Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

Repurposing N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human cytomegalovirus pUL89 endonuclease: Synthesis and biological characterization.

The terminase complex of human cytomegalovirus (HCMV) is required for viral genome packaging and cleavage. Critical to the terminase functions is a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We have previously reported metal-chelating N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human immunodeficiency virus 1 (HIV-1) RNase H. In the current work, we have synthesized new analogs and resynthesized known analogs of two isomeric HtPD subtypes, anti-HtPD (13), and syn-HtPD (14), and characterized them as inhibitors of pUL89-C. Remarkably, the vast majority of analogs strongly inhibited pUL89-C in the biochemical endonuclease assay, with IC50 values in the nM range. In the cell-based antiviral assay, a few analogs inhibited HCMV in low µM concentrations. Selected analogs were further characterized in a biophysical thermal shift assay (TSA) and in silico molecular docking, and the results support pUL89-C as the protein target of these inhibitors. Collectively, the biochemical, antiviral, biophysical, and in silico data reported herein indicate that the isomeric HtPD chemotypes 13-14 can serve as valuable chemical platforms for designing improved inhibitors of HCMV pUL89-C.

The substrate-binding cap of the UDP-diacylglucosamine pyrophosphatase LpxH is highly flexible, enabling facile substrate binding and product release.

Gram-negative bacteria are surrounded by a secondary membrane of which the outer leaflet is composed of the glycolipid lipopolysaccharide (LPS), which guards against hydrophobic toxins, including many antibiotics. Therefore, LPS synthesis in bacteria is an attractive target for antibiotic development. LpxH is a pyrophosphatase involved in LPS synthesis, and previous structures revealed that LpxH has a helical cap that binds its lipid substrates. Here, crystallography and hydrogen-deuterium exchange MS provided evidence for a highly flexible substrate-binding cap in LpxH. Furthermore, molecular dynamics simulations disclosed how the helices of the cap may open to allow substrate entry. The predicted opening mechanism was supported by activity assays of LpxH variants. Finally, we confirmed biochemically that LpxH is inhibited by a previously identified antibacterial compound, determined the potency of this inhibitor, and modeled its binding mode in the LpxH active site. In summary, our work provides evidence that the substrate-binding cap of LpxH is highly dynamic, thus allowing for facile substrate binding and product release between the capping helices. Our results also pave the way for the rational design of more potent LpxH inhibitors.

Triazolopyrimidine and triazolopyridine scaffolds as TDP2 inhibitors.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes cellular resistance to clinically used TOP2 poisons. Inhibiting TDP2 can potentially sensitize cancer cells toward TOP2 poisons. Commercial compound P10A10, to which the structure was assigned as 7-phenyl triazolopyrimidine analogue 6a, was previously identified as a TDP2 inhibitor hit in our virtual and fluorescence-based biochemical screening campaign. We report herein that the hit validation through resynthesis and structure elucidation revealed the correct structure of P10A10 (Chembridge ID 7236827) to be the 5-phenyl triazolopyrimidine regioisomer 7a. Subsequent structure-activity relationship (SAR) via the synthesis of a total of 47 analogues of both the 5-phenyl triazolopyrimidine scaffold (7) and its bioisosteric triazolopyridine scaffold (17) identified four derivatives (7a, 17a, 17e, and 17z) with significant TDP2 inhibition (IC50 < 50 µM), with 17z showing excellent cell permeability and no cytotoxicity.

Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. IMPORTANCE: Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus drug development. We identified and characterized an inhibitor of pUL89 endonuclease activity that also inhibits human cytomegalovirus replication in cell culture. pUL89 endonuclease, therefore, should be explored as a potential target for antiviral development against human cytomegalovirus.

Structure-guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release.

UNLABELLED: Current interferon-based therapy for hepatitis C virus (HCV) infection is inadequate, prompting a shift toward combinations of direct-acting antivirals (DAA) with the first protease-targeted drugs licensed in 2012. Many compounds are in the pipeline yet primarily target only three viral proteins, namely, NS3/4A protease, NS5B polymerase, and NS5A. With concerns growing over resistance, broadening the repertoire for DAA targets is a major priority. Here we describe the complete structure of the HCV p7 protein as a monomeric hairpin, solved using a novel combination of chemical shift and nuclear Overhauser effect (NOE)-based methods. This represents atomic resolution information for a full-length virus-coded ion channel, or "viroporin," whose essential functions represent a clinically proven class of antiviral target exploited previously for influenza A virus therapy. Specific drug-protein interactions validate an allosteric site on the channel periphery and its relevance is demonstrated by the selection of novel, structurally diverse inhibitory small molecules with nanomolar potency in culture. Hit compounds represent a 10,000-fold improvement over prototypes, suppress rimantadine resistance polymorphisms at submicromolar concentrations, and show activity against other HCV genotypes. CONCLUSION: This proof-of-principle that structure-guided design can lead to drug-like molecules affirms p7 as a much-needed new target in the burgeoning era of HCV DAA.

