Target-based drug design strategies to overcome resistance to antiviral agents: opportunities and challenges.

Viral infections have a major impact in human health. Ongoing viral transmission and escalating selective pressure have the potential to favor the emergence of vaccine- and antiviral drug-resistant viruses. Target-based approaches for the design of antiviral drugs can play a pivotal role in combating drug-resistant challenges. Drug design computational tools facilitate the discovery of novel drugs. This review provides a comprehensive overview of current drug design strategies employed in the field of antiviral drug resistance, illustrated through the description of a series of successful applications. These strategies include technologies that enhance compound-target affinity while minimizing interactions with mutated binding pockets. Furthermore, emerging approaches such as virtual screening, targeted protein/RNA degradation, and resistance analysis during drug design have been harnessed to curtail the emergence of drug resistance. Additionally, host targeting antiviral drugs offer a promising avenue for circumventing viral mutation. The widespread adoption of these refined drug design strategies will effectively address the prevailing challenge posed by antiviral drug resistance.

Emerging drug design strategies in anti-influenza drug discovery.

Influenza is an acute respiratory infection caused by influenza viruses (IFV), According to the World Health Organization (WHO), seasonal IFV epidemics result in approximately 3-5 million cases of severe illness, leading to about half a million deaths worldwide, along with severe economic losses and social burdens. Unfortunately, frequent mutations in IFV lead to a certain lag in vaccine development as well as resistance to existing antiviral drugs. Therefore, it is of great importance to develop anti-IFV drugs with high efficiency against wild-type and resistant strains, needed in the fight against current and future outbreaks caused by different IFV strains. In this review, we summarize general strategies used for the discovery and development of antiviral agents targeting multiple IFV strains (including those resistant to available drugs). Structure-based drug design, mechanism-based drug design, multivalent interaction-based drug design and drug repurposing are amongst the most relevant strategies that provide a framework for the development of antiviral drugs targeting IFV.

Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation.

Coupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT properties to improve yields and sensitivity of these applications. The effects of amino acid substitutions in the RT RNase H domain were tested in an engineered HIV-1 group O RT, containing mutations K358R/A359G/S360A and devoid of RNase H activity due to the presence of E478Q (O3MQ RT). Twenty mutant RTs with Lys or Arg at positions interacting with the template-primer (i.e., at positions 473-477, 499-502 and 505) were obtained and characterized. Most of them produced significant amounts of cDNA at 37, 50 and 65 °C, as determined in RT-PCR reactions. However, a big loss of activity was observed with mutants A477K/R, S499K/R, V502K/R and Y505K/R, particularly at 65 °C. Binding affinity experiments confirmed that residues 477, 502 and 505 were less tolerant to mutations. Amino acid substitutions Q500K and Q500R produced a slight increase of cDNA synthesis efficiency at 50 and 65 °C, without altering the KD for model DNA/DNA and RNA/DNA heteroduplexes. Interestingly, molecular dynamics simulations predicted that those mutations inactivate the RNase H activity by altering the geometry of the catalytic site. Proof of this unexpected effect was obtained after introducing Q500K or Q500R in the wild-type HIV-1BH10 RT and mutant K358R/A359G/S360A RT. Our results reveal a novel mechanism of RNase H inactivation that preserves RT DNA binding and polymerization efficiency without substituting RNase H active site residues.

Targeting hepatitis B virus cccDNA levels: Recent progress in seeking small molecule drug candidates.

Hepatitis B virus (HBV) infection is a major global health problem that puts people at high risk of death from cirrhosis and liver cancer. The presence of covalently closed circular DNA (cccDNA) in infected cells is considered to be the main obstacle to curing chronic hepatitis B. At present, the cccDNA cannot be completely eliminated by standard treatments. There is an urgent need to develop drugs or therapies that can reduce HBV cccDNA levels in infected cells. We summarize the discovery and optimization of small molecules that target cccDNA synthesis and degradation. These compounds are cccDNA synthesis inhibitors, cccDNA reducers, core protein allosteric modulators, ribonuclease H inhibitors, cccDNA transcriptional modulators, HBx inhibitors and other small molecules that reduce cccDNA levels.

Current medicinal chemistry strategies in the discovery of novel HIV-1 ribonuclease H inhibitors.

