N-Hydroxypiridinedione: A Privileged Heterocycle for Targeting the HBV RNase H.

Hepatitis B virus (HBV) remains a global health threat. Ribonuclease H (RNase H), part of the virus polymerase protein, cleaves the pgRNA template during viral genome replication. Inhibition of RNase H activity prevents (+) DNA strand synthesis and results in the accumulation of non-functional genomes, terminating the viral replication cycle. RNase H, though promising, remains an under-explored drug target against HBV. We previously reported the identification of a series of N-hydroxypyridinedione (HPD) imines that effectively inhibit the HBV RNase H. In our effort to further explore the HPD scaffold, we designed, synthesized, and evaluated 18 novel HPD oximes, as well as 4 structurally related minoxidil derivatives and 2 barbituric acid counterparts. The new analogs were docked on the RNase H active site and all proved able to coordinate the two Mg2+ ions in the catalytic site. All of the new HPDs effectively inhibited the viral replication in cell assays exhibiting EC50 values in the low µM range (1.1-7.7 µM) with low cytotoxicity, resulting in selectivity indexes (SI) of up to 92, one of the highest reported to date among HBV RNase H inhibitors. Our findings expand the structure-activity relationships on the HPD scaffold, facilitating the development of even more potent anti-HBV agents.

Identification and assessment of the 1,6-dihydroxy-pyridin-2-one moiety as privileged scaffold for HBV ribonuclease H inhibition.

The Hepatitis B Virus (HBV) ribonuclease H (RNase H) although promising remains an unexploited therapeutic target. HBV RNase H inhibition causes premature termination of viral minus-polarity DNA strands, prevents the synthesis of the viral positive-polarity DNA strand, and causes accumulation of RNA:DNA heteroduplexes within viral capsids. As part of our ongoing research to develop more potent anti-HBV RNase H inhibitors, we designed, synthesized and analyzed a library of 18 novel compounds (17 N-hydroyxpyridinedione (HPD) imine derivatives and 1 barbituric acid analogue) as potential leads for HBV treatment development. In cell assays, fourteen HPDs showed significant anti-HBV activity with EC50s from 1.1 to 2.5 µM and selectivity indices (SI) of up to 58. Three of them exhibited more than 3-fold improvement in the SI over the best previous HPD imine (SI = 13). To gain insight to the interaction between the tested compounds and the active site of HBV RNase H, docking experiments were undertaken. In almost all binding poses, the novel HPDs coordinated both active site Mg2+ ions via their oxygen trident. Furthermore, the novel HPDs displayed high cell permeability and solubility as well as good drug-like properties. These results reveal that HPD imines can be significantly active and selective HBV inhibitors, and that the HPD scaffold merits further development towards anti-HBV agents.

Selection pressures on evolution of ribonuclease H explored with rigorous free-energy-based design.

Understanding natural protein evolution and designing novel proteins are motivating interest in development of high-throughput methods to explore large sequence spaces. In this work, we demonstrate the application of multisite λ dynamics (MSλD), a rigorous free energy simulation method, and chemical denaturation experiments to quantify evolutionary selection pressure from sequence-stability relationships and to address questions of design. This study examines a mesophilic phylogenetic clade of ribonuclease H (RNase H), furthering its extensive characterization in earlier studies, focusing on E. coli RNase H (ecRNH) and a more stable consensus sequence (AncCcons) differing at 15 positions. The stabilities of 32,768 chimeras between these two sequences were computed using the MSλD framework. The most stable and least stable chimeras were predicted and tested along with several other sequences, revealing a designed chimera with approximately the same stability increase as AncCcons, but requiring only half the mutations. Comparing the computed stabilities with experiment for 12 sequences reveals a Pearson correlation of 0.86 and root mean squared error of 1.18 kcal/mol, an unprecedented level of accuracy well beyond less rigorous computational design methods. We then quantified selection pressure using a simple evolutionary model in which sequences are selected according to the Boltzmann factor of their stability. Selection temperatures from 110 to 168 K are estimated in three ways by comparing experimental and computational results to evolutionary models. These estimates indicate selection pressure is high, which has implications for evolutionary dynamics and for the accuracy required for design, and suggests accurate high-throughput computational methods like MSλD may enable more effective protein design.

Origin and Evolution of Plant Long Terminal Repeat Retrotransposons with Additional Ribonuclease H.