Discovery of N-benzyl hydroxypyridone carboxamides as a novel and potent antiviral chemotype against human cytomegalovirus (HCMV).

Current drugs for treating human cytomegalovirus (HCMV) infections are limited by resistance and treatment-associated toxicities. In developing mechanistically novel HCMV antivirals, we discovered an N-benzyl hydroxypyridone carboxamide antiviral hit (8a) inhibiting HCMV in submicromolar range. We describe herein the structure-activity relationship (SAR) for 8a, and the characterization of potent analogs for cytotoxicity/cytostatic property, the preliminary mechanism of action, and the absorption, distribution, metabolism and excretion (ADME) properties. The SAR revealed a few pharmacophore features conferring optimal antiviral profile, including the 5-OH, the N-1 benzyl, at least one -CH2- in the linker, and a di-halogen substituted phenyl ring in the amide moiety. In the end, we identified numerous analogs with sub-micromolar antiviral potency and good selectivity index. The preliminary mechanism of action characterization used a pUL89-C biochemical endonuclease assay, a virus entry assay, a time-of-addition assay, and a compound withdrawal assay. ADME profiling measuring aqueous solubility, plasma and liver microsomal stability, and parallel artificial membrane permeability assay (PAMPA) permeability demonstrated largely favorable drug-like properties. Together, these studies validate the N-benzyl hydroxypyridone carboxamide as a viable chemotype for potent and mechanistically distinct antivirals against HCMV.

Novel deazaflavin tyrosyl-DNA phosphodiesterase 2 (TDP2) inhibitors.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a DNA repair enzyme that removes 5'-phosphotyrosyl blockages resulting from topoisomerase II (TOP2)-DNA cleavage complexes trapped by TOP2 inhibitors. TDP2 is a logical target for the development of therapeutics to complement existing treatments based on inhibition of TOP2. There is, however, no TDP2 inhibitor in clinical development at present. Of the reported TDP2 inhibitors, the deazaflavins are the most promising chemical class centered around the lead compound SV-5-153. Recently we reported new subtypes derived within the deazaflavin family with improved membrane permeability properties. In this work we characterize two representative analogues from two new deazaflavin subtypes based on their biochemical TDP2 inhibitory potency and drug-likeness. We demonstrate that the ZW-1288 derivative represents a promising direction for the development of deazaflavins as therapeutic agents. ZW-1288 exhibits potent inhibitory activity at low nanomolar concentrations against recombinant and cellular human TDP2 with profile similar to that of the parent analog SV-5-153 based on high resistance against murine TDP2 and human TDP2 mutated at residue L313H. While expressing weak cytotoxicity on its own, ZW-1288 potentiates the clinical TOP2 inhibitors etoposide (ETP) and mitoxantrone in human prostate DU145 and CCRF-CEM leukemia and chicken lymphoma DT40 cells while not impacting the activity of the topoisomerase I (TOP1) inhibitor camptothecin or the PARP inhibitor olaparib. ZW-1288 increases the uptake of ETP to a lesser extent than SV-5-153 and remained active in TDP2 knockout cells indicating that the deazaflavin TDP2 inhibitors have additional cellular effects that will have to be taken into account for their further development as TDP2 inhibitors.

Novel HIV-1 capsid-targeting small molecules of the PF74 binding site.