During HIV-1 genome replication, the viral reverse transcriptase-associated ribonuclease H (RT-associated RNase H) activity hydrolyzes the RNA strand of RNA/DNA heteroduplex intermediates. As of today, HIV-1 RNase H inhibitors (RHIs) remain at an investigational level, although none of them reached clinical trials. Therefore, RNase H remains as an attractive target for drug design and development. In this paper, we review the current status of medicinal chemistry strategies aimed at the discovery of novel RHIs, while discussing problems encountered in their characterization and further development, thereby providing an update on recent progress in the field.

Design, synthesis, and biological evaluation of novel double-winged galloyl derivatives as HIV-1 RNase H inhibitors.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains as the only enzyme encoded within the viral genome not clinically validated as an antiviral target. We have previously reported that the galloyl derivative II-25 had RNase H inhibitory activity in enzymatic assays but showed weak antiviral activity in phenotypic assays due its large polarity and poor membrane permeability. In this report, we report on a series of II-25 derivatives, obtained by addition of different hydrophobic moieties ("the wings") at the C-2 and C-3 positions of the piperazine ring that showed improved RNase H inhibitory activity. Six compounds showed strong inhibitory activity and were found to be more potent than ß-thujaplicinol in enzymatic assays. The most potent compound was IA-6 and exhibited the best inhibitory activity (IC50 = 0.067 ± 0.02 µM). IA-6 was around 11 and 30 times more potent than II-25 and ß-thujaplicinol, respectively. Molecular modeling studies predict a strong hydrophobic interaction between the furylmethylaminyl group of IA-6 and the side chain of His539, explaining the potent HIV-1 RNase H inhibition. Unfortunately, none of the derivatives showed significant antiviral activity in cell culture. It is worth emphasizing that most of the obtained compounds show low cytotoxicity (CC50 > 20 µM), which confirms the significance of identifying galloyl derivatives as valuable leads for further optimization.

A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing.

RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.

Lethal Mutagenesis of RNA Viruses and Approved Drugs with Antiviral Mutagenic Activity.

In RNA viruses, a small increase in their mutation rates can be sufficient to exceed their threshold of viability. Lethal mutagenesis is a therapeutic strategy based on the use of mutagens, driving viral populations to extinction. Extinction catastrophe can be experimentally induced by promutagenic nucleosides in cell culture models. The loss of HIV infectivity has been observed after passage in 5-hydroxydeoxycytidine or 5,6-dihydro-5-aza-2'-deoxycytidine while producing a two-fold increase in the viral mutation frequency. Among approved nucleoside analogs, experiments with polioviruses and other RNA viruses suggested that ribavirin can be mutagenic, although its mechanism of action is not clear. Favipiravir and molnupiravir exert an antiviral effect through lethal mutagenesis. Both drugs are broad-spectrum antiviral agents active against RNA viruses. Favipiravir incorporates into viral RNA, affecting the G→A and C→U transition rates. Molnupiravir (a prodrug of ß-d-N4-hydroxycytidine) has been recently approved for the treatment of SARS-CoV-2 infection. Its triphosphate derivative can be incorporated into viral RNA and extended by the coronavirus RNA polymerase. Incorrect base pairing and inefficient extension by the polymerase promote mutagenesis by increasing the G→A and C→U transition frequencies. Despite having remarkable antiviral action and resilience to drug resistance, carcinogenic risks and genotoxicity are important concerns limiting their extended use in antiviral therapy.

Novel RNase H Inhibitors Blocking RNA-directed Strand Displacement DNA Synthesis by HIV-1 Reverse Transcriptase.

In retroviruses, strand displacement DNA-dependent DNA polymerization catalyzed by the viral reverse transcriptase (RT) is required to synthesize double-stranded proviral DNA. In addition, strand displacement during RNA-dependent DNA synthesis is critical to generate high-quality cDNA for use in molecular biology and biotechnology. In this work, we show that the loss of RNase H activity due to inactivating mutations in HIV-1 RT (e.g. D443N or E478Q) has no significant effect on strand displacement while copying DNA templates, but has a large impact on DNA polymerization in reactions carried out with RNA templates. Similar effects were observed with ß-thujaplicinol and other RNase H active site inhibitors, including compounds with dual activity (i.e., characterized also as inhibitors of HIV-1 integrase and/or the RT DNA polymerase). Among them, dual inhibitors of HIV-1 RT DNA polymerase/RNase H activities, containing a 7-hydroxy-6-nitro-2H-chromen-2-one pharmacophore were found to be very potent and effective strand displacement inhibitors in RNA-dependent DNA polymerization reactions. These findings might be helpful in the development of transcriptomics technologies to obtain more uniform read coverages when copying long RNAs and for the construction of more representative libraries avoiding biases towards 5' and 3' ends, while providing valuable information for the development of novel antiretroviral agents.

