Structure-Based Drug Design of ADRA2A Antagonists Derived from Yohimbine.

Yohimbine, a natural indole alkaloid and a nonselective adrenoceptor antagonist, possesses potential benefits in treating inflammatory disorders and sepsis. Nevertheless, its broader clinical use faces challenges due to its low receptor selectivity. A structure-activity relationship study of novel yohimbine analogues identified amino esters of yohimbic acid as potent and selective ADRA2A antagonists. Specifically, amino ester 4n, in comparison to yohimbine, showed a 6-fold higher ADRA1A/ADRA2A selectivity index (SI > 556 for 4n) and a 25-fold higher ADRA2B/ADRA2A selectivity index. Compound 4n also demonstrated high plasma and microsomal stability, moderate-to-low membrane permeability determining its limited ability to cross the blood-brain barrier, and negligible toxicity on nontumor normal human dermal fibroblasts. Compound 4n represents an important complementary pharmacological tool to study the involvement of adrenoceptor subtypes in pathophysiologic conditions such as inflammation and sepsis and a novel candidate for further preclinical development to treat ADRA2A-mediated pathologies.

Rational Design of Highly Potent SARS-CoV-2 nsp14 Methyltransferase Inhibitors.

The search for new drugs against COVID-19 and its causative agent, SARS-CoV-2, is one of the major trends in the current medicinal chemistry. Targeting capping machinery could be one of the therapeutic concepts based on a unique mechanism of action. Viral RNA cap synthesis involves two methylation steps, the first of which is mediated by the nsp14 protein. Here, we rationally designed and synthesized a series of compounds capable of binding to both the S-adenosyl-l-methionine and the RNA-binding site of SARS-CoV-2 nsp14 N7-methyltransferase. These hybrid molecules showed excellent potency, high selectivity toward various human methyltransferases, nontoxicity, and high cell permeability. Despite the outstanding activity against the enzyme, our compounds showed poor antiviral performance in vitro. This suggests that the activity of this viral methyltransferase has no significant effect on virus transcription and replication at the cellular level. Therefore, our compounds represent unique tools to further explore the role of the SARS-CoV-2 nsp14 methyltransferase in the viral life cycle and the pathogenesis of COVID-19.

Design, Synthesis, Biological Evaluation, and Crystallographic Study of Novel Purine Nucleoside Phosphorylase Inhibitors.

Purine nucleoside phosphorylase (PNP) is a well-known molecular target with potential therapeutic applications in the treatment of T-cell malignancies and/or bacterial/parasitic infections. Here, we report the design, development of synthetic methodology, and biological evaluation of a series of 30 novel PNP inhibitors based on acyclic nucleoside phosphonates bearing a 9-deazahypoxanthine nucleobase. The strongest inhibitors exhibited IC50 values as low as 19 nM (human PNP) and 4 nM (Mycobacterium tuberculosis (Mt) PNP) and highly selective cytotoxicity toward various T-lymphoblastic cell lines with CC50 values as low as 9 nM. No cytotoxic effect was observed on other cancer cell lines (HeLa S3, HL60, HepG2) or primary PBMCs for up to 10 µM. We report the first example of the PNP inhibitor exhibiting over 60-fold selectivity for the pathogenic enzyme (MtPNP) over hPNP. The results are supported by a crystallographic study of eight enzyme-inhibitor complexes and by ADMET profiling in vitro and in vivo.

Helquat dyes targeting G-quadruplexes as a new class of anti-HIV-1 inhibitors.

The secondary structure of nucleic acids containing quartets of guanines, termed G-quadruplexes, is known to regulate the transcription of many genes. Several G-quadruplexes can be formed in the HIV-1 long terminal repeat promoter region and their stabilization results in the inhibition of HIV-1 replication. Here, we identified helquat-based compounds as a new class of anti-HIV-1 inhibitors that inhibit HIV-1 replication at the stage of reverse transcription and provirus expression. Using Taq polymerase stop and FRET melting assays, we have demonstrated their ability to stabilize G-quadruplexes in the HIV-1 long-terminal repeat sequence. Moreover, these compounds were not binding to the general G-rich region, but rather to G-quadruplex-forming regions. Finally, docking and molecular dynamics calculations indicate that the structure of the helquat core greatly affects the binding mode to the individual G-quadruplexes. Our findings can provide useful information for the further rational design of inhibitors targeting G-quadruplexes in HIV-1.

5'-Phosphonate modified oligoadenylates as potent activators of human RNase L.

The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5'-phosphorylated 2',5'-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5'-phosphate end on the activation of human RNase L, four sets of 5'-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5'-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.

Synthesis of phosphonate derivatives of 2'-deoxy-2'-fluorotetradialdose d-nucleosides and tetradialdose d-nucleosides.