The PF74 binding site in HIV-1 capsid protein (CA) is a compelling antiviral drug target. Although PF74 confers mechanistically distinct antiviral phenotypes by competing against host factors for CA binding, it suffers from prohibitively low metabolic stability. Therefore, there has been increasing interest in designing novel sub-chemotypes of PF74 with similar binding mode and improved metabolic stability. We report herein our efforts to explore the inter-domain interacting indole moiety for designing novel CA-targeting small molecules. Our design includes simple substitution on the indole ring, and more importantly, novel sub-chemotypes with the indole moiety replaced with a few less electron-rich rings. All 56 novel analogs were synthesized and evaluated for antiviral activity, cytotoxicity, and impact on CA hexamer stability. Selected analogs were tested for metabolic stability in liver microsomes. Molecular modeling was performed to verify compound binding to the PF74 site. In the end, 5-hydroxyindole analogs (8,9 and 12) showed improved potency (up to 20-fold) over PF74. Of the novel sub-chemotypes, α- and ß-naphthyl analogs (33 and 27) exhibited sub micromolar antiviral potencies comparable to that of PF74. Interestingly, although only moderately inhibiting HIV-1 (single-digit micromolar EC50s), analogs of the 2-indolone sub-chemotype consistently lowered the melting point (Tm) of CA hexamers, some with improved metabolic stability over PF74.

Toward Structurally Novel and Metabolically Stable HIV-1 Capsid-Targeting Small Molecules.

HIV-1 capsid protein (CA) plays an important role in many steps of viral replication and represents an appealing antiviral target. Several CA-targeting small molecules of various chemotypes have been studied, but the peptidomimetic PF74 has drawn particular interest due to its potent antiviral activity, well-characterized binding mode, and unique mechanism of action. Importantly, PF74 competes against important host factors for binding, conferring highly desirable antiviral phenotypes. However, further development of PF74 is hindered by its prohibitively poor metabolic stability, which necessitates the search for structurally novel and metabolically stable chemotypes. We have conducted a pharmacophore-based shape similarity search for compounds mimicking PF74. We report herein the analog synthesis and structure-activity relationship (SAR) of two hits from the search, and a third hit designed via molecular hybridization. All analogs were characterized for their effect on CA hexamer stability, antiviral activity, and cytotoxicity. These assays identified three active compounds that moderately stabilize CA hexamer and inhibit HIV-1. The most potent analog (10) inhibited HIV-1 comparably to PF74 but demonstrated drastically improved metabolic stability in liver microsomes (31 min vs. 0.7 min t1/2). Collectively, the current studies identified a structurally novel and metabolically stable PF74-like chemotype for targeting HIV-1 CA.

Rationally derived inhibitors of hepatitis C virus (HCV) p7 channel activity reveal prospect for bimodal antiviral therapy.

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or 'viroporins', contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.

Novel Deazaflavin Analogues Potently Inhibited Tyrosyl DNA Phosphodiesterase 2 (TDP2) and Strongly Sensitized Cancer Cells toward Treatment with Topoisomerase II (TOP2) Poison Etoposide.

Topoisomerase II (TOP2) poisons as anticancer drugs work by trapping TOP2 cleavage complexes (TOP2cc) to generate DNA damage. Repair of such damage by tyrosyl DNA phosphodiesterase 2 (TDP2) could render cancer cells resistant to TOP2 poisons. Inhibiting TDP2, thus, represents an attractive mechanism-based chemosensitization approach. Currently known TDP2 inhibitors lack cellular potency and/or permeability. We report herein two novel subtypes of the deazaflavin TDP2 inhibitor core. By introducing an additional phenyl ring to the N-10 phenyl ring (subtype 11) or to the N-3 site of the deazaflavin scaffold (subtype 12), we have generated novel analogues with considerably improved biochemical potency and/or permeability. Importantly, many analogues of both subtypes, particularly compounds 11a, 11e, 12a, 12b, and 12h, exhibited much stronger cancer cell sensitizing effect than the best previous analogue 4a toward the treatment with etoposide, suggesting that these analogues could serve as effective cellular probes.

Pharmacophore-based design of novel 3-hydroxypyrimidine-2,4-dione subtypes as inhibitors of HIV reverse transcriptase-associated RNase H: Tolerance of a nonflexible linker.

The pharmacophore of active site inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated RNase H typically entails a flexible linker connecting the chelating core and the hydrophobic aromatics. We report herein that novel 3-hydroxypyrimidine-2,4-dione (HPD) subtypes with a nonflexible C-6 carbonyl linkage exhibited potent and selective biochemical inhibitory profiles with strong RNase H inhibition at low nM, weak to moderate integrase strand transfer (INST) inhibition at low µM, and no to marginal RT polymerase (pol) inhibition up to 10 µM. A few analogues also demonstrated significant antiviral activity without cytotoxicity. The overall inhibitory profile is comparable to or better than that of previous HPD subtypes with a flexible C-6 linker, suggesting that the nonflexible carbonyl linker can be tolerated in the design of novel HIV RNase H active site inhibitors.