Retroviruses originated from long terminal repeat retrotransposons (LTR-RTs) through several structural adaptations. One such modification was the arrangement of an additional ribonuclease H (aRH) domain next to native RH, followed by degradation and subfunctionalization of the latter. We previously showed that this retrovirus-like structure independently evolved in Tat LTR-RTs in flowering plants, proposing its origin from sequential rearrangements of ancestral Tat structures identified in lycophytes and conifers. However, most nonflowering plant genome assemblies were not available at that time, therefore masking the history of aRH acquisition by Tat and challenging our hypothesis. Here, we revisited Tat's evolution scenario upon the aRH acquisition by covering most of the extant plant phyla. We show that Tat evolved and obtained aRH in an ancestor of land plants. Importantly, we found the retrovirus-like structure in clubmosses, hornworts, ferns, and gymnosperms, suggesting its ancient origin, broad propagation, and yet-to-be-understood benefit for the LTR-RTs' adaptation.

Construction and characterization of ribonuclease H2 C subunit-knockout NIH3T3 cells.

Mammalian ribonuclease (RNase) H2 is a trimer consisting of catalytic A and accessory B and C subunits. RNase H2 is involved in the removal of misincorporated ribonucleotides from genomic DNA. In humans, mutations in RNase H2 gene cause a severe neuroinflammatory disorder, Aicardi-Goutières syndrome (AGS). Here, we constructed RNase H2 C subunit (RH2C)-knockout mouse fibroblast NIH3T3 cells. Compared with the wild-type NIH3T3 cells, the knockout cells exhibited a decreased single ribonucleotide-hydrolyzing activity and an increased accumulation of ribonucleotides in genomic DNA. Transient expression of wild-type RH2C in the knockout cells increased this activity and decreased this ribonucleotide accumulation. Same events were observed when RH2C variants with an AGS-causing mutation, R69W or K145I, were expressed. These results corresponded with our previous results on the RNase H2 A subunit (RH2A)-knockout NIH3T3 cells and the expression of wild-type RH2A or RH2A variants with an AGS-causing mutation, N213I and R293H, in the RH2A-knockout cells.

Advancing beyond reverse transcriptase inhibitors: The new era of hepatitis B polymerase inhibitors.

Hepatitis B virus (HBV) is a genetically diverse blood-borne virus responsible for chronic hepatitis B. The HBV polymerase plays a key role in viral genome replication within the human body and has been identified as a potential drug target for chronic hepatitis B therapeutics. However, available nucleotide reverse transcriptase inhibitors only target the reverse transcriptase domain of the HBV polymerase; they also pose resistance issues and require lifelong treatment that can burden patients financially. In this study, various chemical classes are reviewed that have been developed to target different domains of the HBV polymerase: Terminal protein, which plays a vital role in the formation of the viral DNA; Reverse transcriptase, which is responsible for the synthesis of the viral DNA from RNA, and; Ribonuclease H, which is responsible for degrading the RNA strand in the RNA-DNA duplex formed during the reverse transcription process. Host factors that interact with the HBV polymerase to achieve HBV replication are also reviewed; these host factors can be targeted by inhibitors to indirectly inhibit polymerase functionality. A detailed analysis of the scope and limitations of these inhibitors from a medicinal chemistry perspective is provided. The structure-activity relationship of these inhibitors and the factors that may affect their potency and selectivity are also examined. This analysis will be useful in supporting the further development of these inhibitors and in designing new inhibitors that can inhibit HBV replication more efficiently.

Recent advances on dual inhibitors targeting HIV reverse transcriptase associated polymerase and ribonuclease H.

Reverse transcriptase (RT) plays an indispensable role in the replication of human immunodeficiency virus (HIV) through its associated polymerase and ribonuclease H (RNase H) activities during the viral RNA genome transformation into proviral DNA. Due to the fact that HIV is a highly mutagenic virus and easily resistant to single-target RT inhibitors, dual inhibitors targeting HIV RT associated polymerase and RNase H have been developed. These dual inhibitors have the advantages of increasing efficacy, reducing drug resistance, drug-drug interactions, and cytotoxicity, as well as improving patient compliance. In this review, we summarize recent advances in polymerase/RNase H dual inhibitors focusing on drug design strategies, and structure-activity relationships and share new insights into developing anti-HIV drugs.

A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence anti-quenching.

Balancing the risks and benefits of organophosphate pesticides (OPs) on human and environmental health relies partly on their accurate measurement. A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs (triazophos, parathion, and chlorpyrifos) in apples, turnips, cabbages, and rice. Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs. DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification. The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore. The resulting fluorescence signal enables multiplexed quantification of triazophos, parathion, and chlorpyrifos residues over the concentration range of 0.01-25, 0.01-50, and 0.1-50 ng/mL with limits of detection of 0.014, 0.011, and 0.126 ng/mL, respectively. The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%, which correlate well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed bio-barcode immunoassay is stable, reproducible and reliable, and is able to detect low residual levels of multi-residue OPs in agricultural products.

