Geometric Isomer of Guanabenz Confers Hepatoprotection to a Murine Model of Acetaminophen Toxicity.

Overdose of acetaminophen, a widely used antipyretic and analgesic drug, is one of the leading causes of drug-induced acute liver injury in the United States and worldwide. Phase-I metabolism of acetaminophen generates the toxic N-acetyl-p-benzoquinone imine (NAPQI) intermediate. Reactions of NAPQI with a wide range of biomolecules cause increased oxidative stress, endoplasmic reticulum (ER) stress, inflammation, and mitochondrial dysfunction, some of the cellular events contributing toward liver toxicity. Previously, we evaluated the potential of an FDA-approved, ER stress-modulating antihypertensive drug, Wytensin (trans-guanabenz, E-GA), as an antidote for acetaminophen hepatotoxicity. E-GA prevented elevation of the liver enzyme alanine aminotransferase (ALT), even when administered up to 6 h after acetaminophen overdose, and exhibited synergistic analgesic interactions. However, the commercially available guanabenz exists solely as a trans-isomer and suffers from sedative side effects resulting from the inhibition of central α2A-adrenergic receptors in locus coeruleus. Here, we studied the utility of the relatively unexplored cis-isomer of guanabenz as a treatment option for acetaminophen-induced liver toxicity. cis(Z)-Guanabenz acetate (Z-GA) lacks interaction with α2A-adrenoreceptors and is thus devoid of sedative, blood-pressure-lowering side effects of E-GA. Treatment of mice with Z-GA (10 mg/kg) before acetaminophen overdose and up to 6 h post APAP administration prevented liver injury and suppressed the elevation of serum ALT levels. Mechanistically, hepatoprotective effects of both isomers are similar and partly attributed to attenuation of the ER stress and oxidative stress in the liver. The results of this study suggest that Z-GA may be a safer, effective antidote for the clinical management of acute liver injury resulting from acetaminophen overdose. It also raises a tantalizing possibility of a prophylactic combination of the geometric isomer of the approved drug guanabenz with acetaminophen in a clinical setting.

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.

Teacher victimization and teachers' subjective well-being: Does school climate matter?

Guided by the job demands-resources model, we examined the multilevel associations between victimization experience with student violence directed against teachers, school climate, and teachers' subjective well-being (i.e., school connectedness and teaching efficacy) among 1711 teachers (7th-12th grade) from 58 middle and high schools in China. Hierarchical linear modeling analyses revealed that teachers who reported more frequent teacher victimization perceived a lower level of teaching efficacy; however, teachers in schools with a higher level of teacher victimization scores at the school level perceived a higher level of teaching efficacy. Although school climate was positively related to teacher well-being at both teacher and school levels, the negative association between teacher victimization and teachers' subjective well-being at the teacher level was exacerbated in schools with a more positive school climate at the school level. The significant cross-level moderating effect of school-level school climate in the association between teacher-level victimization and subjective well-being was consistent with the "healthy context paradox" but contradicted with the "emotion contagion hypothesis." Our findings support the risk influence of teacher victimization and the promotive role of positive school climate on teachers' subjective well-being. Our results also indicate that teachers in schools with a more positive and collective perception of school climate tend to be more attuned to the negative influences of teacher victimization on their subjective well-being than teachers in schools with a less positive and collective perception of school climate.

Guanabenz Attenuates Acetaminophen-Induced Liver Toxicity and Synergizes Analgesia in Mice.

Endoplasmic reticulum (ER) stress has been shown to be involved in the hepatotoxicity of acetaminophen (APAP). Guanabenz (GA), a widely known antihypertensive drug, is reported to exhibit an anti-ER stress effect. In this study, we investigated the potential of GA as an antidote against APAP-induced hepatotoxicity. The underlying biochemical mechanisms for the hepatoprotective effect of GA were explored. Here we found that treatment of mice with GA (10 mg/kg) before APAP overdose dramatically prevented APAP-induced liver enzyme elevation and resultant toxicity in mice, as indicated by suppression of elevated serum alanine aminotransferase (ALT) levels and liver histological analysis. Importantly, delayed administration of GA within 6 h after APAP overdose also showed an almost equivalent protective effect against APAP liver toxicity. Mechanistically, several pathways are involved in the protective effect of GA against APAP-induced live toxicity, including attenuation of ER stress and oxidative stress, increased levels of nontoxic phase I and II metabolites of APAP, decrease in the formation of toxic N-acetyl-p-benzoquinone imine (NAPQI), and its subsequent protein binding. Importantly, combination of GA with APAP exhibited synergistic interaction in the latter's analgesic activity, while sparing its antipyretic action. These findings provide the preclinical evidence of GA as a promising antidote for treatment of APAP-induced liver toxicity and raise a possibility of its combination with APAP in clinical settings.