Analogs of nucleosides and nucleotides represent a promising pool of potential therapeutics. This work describes a new synthetic route leading to 2'-deoxy-2'-fluorotetradialdose D-nucleoside phosphonates. Moreover, a new universal synthetic route leading to tetradialdose d-nucleosides bearing purine nucleobases is also described. All new compounds were tested as triphosphate analogs for inhibitory potency against a variety of viral polymerases. The fluorinated nucleosides were transformed to phosphoramidate prodrugs and evaluated in cell cultures against various viruses including influenza and SARS-CoV-2.

Structurally Redesigned Bioorthogonal Reagents for Mitochondria-Specific Prodrug Activation.

The development of abiotic chemical reactions that can be performed in an organelle-specific manner can provide new opportunities in drug delivery and cell and chemical biology. However, due to the complexity of the cellular environment, this remains a significant challenge. Here, we introduce structurally redesigned bioorthogonal tetrazine reagents that spontaneously accumulate in mitochondria of live mammalian cells. The attributes leading to their efficient accumulation in the organelle were optimized to include the right combination of lipophilicity and positive delocalized charge. The best performing mitochondriotropic tetrazines enable subcellular chemical release of TCO-caged compounds as we show using fluorogenic substrates and mitochondrial uncoupler niclosamide. Our work demonstrates that a shrewd redesign of common bioorthogonal reagents can lead to their transformation into organelle-specific probes, opening the possibility to activate prodrugs and manipulate biological processes at the subcellular level by using purely chemical tools.

Antiviral Activity of 7-Substituted 7-Deazapurine Ribonucleosides, Monophosphate Prodrugs, and Triphoshates against Emerging RNA Viruses.

A series of 7-deazaadenine ribonucleosides bearing alkyl, alkenyl, alkynyl, aryl, or hetaryl groups at position 7 as well as their 5'-O-triphosphates and two types of monophosphate prodrugs (phosphoramidates and S-acylthioethanol esters) were prepared and tested for antiviral activity against selected RNA viruses (Dengue, Zika, tick-borne encephalitis, West Nile, and SARS-CoV-2). The modified triphosphates inhibited the viral RNA-dependent RNA polymerases at micromolar concentrations through the incorporation of the modified nucleotide and stopping a further extension of the RNA chain. 7-Deazaadenosine nucleosides bearing ethynyl or small hetaryl groups at position 7 showed (sub)micromolar antiviral activities but significant cytotoxicity, whereas the nucleosides bearing bulkier heterocycles were still active but less toxic. Unexpectedly, the monophosphate prodrugs were similarly or less active than the corresponding nucleosides in the in vitro antiviral assays, although the bis(S-acylthioethanol) prodrug 14h was transported to the Huh7 cells and efficiently released the nucleoside monophosphate.

Kinetic Target-Guided Synthesis of Small-Molecule G-Quadruplex Stabilizers.

The formation of a G-quadruplex motif in the promoter region of the c-MYC protooncogene prevents its expression. Accordingly, G-quadruplex stabilization by a suitable ligand may be a viable approach for anticancer therapy. In our study, we used the 4-(4-methylpiperazin-1-yl)aniline molecule, previously identified as a fragment library screen hit, as a template for the SAR-guided design of a new small library of clickable fragments and subjected them to click reactions, including kinetic target-guided synthesis in the presence of a G-quadruplex forming oligonucleotide Pu24. We tested the clickable fragments and products of click reactions for their G-quadruplex stabilizing activity and determined their mode of binding to the c-MYC G-quadruplex by NMR spectroscopy. The enhanced stabilizing potency of click products in biology assays (FRET, Polymerase extension assay) matched the increased yields of in situ click reactions. In conclusion, we identified the newly synthesized click products of bis-amino derivatives of 4-(4-methylpiperazin-1-yl)aniline as potent stabilizers of c-MYC G-quadruplex, and their further evolution may lead to the development of an efficient tool for cancer treatment.

Adenosine triphosphate analogs can efficiently inhibit the Zika virus RNA-dependent RNA polymerase.

We describe the expression and purification of an active recombinant Zika virus RNA-dependent RNA polymerase (RdRp). Next, we present the development and optimization of an in vitro assay to measure its activity. We then applied the assay to selected triphosphate analogs and discovered that 2'-C-methylated nucleosides exhibit strong inhibitory activity. Surprisingly, also carbocyclic derivatives with the carbohydrate locked in a North-like conformation as well as a ribonucleotide with a South conformation exhibited strong activity. Our results suggest that the traditional 2'-C-methylated nucleosides pursued in the race for anti-HCV treatment can be superseded by brand new scaffolds in the case of the Zika virus.

The AtRAD21.1 and AtRAD21.3 Arabidopsis cohesins play a synergistic role in somatic DNA double strand break damage repair.