A Novel, Cleaved Probe-Based Reverse Transcription Loop-Mediated Isothermal Amplification Method for Specific and Sensitive Detection of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) causes watery diarrhea, vomiting, and 30-40% mortality in newborn piglets. A simple, rapid, and sensitive method for PDCoV detection is valuable in its surveillance and control. Here, we developed a novel, cleaved probe-based reverse transcription loop-mediated isothermal amplification (CP-RT-LAMP) method for PDCoV detection. A cleaved probe with a ribonucleotide insertion that targeted the N gene of PDCoV was designed. During the reaction, the enzyme ribonuclease H2 is activated only when the cleaved probe is perfectly complementary to the template, leading to the hydrolytic release of a quencher moiety and signal output. This method can be easily used on a real-time fluorescence quantitative equipment or an on-site isothermal instrument combined with a smartphone. The specificity assay showed no cross-reactivity with other porcine enteric pathogens. This method had a detection limit of 25 copies/µL, suggesting comparable sensitivity with reverse transcription quantitative PCR (RT-qPCR). In detecting 100 clinical samples (48 fecal swab specimens and 52 intestinal specimens), the detection rate of the CP-RT-LAMP method (26%) was higher than that of RT-qPCR (17%). Thus, it is a highly specific and sensitive diagnostic method for PDCoV, with a great application potential for monitoring PDCoV in the laboratory or point-of-care testing in the field.

Case Report: Rare Homozygous RNASEH1 Mutations Associated With Adult-Onset Mitochondrial Encephalomyopathy and Multiple Mitochondrial DNA Deletions.

Mitochondrial DNA (mtDNA) maintenance disorders embrace a broad range of clinical syndromes distinguished by the evidence of mtDNA depletion and/or deletions in affected tissues. Among the nuclear genes associated with mtDNA maintenance disorders, RNASEH1 mutations produce a homogeneous phenotype, with progressive external ophthalmoplegia (PEO), ptosis, limb weakness, cerebellar ataxia, and dysphagia. The encoded enzyme, ribonuclease H1, is involved in mtDNA replication, whose impairment leads to an increase in replication intermediates resulting from mtDNA replication slowdown. Here, we describe two unrelated Italian probands (Patient 1 and Patient 2) affected by chronic PEO, ptosis, and muscle weakness. Cerebellar features and severe dysphagia requiring enteral feeding were observed in one patient. In both cases, muscle biopsy revealed diffuse mitochondrial abnormalities and multiple mtDNA deletions. A targeted next-generation sequencing analysis revealed the homozygous RNASEH1 mutations c.129-3C>G and c.424G>A in patients 1 and 2, respectively. The c.129-3C>G substitution has never been described as disease-related and resulted in the loss of exon 2 in Patient 1 muscle RNASEH1 transcript. Overall, we recommend implementing the use of high-throughput sequencing approaches in the clinical setting to reach genetic diagnosis in case of suspected presentations with impaired mtDNA homeostasis.

Alpha-hydroxytropolones are noncompetitive inhibitors of human RNase H1 that bind to the active site and modulate substrate binding.

The ribonucleases H (RNases H) of HIV and hepatitis B virus are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. Eukaryotic ribonuclease H1 (RNase H1) is an essential protein and a probable off-target enzyme for viral RNase H inhibitors. α-hydroxytropolones (αHTs) are a class of anti-RNase H inhibitors that can inhibit the HIV, hepatitis B virus, and human RNases H1; however, it is unclear how these inhibitors could be developed to distinguish between these enzymes. To accelerate the development of selective RNase H inhibitors, we performed biochemical and kinetic studies on the human enzyme, which was recombinantly expressed in Escherichia coli. Size-exclusion chromatography showed that free RNase H1 is monomeric and forms a 2:1 complex with a substrate of 12 bp. FRET heteroduplex cleavage assays were used to test inhibition of RNase H1 in steady-state kinetics by two structurally diverse αHTs, 110 and 404. We determined that turnover rate was reduced, but inhibition was not competitive with substrate, despite inhibitor binding to the active site. Given the compounds' reversible binding to the active site, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which, by binding to the active site, αHTs stabilize an inactive enzyme-substrate-inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid the development of RNase H inhibitors selective for the viral enzymes.

A novel fluorescence amplification strategy combining cascade primer exchange reaction with CRISPR/Cas12a system for ultrasensitive detection of RNase H activity.