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.

Teacher victimization by students in China: A multilevel analysis.

The goal of this study is to examine individual-level and school-level predictors of teacher victimization (TV) by students in China based upon the multilevel social-ecological framework. A sample of 1711 teachers (7-12th grade) from 58 schools from eight provinces in mainland China completed measures of teacher victimization (i.e., physical TV, verbal TV, social TV, cyber TV, sexual harassment, and personal property offenses) by students, school-wide bullying and disciplinary practices, and demographics. In the present sample, 25.1% of teachers reported that they experienced at least one of the six forms of victimization from students in the past school year. Prevalence of teacher victimization ranged from 4.0% (physical victimization) to 16.8% (social victimization). Male teachers were more likely to experience all forms of TV and homeroom teachers were more likely than non-homeroom teachers to experience social TV. Schools with fewer students, but higher number of teachers, also had higher levels of TV. Student bullying and punitive disciplinary practices at the teacher level were associated with higher levels of most forms of TV. Implications of these findings are discussed.

A fluorescence-based high-throughput assay to identify inhibitors of tyrosylprotein sulfotransferase activity.

Tyrosylprotein sulfotransferases (TPSTs) are Golgi-resident enzymes that catalyze the transfer of a sulfuryl group to the side chain hydroxyl of tyrosine residues. Sulfotyrosine residues are involved in protein-protein interactions in the extracellular space. These interactions are important for chemokines to bind cognate receptor, for cell adhesion and trafficking, and for pathogen entry into cells. To better understand the role of TPSTs in cellular processes and disease states, we are interested in identifying small molecules to modulate TPST activity in experimental systems. Towards that end, we developed a fluorescent peptide assay for TPST2 activity. Here, we demonstrate that this assay can be used to screen the 1280 compound LOPAC library in a 384-well format and in a high-throughput manner. We identified 19 primary hits for a hit rate of 1.5%. Three of the primary hits were verified by dose-response assay and confirmed as inhibitors by a secondary mass spectrometry assay for TPST activity. One hit, suramin, possessed inhibitory properties consistent with a competitive inhibitor of substrate binding and molecular docking revealed a good fit into the TPST2 substrate-binding pocket. This assay can be used to screen larger libraries to identify small molecules that inhibit TPST sulfotransferase activity.

Dual anti-HIV mechanism of clofarabine.

BACKGROUND: HIV-1 replication kinetics inherently depends on the availability of cellular dNTPs for viral DNA synthesis. In activated CD4(+) T cells and other rapidly dividing cells, the concentrations of dNTPs are high and HIV-1 reverse transcription occurs in an efficient manner. In contrast, nondividing cells such as macrophages have lower dNTP pools, which restricts efficient reverse transcription. Clofarabine is an FDA approved ribonucleotide reductase inhibitor, which has shown potent antiretroviral activity in transformed cell lines. Here, we explore the potency, toxicity and mechanism of action of clofarabine in the human primary HIV-1 target cells: activated CD4(+) T cells and macrophages. RESULTS: Clofarabine is a potent HIV-1 inhibitor in both activated CD4(+) T cells and macrophages. Due to its minimal toxicity in macrophages, clofarabine displays a selectivity index over 300 in this nondividing cell type. The anti-HIV-1 activity of clofarabine correlated with a significant decrease in both cellular dNTP levels and viral DNA synthesis. Additionally, we observed that clofarabine triphosphate was directly incorporated into DNA by HIV-1 reverse transcriptase and blocked processive DNA synthesis, particularly at the low dNTP levels found in macrophages. CONCLUSIONS: Taken together, these data provide strong mechanistic evidence that clofarabine is a dual action inhibitor of HIV-1 replication that both limits dNTP substrates for viral DNA synthesis and directly inhibits the DNA polymerase activity of HIV-1 reverse transcriptase.

5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2'-Deoxycytidine.

5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2'-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses.

Eeyarestatin I derivatives with improved aqueous solubility.

Inhibition of p97 (also known as valosin-containing protein (VCP)), has been validated as a promising strategy for cancer therapy. Eeyarestatin I (EerI) blocks p97 through a novel mechanism of action and has favorable anti-cancer activities against cultured cancer cells. However, its poor aqueous solubility severely limits its in vivo applications. To circumvent this problem, we have identified EerI derivatives that possess improved aqueous solubility by introducing a single solubilizing group. These modified compounds preserved endoplasmic reticulum (ER) stress-inducing and antiproliferative activities as well as generally good in vitro metabolic properties, suggesting that these EerI derivatives could serve as candidates for further optimization.

Design and synthesis of an activity-based protein profiling probe derived from cinnamic hydroxamic acid.