BACKGROUND: The RAD21 cohesin plays, besides its well-recognised role in chromatid cohesion, a role in DNA double strand break (dsb) repair. In Arabidopsis there are three RAD21 paralog genes (AtRAD21.1, AtRAD21.2 and AtRAD21.3), yet only AtRAD21.1 has been shown to be required for DNA dsb damage repair. Further investigation of the role of cohesins in DNA dsb repair was carried out and is here reported. RESULTS: We show for the first time that not only AtRAD21.1 but also AtRAD21.3 play a role in somatic DNA dsb repair. Comet data shows that the lack of either cohesins induces a similar high basal level of DNA dsb in the nuclei and a slower DNA dsb repair kinetics in both cohesin mutants. The observed AtRAD21.3 transcriptional response to DNA dsb induction reinforces further the role of this cohesin in DNA dsb repair. The importance of AtRAD21.3 in DNA dsb damage repair, after exposure to DNA dsb damage inducing agents, is notorious and recognisably evident at the phenotypical level, particularly when the AtRAD21.1 gene is also disrupted. CONCLUSIONS: Our data demonstrates that both Arabidopsis cohesin (AtRAD21.1 and AtRAD21.3) play a role in somatic DNA dsb repair. Furthermore, the phenotypical data from the atrad21.1 atrad21.3 double mutant indicates that these two cohesins function synergistically in DNA dsb repair. The implications of this data are discussed.

Genotoxin induced mutagenesis in the model plant Physcomitrella patens.

The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype.

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens.

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.

GMI1, a structural-maintenance-of-chromosomes-hinge domain-containing protein, is involved in somatic homologous recombination in Arabidopsis.

DNA double-strand breaks (DSBs) pose one of the most severe threats to genome integrity, potentially leading to cell death. After detection of a DSB, the DNA damage and repair response is initiated and the DSB is repaired by non-homologous end joining and/or homologous recombination. Many components of these processes are still unknown in Arabidopsis thaliana. In this work, we characterized γ-irradiation and mitomycin C induced 1 (GMI1), a member of the SMC-hinge domain-containing protein family. RT-PCR analysis and promoter-GUS fusion studies showed that γ-irradiation, the radio-mimetic drug bleocin, and the DNA cross-linking agent mitomycin C strongly enhance GMI1 expression particularly in meristematic tissues. The induction of GMI1 by γ-irradiation depends on the signalling kinase Ataxia telangiectasia-mutated (ATM) but not on ATM and Rad3-related (ATR). Epistasis analysis of single and double mutants demonstrated that ATM acts upstream of GMI1 while the atr gmi1-2 double mutant was more sensitive than the respective single mutants. Comet assay revealed a reduced rate of DNA double-strand break repair in gmi1 mutants during the early recovery phase after exposure to bleocin. Moreover, the rate of homologous recombination of a reporter construct was strongly reduced in gmi1 mutant plants upon exposure to bleocin or mitomycin C. GMI1 is the first member of its protein family known to be involved in DNA repair.

DNA ligase 1 deficient plants display severe growth defects and delayed repair of both DNA single and double strand breaks.

BACKGROUND: DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability. RESULTS: Knockout mutants of atlig1 are lethal. Therefore, RNAi lines with reduced levels of AtLIG1 were generated to allow the roles and importance of Arabidopsis DNA ligase 1 in DNA metabolism to be elucidated. Viable plants were fertile but displayed a severely stunted and stressed growth phenotype. Cell size was reduced in the silenced lines, whilst flow cytometry analysis revealed an increase of cells in S-phase in atlig1-RNAi lines relative to wild type plants. Comet assay analysis of isolated nuclei showed atlig1-RNAi lines displayed slower repair of single strand breaks (SSBs) and also double strand breaks (DSBs), implicating AtLIG1 in repair of both these lesions. CONCLUSION: Reduced levels of Arabidopsis DNA ligase 1 in the silenced lines are sufficient to support plant development but result in retarded growth and reduced cell size, which may reflect roles for AtLIG1 in both replication and repair. The finding that DNA ligase 1 plays an important role in DSB repair in addition to its known function in SSB repair, demonstrates the existence of a previously uncharacterised novel pathway, independent of the conserved NHEJ. These results indicate that DNA ligase 1 functions in both DNA replication and in repair of both ss and dsDNA strand breaks in higher plants.

Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance.

DNA double strand breaks (DSBs) are one of the most cytotoxic forms of DNA damage and must be repaired by recombination, predominantly via non-homologous joining of DNA ends (NHEJ) in higher eukaryotes. However, analysis of DSB repair kinetics of plant NHEJ mutants atlig4-4 and atku80 with the neutral comet assay shows that alternative DSB repair pathways are active. Surprisingly, these kinetic measurements show that DSB repair was faster in the NHEJ mutant lines than in wild-type Arabidopsis. Here we provide the first characterization of this KU-independent, rapid DSB repair pathway operating in Arabidopsis. The alternate pathway that rapidly removes the majority of DSBs present in nuclear DNA depends upon structural maintenance of chromosomes (SMC) complex proteins, namely MIM/AtRAD18 and AtRAD21.1. An absolute requirement for SMC proteins and kleisin for rapid repair of DSBs in Arabidopsis opens new insight into the mechanism of DSB removal in plants.