Ribonuclease H (RNase H), which plays a vital role in various cellular processes, is to be closely related to the emergence of many diseases. As an essential therapeutic target, it shows great prospects in the development of associated drugs. Herein, a DNA-RNA chimeric hairpin (DR HP) was designed to introduce a new signal amplification strategy based on cascade primer exchange reaction (cPER) and CRISPR/Cas12a system for sensitive and specific analysis of RNase H activity. In the presence of RNase H, the RNA fragment of DR HP was specifically degraded and the blocked primer DNA was released. The process of enzymatic hydrolysis of substrate hairpin and cyclic signal amplification was completed in a one-step method under isothermal conditions, enriching many activator strands to initiate trans-cleavage of CRISPR/Cas system, thereby restoring the fluorescence signal. Under optimized conditions, the developed strategy exhibited a good linear relationship ranging from 0.005 to 0.1U/mL and offered a detection limit of 0.00061U/mL. Moreover, this method was used for RNase H activity assay in complicated human serum and real cell lysates with good stability and repeatability, and was also demonstrated to apply for RNase H inhibitors screening and inhibitory capability assessment. Therefore, the proposed system is a promising platform not only for determination of RNase H activity, but open up new thoughts for the biological enzyme research and inhibitor screening.

Recent Advances in Computer-aided Antiviral Drug Design Targeting HIV-1 Integrase and Reverse Transcriptase Associated Ribonuclease H.

Acquired immunodeficiency syndrome (AIDS) has been a chronic, life-threatening disease for a long time. Though, a broad range of antiretroviral drug regimens is applicable for the successful suppression of virus replication in human immunodeficiency virus type 1 (HIV-1) infected people. The mutation-induced drug resistance problems during the treatment of AIDS forced people to continuously look for new antiviral agents. HIV-1 integrase (IN) and reverse transcriptase associated ribonuclease (RT-RNase H), two pivotal enzymes in HIV-1 replication progress, have gained popularity as druggable targets for designing novel HIV-1 antiviral drugs. During the development of HIV-1 IN and/or RT-RNase H inhibitors, computer-aided drug design (CADD), including homology modeling, pharmacophore, docking, molecular dynamics (MD) simulation and binding free energy calculation, represent a significant tool to accelerate the discovery of new drug candidates and reduce costs in antiviral drug development. In this review, we summarized the recent advances in the design of single- and dual-target inhibitors against HIV-1 IN or/and RT-RNase H as well as the prediction of mutation-induced drug resistance based on computational methods. We highlighted the results of the reported literatures and proposed some perspectives on the design of novel and more effective antiviral drugs in the future.

The hepatitis B virus polymerase.

Hepatitis B virus (HBV) is a hepatotropic, partially double-stranded DNA virus that replicates by reverse transcription and is a major cause of chronic liver disease and hepatocellular carcinoma. Reverse transcription is catalyzed by the four-domain multifunctional HBV polymerase (P) protein that has protein-priming, RNA- and DNA-dependent DNA synthesis (i.e., reverse transcriptase), and ribonuclease H activities. P also likely promotes the three strand transfers that occur during reverse transcription, and it may participate in immune evasion by HBV. Reverse transcription is primed by a tyrosine residue in the amino-terminal domain of P, and P remains covalently attached to the product DNA throughout reverse transcription. The reverse transcriptase activity of P is the target for the nucleos(t)ide analog drugs that dominate HBV treatment, and P is the target of ongoing efforts to develop new drugs against both the reverse transcriptase and ribonuclease H activities. Despite the unusual reverse transcription pathway catalyzed by P and the importance of P to HBV therapy, understanding the enzymology and structure of HBV P severely lags that of the retroviral reverse transcriptases due to substantial technical challenges to studying the enzyme. Obtaining a better understanding of P will broaden our appreciation of the diversity among reverse transcribing elements in nature, and will help improve treatment for people chronically infected with HBV.

Retroviral RNase H: Structure, mechanism, and inhibition.

All retroviruses encode the enzyme, reverse transcriptase (RT), which is involved in the conversion of the single-stranded viral RNA genome into double-stranded DNA. RT is a multifunctional enzyme and exhibits DNA polymerase and ribonuclease H (RNH) activities, both of which are essential to the reverse-transcription process. Despite the successful development of polymerase-targeting antiviral drugs over the last three decades, no bona fide inhibitor against the RNH activity of HIV-1 RT has progressed to clinical evaluation. In this review article, we describe the retroviral RNH function and inhibition, with primary consideration of the structural aspects of inhibition.

Exploring New Scaffolds for the Dual Inhibition of HIV-1 RT Polymerase and Ribonuclease Associated Functions.

Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. Pursuing on our investigation on potential anti HIV multi-target agents we have designed and synthesized a small library of biphenylhydrazo 4-arylthiazoles derivatives and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity. In particular, compound 2-{3-[(2-{4-[4-(hydroxynitroso)phenyl]-1,3-thiazol-2-yl} hydrazin-1-ylidene) methyl]-4-methoxyphenyl} benzamide bromide (EMAC2063) was the most potent towards RNaseH (IC50 = 4.5 mM)- and RDDP (IC50 = 8.0 mM) HIV RT-associated functions.

Small-molecule Inhibitors of HIV-1 Reverse Transcriptase-Associated Ribonuclease H Function: Challenges and Recent Developments.

Multiple combinations of antiretroviral drugs have remarkably improved the treatment of HIV-1 infection. However, life-long treatments and drug resistance are still an open issue that requires continuous efforts for the identification of novel antiviral drugs. BACKGROUND: The reverse transcriptase-associated ribonuclease H (RNase H) hydrolyzes the HIV genome to allow synthesizing viral DNA. Currently, no RNase H inhibitors (RHIs) have reached the clinical phase. Therefore, RNase H can be defined as an attractive target for drug design. OBJECTIVE: Despite the wealth of information available for RNase H domain, the development of RHIs with high specificity and low cellular toxicity has been disappointing. However, it is now becoming increasingly evident that reverse transcriptase is a highly versatile enzyme, undergoing major structural alterations to complete its catalysis, and that exists a close spatial and temporal interplay between reverse transcriptase polymerase and RNase H domains. This review sums up the present challenges in targeting RNase H encompassing the challenges in selectively inhibiting RNase H vs polymerase and/or HIV-1 integrase and the weak antiviral activity of active site inhibitors, probably for a substrate barrier that impedes small molecules to reach the targeted site. Moreover, the focus is given on the most recent progress in the field of medicinal chemistry that has led to the identification of several small molecules as RHIs in the last few years. CONCLUSION: RHIs could be a new class of drugs with a novel mechanism of action highly precious for the treatment of resistant HIV strains.

Enzyme-triggered DNA nanomimosa: A ratiometric nanoprobe for RNase H activity sensing in living cells.

Since ribonuclease H (RNase H) exhibits its importance in a variety of cellular processes. It is necessary to establish strategy for RNase H detection. In this work, we are enlightened by mimosa, a natural plant which can fold in response to stimuli, to construct a DNA tetrahedron-based nanoprobe, termed DNA nanomimosa, to sensing RNase H activity based on fluorescent resonance energy transfer (FRET). The DNA nanomimosa was self-assembled from four DNA chains and one RNA chain. One of the four DNA chains contains a FRET-paired fluorophores-labeled hairpin DNA structures which is unfolded by the RNA chain through hybridization. Without RNase H, the RNA chain separate the two FRET-paired fluorophores in hairpin DNA structure, giving a feeble FRET signal. However, the presence of RNase H can selectively digest the RNA strand in RNA/unfolded-hairpin DNA section, resulting in the hairpin DNA configuration changed from "unfolded" state to "folded" state and further turn on the FRET signal. The DNA nanomimosa can be applied to achieve the determination of RNase H activity by recording the emission intensity of donor and acceptor fluorophores. This strategy shows a low detection limit by 0.017 U/mL, good specificity, and distinct advantages like the self-delivery ability, good biocompatibility, and the capacity to minimize the effects of fluctuations. This design provides a potential application in ribonuclease research and could be expanded for other biomedical research and clinical diagnostics.

Current state of diagnostic, screening and surveillance testing methods for COVID-19 from an analytical chemistry point of view.

Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.

A mid-throughput HBV replication inhibition assay capable of detecting ribonuclease H inhibitors.

The hepatitis B virus (HBV) ribonuclease H (RNaseH) is a promising but unexploited drug target. Inhibiting the RNaseH blocks viral reverse transcription by truncating the minus-polarity DNA strand, causing accumulation of RNA:DNA heteroduplexes, and abrogating plus-polarity DNA synthesis. Screening for RNaseH inhibitors is complicated by the presence of the minus-polarity DNA strand even when replication is fully inhibited because this residual DNA can be detected by standard screening assays that measure reduction in total HBV DNA accumulation. We previously developed a strand-preferential qPCR assay that detects RNaseH replication inhibitors by measuring preferential suppression of the viral plus-polarity DNA strand. However, this assay employed cells grown in 6- or 12-well plates and hence was of very low throughput. Here, we adapted the assay to a 96-well format and conducted a proof-of-principle screen of 727 compounds. The newly developed assay is a valuable tool for anti-HBV drug discovery, particularly when screening for RNaseH inhibitors.