In our continued effort to discover new anti-hepatitis C virus (HCV) agents, we validated the anti-replicon activity of compound 1, a potent and selective anti-HCV hydroxamic acid recently reported by us. Generally favorable physicochemical and in vitro absorption, distribution, metabolism, and excretion (ADME) properties exhibited by 1 made it an ideal parent compound from which activity-based protein profiling (ABPP) probe 3 was designed and synthesized. Evaluation of probe 3 revealed that it possessed necessary anti-HCV activity and selectivity. Therefore, we have successfully obtained compound 3 as a suitable ABPP probe to identify potential molecular targets of compound 1. Probe 3 and its improved analogs are expected to join a growing list of ABPP probes that have made important contributions to not only the studies of biochemical and cellular functions but also discovery of selective inhibitors of protein targets.

Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase.

Although many compounds have been approved for the treatment of human immunodeficiency type-1 (HIV-1) infection, additional anti-HIV-1 drugs (particularly those belonging to new drug classes) are still needed due to issues such as long-term drug-associated toxicities, transmission of drug-resistant variants, and development of multi-class resistance. Lethal mutagenesis represents an antiviral strategy that has not yet been clinically translated for HIV-1 and is based on the use of small molecules to induce excessive levels of deleterious mutations within the viral genome. Here, we show that 5-azacytidine (5-aza-C), a ribonucleoside analog that induces the lethal mutagenesis of HIV-1, and multiple inhibitors of the enzyme ribonucleotide reductase (RNR) interact in a synergistic fashion to more effectively reduce the infectivity of HIV-1. In these drug combinations, RNR inhibitors failed to significantly inhibit the conversion of 5-aza-C to 5-aza-2'-deoxycytidine, suggesting that 5-aza-C acts primarily as a deoxyribonucleoside even in the presence of RNR inhibitors. The mechanism of antiviral synergy was further investigated for the combination of 5-aza-C and one specific RNR inhibitor, resveratrol, as this combination improved the selectivity index of 5-aza-C to the greatest extent. Antiviral synergy was found to be primarily due to the reduced accumulation of reverse transcription products rather than the enhancement of viral mutagenesis. To our knowledge, these observations represent the first demonstration of antiretroviral synergy between a ribonucleoside analog and RNR inhibitors, and encourage the development of additional ribonucleoside analogs and RNR inhibitors with improved antiretroviral activity.

Discovery of 1-hydroxypyridine-2-thiones as selective histone deacetylase inhibitors and their potential application for treating leukemia.

Histone deacetylase (HDAC) is a validated target for pursuing anticancer agents. However, obtaining a selective inhibitor against a given HDAC member remains a significant challenge. We report here the use of 1-hydroxypyridine-2-thione (1HPT) as a key pharmacophore for zinc-binding can result in highly selective HDAC inhibitors. 1HPT-6-carboxylic acid exhibits selective inhibition of HDAC6 with an IC50 of 150 nM that corresponds to a remarkable 0.9 ligand efficiency. Two analogs with simple amino acids shows nearly 600-fold selectivity among the eleven zinc-dependent HDACs. At low micromolar concentration these compounds inhibit the growth of HDAC8-overexpressing chronic myelogenous leukemia cells and specific form of acute myelogenous leukemia cells. Their potential mode of binding was examined by molecular docking and their stability was assessed in mouse and human plasma. Together the results suggest 1HPT analogs exhibit promising therapeutic potential for further development as anticancer agents to treat leukemia.

Cancer sensitizing effect of deazaflavin analogs is associated with increased intracellular drug accumulation.

As part of our efforts geared towards developing mechanism-based cancer sensitizing agents, we have previously synthesized and characterized novel deazaflavin analogs as potent tyrosyl DNA phosphodiesterase 2 (TDP2) inhibitors for combination treatments with topoisomerase II (TOP2) poisons. Interestingly, the sensitizing effect of a few analogs toward TOP2 poison etoposide (ETP) was associated with a significant increase in intracellular drug accumulation, which could be an alternative mechanism to boost the clinical efficacy of ETP in cancer chemotherapies. Hence, we evaluated more deazaflavin TDP2 inhibitors for their impact on drug retention in cancer cells. We found that all but one tested TDP2 inhibitors substantially increased the ETP retention in DT40 cells. Particularly, we identified an exceptionally potent analog, ZW-1226, which at 3 nM increased the intracellular ETP by 13-fold. Significantly, ZW-1226 also stimulated cellular accumulation of two other anticancer drugs, TOP2 poison teniposide and antifolate pemetrexed, and produced an effect more pronounced than those of ABC transporter inhibitors verapamil and elacridar in human leukemic CCRF-CEM cells toward ETP. Lastly, ZW-1226 potentiated the action of ETP in the sensitive human CCRF-CEM cells and a few resistant non-small-cell lung cancer (NSCLC) cells, including H460 and H838 cells. Collectively, the results of this study strongly suggest that deazaflavin analog ZW-1226 could be an effective cancer sensitizing agent which warrants further investigation.