Update and latest advances in antiretroviral therapy.

Since the first cases of AIDS appeared in 1981, human immunodeficiency virus type 1 (HIV-1) infection has reached pandemic proportions. Forty years later, research has led to the approval of more than 30 antiretroviral drugs, while combination therapies have turned HIV-1 infection into a chronic, but manageable disease. Still, drug toxicity and acquired and transmitted drug resistance remain as major threats to therapy success. In this review, we provide an overview on currently available anti-HIV drugs and the latest developments in antiretroviral therapy, focused on new antiretroviral agents acting on known and unexploited antiviral targets, prevention therapies aimed to improve available drug combinations, and research on new long-acting therapies, particularly those involving novel drug candidates such as lenacapavir or islatravir.

Discovery, optimization, and target identification of novel coumarin derivatives as HIV-1 reverse transcriptase-associated ribonuclease H inhibitors.

Despite significant advances in antiretroviral therapy, acquired immunodeficiency syndrome remains as one of the leading causes of death worldwide. New antiretroviral drugs combined with updated treatment strategies are needed to improve convenience, tolerability, safety, and antiviral efficacy of available therapies. In this work, a focused library of coumarin derivatives was exploited by cell phenotypic screening to discover novel inhibitors of HIV-1 replication. Five compounds (DW-3, DW-4, DW-11, DW-25 and DW-31) showed moderate activity against wild-type and drug-resistant strains of HIV-1 (IIIB and RES056). Four of those molecules were identified as inhibitors of the viral RT-associated RNase H. Structural modification of the most potent DW-3 and DW-4 led to the discovery of compound 8a. This molecule showed increased potency against wild-type HIV-1 strain (EC50 = 3.94 ± 0.22 µM) and retained activity against a panel of mutant strains, showing EC50 values ranging from 5.62 µM to 202 µM. In enzymatic assays, 8a was found to inhibit the viral RNase H with an IC50 of 12.3 µM. Molecular docking studies revealed that 8a could adopt a binding mode similar to that previously reported for other active site HIV-1 RNase H inhibitors.

An Update on Antiretroviral Therapy.

Human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS) still claim many lives across the world. However, research efforts during the last 40 years have led to the approval of over 30 antiretroviral drugs and the introduction of combination therapies that have turned HIV infection into a chronic but manageable disease. In this chapter, we provide an update on current available drugs and treatments, as well as future prospects towards reducing pill burden and developing long-acting drugs and novel antiretroviral therapies. In addition, we summarize efforts to cure HIV, including pharmaceutical strategies focused on the elimination of the virus.

Search, Identification, and Design of Effective Antiviral Drugs Against Pandemic Human Coronaviruses.

Recent coronavirus outbreaks of SARS-CoV-1 (2002-2003), MERS-CoV (since 2012), and SARS-CoV-2 (since the end of 2019) are examples of how viruses can damage health care and generate havoc all over the world. Coronavirus can spread quickly from person to person causing high morbidity and mortality. Unfortunately, the antiviral armamentarium is insufficient to fight these infections. In this chapter, we provide a detailed summary of the current situation in the development of drugs directed against pandemic human coronaviruses. Apart from the recently licensed remdesivir, other antiviral agents discussed in this review include molecules targeting viral components (e.g., RNA polymerase inhibitors, entry inhibitors, or protease inhibitors), compounds interfering with virus-host interactions, and drugs identified in large screening assays, effective against coronavirus replication, but with an uncertain mechanism of action.

Decoding molnupiravir-induced mutagenesis in SARS-CoV-2.

Molnupiravir, a prodrug of the nucleoside derivative ß-D-N4-hydroxycytidine (NHC), is currently in clinical trials for COVID-19 therapy. However, the biochemical mechanisms involved in molnupiravir-induced mutagenesis had not been explored. In a recent study, Gordon et al. demonstrated that NHC can be incorporated into viral RNA and subsequently extended and used as template for RNA-dependent RNA synthesis, proposing a mutagenesis model consistent with available virological evidence. Their study uncovers molecular mechanisms by which molnupiravir drives SARS-CoV-2 into error catastrophe.

Medicinal chemistry strategies for discovering antivirals effective against drug-resistant viruses.

