugs and the development of novel strategies are ongoing.