5-Aminothiophene-2,4-dicarboxamide analogues as hepatitis B virus capsid assembly effectors.

Chronic hepatitis B virus (HBV) infection represents a major health threat. Current FDA-approved drugs do not cure HBV. Targeting HBV core protein (Cp) provides an attractive approach toward HBV inhibition and possibly infection cure. We have previously identified and characterized a 5-amino-3-methylthiophene-2,4-dicarboxamide (ATDC) compound as a structurally novel hit for capsid assembly effectors (CAEs). We report herein hit validation through studies on absorption, distribution, metabolism and excretion (ADME) properties and pharmacokinetics (PK), and hit optimization via analogue synthesis aiming to probe the structure-activity relationship (SAR) and structure-property relationship (SPR). In the end, these medicinal chemistry efforts led to the identification of multiple analogues strongly binding to Cp, potently inhibiting HBV replication in nanomolar range without cytotoxicity, and exhibiting good oral bioavailability (F). Two of our analogues, 19o (EC50 = 0.11 µM, CC50 > 100 µM, F = 25%) and 19k (EC50 = 0.31 µM, CC50 > 100 µM, F = 46%), displayed overall lead profiles superior to reported CAEs 7-10 used in our studies.

Hydroxypyridonecarboxylic Acids as Inhibitors of Human Cytomegalovirus pUL89 Endonuclease.

Human cytomegalovirus (HCMV) infection poses a major health threat to immunocompromised individuals. Until recently, treatment of HCMV infection has relied solely on polymerase inhibitors that have safety and resistance issues. pUL89 provides the enzymatic functions for the HCMV terminase complex in viral DNA packaging and is an attractive target for developing a new class of HCMV drugs. However, inhibitors of the endonuclease activity of the C terminus of pUL89 (pUL89-C) were unknown before our recently characterized hydroxypyridonecarboxylic acid (HPCA) hit 7 r (1-(3-chloro-4-fluorobenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid; numbered as 10 k in our previous publication: Y. Wang, L. Mao, J. Kankanala, Z. Wang, R. J. Geraghty, J. Virol. 2017, 91, e02152-16). Herein, we explored the structure-activity relationship (SAR) of the HPCA chemotype mainly with regard to the N1 site through the synthesis of 35 analogues. The SAR studies, along with molecular modeling, identified a possible pharmacophore model minimally consisting of a chelating triad and a hydrophobic phenyl or biphenyl methyl substituent at N1. Lastly, our best compounds consistently inhibited pUL89-C in the low micromolar range in biochemical assays and exhibited strong antiviral activity without cytotoxicity, laying a solid medicinal chemistry foundation for further HCMV drug discovery efforts targeting pUL89-C.

New fluorescence-based high-throughput screening assay for small molecule inhibitors of tyrosyl-DNA phosphodiesterase 2 (TDP2).

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes resistance to TOP2-targeted cancer therapy. Inhibiting TDP2 could sensitize cancer cells toward TOP2 inhibitors. However, potent TDP2 inhibitors with favorable physicochemical properties are not yet reported. Therefore, there is a need to search for novel molecular scaffolds capable of inhibiting TDP2. We report herein a new simple, robust, homogenous mix-and-read fluorescence biochemical assay based using humanized zebrafish TDP2 (14M_zTDP2), which provides biochemical and molecular structure basis for TDP2 inhibitor discovery. The assay was validated by screening a preselected library of 1600 compounds (Z' ≥ 0.72) in a 384-well format, and by running in parallel gel-based assays with fluorescent DNA substrates. This library was curated via virtual high throughput screening (vHTS) of 460,000 compounds from Chembridge Library, using the crystal structure of the novel surrogate protein 14M_zTDP2. From this primary screening, we selected the best 32 compounds (2% of the library) to further assess their TDP2 inhibition potential, leading to the IC50 determination of 10 compounds. Based on the dose-response curve profile, pan-assay interference compounds (PAINS) structure identification, physicochemical properties and efficiency parameters, two hit compounds, 11a and 19a, were tested using a novel secondary fluorescence gel-based assay. Preliminary structure-activity relationship (SAR) studies identified guanidine derivative 12a as an improved hit with a 6.4-fold increase in potency over the original HTS hit 11a. This study highlights the importance of the development of combination approaches (biochemistry, crystallography and high throughput screening) for the discovery of TDP2 inhibitors.