During the last forty years we have witnessed impressive advances in the field of antiviral drug discovery culminating with the introduction of therapies able to stop human immunodeficiency virus (HIV) replication, or cure hepatitis C virus infections in people suffering from liver disease. However, there are important viral diseases without effective treatments, and the emergence of drug resistance threatens the efficacy of successful therapies used today. In this review, we discuss strategies to discover antiviral compounds specifically designed to combat drug resistance. Currently, efforts in this field are focused on targeted proteins (e.g. multi-target drug design strategies), but also on drug conformation (either improving drug positioning in the binding pocket or introducing conformational constraints), in the introduction or exploitation of new binding sites, or in strengthening interaction forces through the introduction of multiple hydrogen bonds, covalent binding, halogen bonds, additional van der Waals forces or multivalent binding. Among the new developments, proteolysis targeting chimeras (PROTACs) have emerged as a valid approach taking advantage of intracellular mechanisms involving protein degradation by the ubiquitin-proteasome system. Finally, several molecules targeting host factors (e.g. human dihydroorotate dehydrogenase and DEAD-box polypeptide 3) have been identified as broad-spectrum antiviral compounds. Implementation of herein described medicinal chemistry strategies are expected to contribute to the discovery of new drugs effective against current and future threats due to emerging and re-emerging viral pandemics.

Analysis and Molecular Determinants of HIV RNase H Cleavage Specificity at the PPT/U3 Junction.

HIV reverse transcriptases (RTs) convert viral genomic RNA into double-stranded DNA. During reverse transcription, polypurine tracts (PPTs) resilient to RNase H cleavage are used as primers for plus-strand DNA synthesis. Nonnucleoside RT inhibitors (NNRTIs) can interfere with the initiation of plus-strand DNA synthesis by enhancing PPT removal, while HIV RT connection subdomain mutations N348I and N348I/T369I mitigate this effect by altering RNase H cleavage specificity. Now, we demonstrate that among approved nonnucleoside RT inhibitors (NNRTIs), nevirapine and doravirine show the largest effects. The combination N348I/T369I in HIV-1BH10 RT has a dominant effect on the RNase H cleavage specificity at the PPT/U3 site. Biochemical studies showed that wild-type HIV-1 and HIV-2 RTs were able to process efficiently and accurately all tested HIV PPT sequences. However, the cleavage accuracy at the PPT/U3 junction shown by the HIV-2EHO RT was further improved after substituting the sequence YQEPFKNLKT of HIV-1BH10 RT (positions 342-351) for the equivalent residues of the HIV-2 enzyme (HQGDKILKV). Our results highlight the role of ß-sheets 17 and 18 and their connecting loop (residues 342-350) in the connection subdomain of the large subunit, in determining the RNase H cleavage window of HIV RTs.

Novel indolylarylsulfone derivatives as covalent HIV-1 reverse transcriptase inhibitors specifically targeting the drug-resistant mutant Y181C.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are widely used in combination therapies against HIV-1. However, emergent and transmitted drug resistance compromise their efficacy in the clinical setting. Y181C is selected in patients receiving nevirapine, etravirine and rilpivirine, and together with K103N is the most prevalent NNRTI-associated mutation in HIV-infected patients. Herein, we report on the design, synthesis and biological evaluation of a novel series of indolylarylsulfones bearing acrylamide or ethylene sulfonamide reactive groups as warheads to inactivate Cys181-containing HIV-1 RT via a Michael addition reaction. Compounds I-7 and I-9 demonstrated higher selectivity towards the Y181C mutant than against the wild-type RT, in nucleotide incorporation inhibition assays. The larger size of the NNRTI binding pocket in the mutant enzyme facilitates a better fit for the active compounds, while stacking interactions with Phe227 and Pro236 contribute to inhibitor binding. Mass spectrometry data were consistent with the covalent modification of the RT, although off-target reactivity constitutes a major limitation for further development of the described inhibitors.

Reverse Transcriptase: From Transcriptomics to Genome Editing.

Reverse transcriptases (RTs) are enzymes that can generate a complementary strand of DNA (cDNA) from RNA. Coupled with PCR, RTs have been widely used to detect RNAs and to clone expressed genes. Classical retroviral RTs have been improved by protein engineering. These enzymes and newly characterized RTs are key elements in the development of next-generation sequencing techniques that are now being applied to the study of transcriptomics. In addition, engineered RTs fused to a CRISPR/Cas9 nickase have recently shown great potential as tools to manipulate eukaryotic genomes. In this review, we discuss the properties and uses of wild type and engineered RTs in biotechnological applications, from conventional RT-PCR to recently introduced prime editing.