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.

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.

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.

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.

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.

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.

Key Role of the Local Hydrophobicity in the East Patch of Plastocyanins on Their Thermal Stability and Redox Properties.

Understanding the molecular basis of the thermal stability and functionality of redox proteins has important practical applications. Here, we show a distinct thermal dependence of the spectroscopic and electrochemical properties of two plastocyanins from the thermophilic cyanobacterium Phormidium laminosum and their mesophilic counterpart from Synechocystis sp. PCC 6803, despite the similarity of their molecular structures. To explore the origin of these differences, we have mimicked the local hydrophobicity in the east patch of the thermophilic protein by replacing a valine of the mesophilic plastocyanin by isoleucine. Interestingly, the resulting mutant approaches the thermal stability, redox thermodynamics, and dynamic coupling of the flexible site motions of the thermophilic protein, indicating the existence of a close connection between the hydrophobic packing of the east patch region of plastocyanin and the functional control and stability of the oxidized and reduced forms of the protein.

Electrostatic strain and concerted motions in the transient complex between plastocyanin and cytochrome f from the cyanobacterium Phormidium laminosum.

Many fleeting macromolecular interactions, like those being involved in electron transport, are essential in biology. However, little is known about the behaviour of the partners and their dynamics within their short-lived complex. To tackle such issue, we have performed molecular dynamics simulations on an electron transfer complex formed by plastocyanin and cytochrome f from the cyanobacterium Phormidium laminosum. Besides simulations of the isolated partners, two independent trajectories of the complex were calculated, starting from the two different conformations in the NMR ensemble. The first one leads to a more stable ensemble with a shorter distance between the metal sites of the two partners. The second experiences a significant drift of the complex conformation. Analyses of the distinct calculations show that the conformation of cytochrome f is strained upon binding of its partner, and relaxes upon its release. Interestingly, the principal component analysis of the trajectories indicates that plastocyanin displays a concerted motion with the small domain of cytochrome f that can be attributed to electrostatic interactions between the two proteins.