6-Arylthio-3-hydroxypyrimidine-2,4-diones potently inhibited HIV reverse transcriptase-associated RNase H with antiviral activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not targeted by current drugs. Although a few chemotypes have been reported to inhibit HIV RNase H in biochemical assays, their general lack of significant antiviral activity in cell culture necessitates continued efforts in identifying highly potent RNase H inhibitors to confer antiviral activity. We report herein the design, synthesis, biochemical and antiviral evaluations of a new 6-arylthio subtype of the 3-hydroxypyrimidine-2,4-dione (HPD) chemotype. In biochemical assays these new analogues inhibited RT RNase H in single-digit nanomolar range without inhibiting RT polymerase (pol) at concentrations up to 10 µM, amounting to exceptional biochemical inhibitory selectivity. Many analogues also inhibited integrase strand transfer (INST) activity in low to sub micromolar range. More importantly, most analogues inhibited HIV in low micromolar range without cytotoxicity. In the end, compound 13j (RNase H IC50 = 0.005 µM; RT pol IC50 = 10 µM; INST IC50 = 4.0 µM; antiviral EC50 = 7.7 µM; CC50 > 100 µM) represents the best analogues within this series. These results characterize the new 6-arylthio-HPD subtype as a promising scaffold for HIV RNase H inhibitor discovery.

6-Biphenylmethyl-3-hydroxypyrimidine-2,4-diones potently and selectively inhibited HIV reverse transcriptase-associated RNase H.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains an unvalidated drug target. Reported HIV RNase H inhibitors generally lack significant antiviral activity. We report herein the design, synthesis, biochemical and antiviral evaluations of a new 6-biphenylmethyl subtype of the 3-hydroxypyrimidine-2,4-dione (HPD) chemotype. In biochemical assays, analogues of this new subtype potently inhibited RT RNase H in low nanomolar range without inhibiting RT polymerase (pol) or integrase strand transfer (INST) at the highest concentrations tested. In cell-based assays, a few analogues inhibited HIV in low micromolar range without cytotoxicity at concentrations up to 100 µM.

Design, synthesis and biological evaluations of N-Hydroxy thienopyrimidine-2,4-diones as inhibitors of HIV reverse transcriptase-associated RNase H.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) is the only HIV enzymatic function not targeted by current antiviral drugs. Although various chemotypes have been reported to inhibit HIV RNase H, few have shown significant antiviral activities. We report herein the design, synthesis and biological evaluation of a novel N-hydroxy thienopyrimidine-2,3-dione chemotype (11) which potently and selectively inhibited RNase H with considerable potency against HIV-1 in cell culture. Current structure-activity-relationship (SAR) identified analogue 11d as a nanomolar inhibitor of RNase H (IC50 = 0.04 µM) with decent antiviral potency (EC50 = 7.4 µM) and no cytotoxicity (CC50 > 100 µM). In extended biochemical assays compound 11d did not inhibit RT polymerase (pol) while inhibiting integrase strand transfer (INST) with 53 fold lower potency (IC50 = 2.1 µM) than RNase H inhibition. Crystallographic and molecular modeling studies confirmed the RNase H active site binding mode.

Double-Winged 3-Hydroxypyrimidine-2,4-diones: Potent and Selective Inhibition against HIV-1 RNase H with Significant Antiviral Activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function yet to be exploited as an antiviral target. One of the possible challenges may be that targeting HIV RNase H is confronted with a steep substrate barrier. We have previously reported a 3-hydroxypyrimidine-2,4-dione (HPD) subtype that potently and selectively inhibited RNase H without inhibiting HIV in cell culture. We report herein a critical redesign of the HPD chemotype featuring an additional wing at the C5 position that led to drastically improved RNase H inhibition and significant antiviral activity. Structure-activity relationship (SAR) concerning primarily the length and flexibility of the two wings revealed important structural features that dictate the potency and selectivity of RNase H inhibition as well as the observed antiviral activity. Our current medicinal chemistry data also revealed that the RNase H biochemical inhibition largely correlated the antiviral activity.