Modulation of endogenous opioid signaling by inhibitors of puromycin-sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin-sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

Deletion of Glyoxalase 1 Exacerbates Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) overdose triggers a cascade of intracellular oxidative stress events, culminating in acute liver injury. The clinically used antidote, N-acetylcysteine (NAC), has a narrow therapeutic window, and early treatment is essential for a satisfactory therapeutic outcome. For more versatile therapies that can be effective even at late presentation, the intricacies of APAP-induced hepatotoxicity must be better understood. Accumulation of advanced glycation end products (AGEs) and the consequent activation of the receptor for AGEs (RAGE) are considered one of the key mechanistic features of APAP toxicity. Glyoxalase 1 (Glo-1) regulates AGE formation by limiting the levels of methylglyoxal (MEG). In this study, we studied the relevance of Glo-1 in the APAP-mediated activation of RAGE and downstream cell death cascades. Constitutive Glo-1-knockout mice (GKO) and a cofactor of Glo-1, ψ-GSH, were used as tools. Our findings showed elevated oxidative stress resulting from the activation of RAGE and hepatocyte necrosis through steatosis in GKO mice treated with high-dose APAP compared to wild-type controls. A unique feature of the hepatic necrosis in GKO mice was the appearance of microvesicular steatosis as a result of centrilobular necrosis, rather than the inflammation seen in the wild type. The GSH surrogate and general antioxidant ψ-GSH alleviated APAP toxicity irrespective of the Glo-1 status, suggesting that oxidative stress is the primary driver of APAP toxicity. Overall, the exacerbation of APAP hepatotoxicity in GKO mice suggests the importance of this enzyme system in antioxidant defense against the initial stages of APAP overdose.

Modulation of endogenous opioid signaling by inhibitors of puromycin sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

Deletion of Glyoxalase 1 exacerbates acetaminophen-induced hepatotoxicity in mice.

Acetaminophen (APAP) overdose triggers a cascade of intracellular oxidative stress events culminating in acute liver injury. The clinically used antidote, N-acetylcysteine (NAC) has a narrow therapeutic window and early treatment is essential for satisfactory therapeutic outcome. For more versatile therapies that can be effective even at late-presentation, the intricacies of APAP-induced hepatotoxicity must be better understood. Accumulation of advanced glycation end-products (AGEs) and consequent activation of the receptor for AGEs (RAGE) are considered one of the key mechanistic features of APAP toxicity. Glyoxalase-1 (Glo-1) regulates AGE formation by limiting the levels of methylglyoxal (MEG). In this study, we studied the relevance of Glo-1 in APAP mediated activation of RAGE and downstream cell-death cascades. Constitutive Glo-1 knockout mice (GKO) and a cofactor of Glo-1, ψ-GSH, were employed as tools. Our findings show elevated oxidative stress, activation of RAGE and hepatocyte necrosis through steatosis in GKO mice treated with high-dose APAP compared to wild type controls. A unique feature of the hepatic necrosis in GKO mice is the appearance of microvesicular steatosis as a result of centrilobular necrosis, rather than inflammation seen in wild type. The GSH surrogate and general antioxidant, ψ-GSH alleviated APAP toxicity irrespective of Glo-1 status, suggesting that oxidative stress being the primary driver of APAP toxicity. Overall, exacerbation of APAP hepatotoxicity in GKO mice suggests the importance of this enzyme system in antioxidant defense against initial stages of APAP overdose.

4,5-Dihydroxypyrimidine Methyl Carboxylates, Carboxylic Acids, and Carboxamides as Inhibitors of Human Cytomegalovirus pUL89 Endonuclease.

Human cytomegalovirus (HCMV) terminase complex entails a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We report herein the design, synthesis, and characterization of dihydroxypyrimidine (DHP) acid (14), methyl ester (13), and amide (15) subtypes as inhibitors of HCMV pUL89-C. All analogs synthesized were tested in an endonuclease assay and a thermal shift assay (TSA) and subjected to molecular docking to predict binding affinity. Although analogs inhibiting pUL89-C in the sub-µM range were identified from all three subtypes, acids (14) showed better overall potency, substantially larger thermal shift, and considerably better docking scores than esters (13) and amides (15). In the cell-based antiviral assay, six analogs inhibited HCMV with moderate activities (EC50 = 14.4-22.8 µM). The acid subtype (14) showed good in vitro ADME properties, except for poor permeability. Overall, our data support the DHP acid subtype (14) as a valuable scaffold for developing antivirals targeting HCMV pUL89-C.

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.