Halogen Bonding in Haspin-Halogenated Tubercidin Complexes: Molecular Dynamics and Quantum Chemical Calculations.

Haspin, an atypical serine/threonine protein kinase, is a potential target for cancer therapy. 5-iodotubercidin (5-iTU), an adenosine derivative, has been identified as a potent Haspin inhibitor in vitro. In this paper, quantum chemical calculations and molecular dynamics (MD) simulations were employed to identify and quantitatively confirm the presence of halogen bonding (XB), specifically halogen∙∙∙π (aromatic) interaction between halogenated tubercidin ligands with Haspin. Consistent with previous theoretical finding, the site specificity of the XB binding over the ortho-carbon is identified in all cases. A systematic increase of the interaction energy down Group 17, based on both quantum chemical and MD results, supports the important role of halogen bonding in this series of inhibitors. The observed trend is consistent with the experimental observation of the trend of activity within the halogenated tubercidin ligands (F < Cl < Br < I). Furthermore, non-covalent interaction (NCI) plots show that cooperative non-covalent interactions, namely, hydrogen and halogen bonds, contribute to the binding of tubercidin ligands toward Haspin. The understanding of the role of halogen bonding interaction in the ligand-protein complexes may shed light on rational design of potent ligands in the future.

Exploring the thermodynamic, kinetic and inhibitory mechanisms of 5-iTU targeting mitotic kinase haspin by integrated molecular dynamics.

As a human mitotic kinase, haspin is considered as a promising target for various diseases including cancers. However, no inhibitors targeting haspin have entered clinical trials presently. 5-iTU (5-iodotubercidin) is a useful and classical chemical probe for the investigation of haspin activity, but its inhibitory mechanism remains unclear. In this study, integrated molecular dynamics (MD) of conventional MD, extended adaptive biasing force (eABF), random acceleration MD and well-tempered metadynamics were applied to investigate the thermodynamic and kinetic features of 5-iTU and three derivatives targeting haspin. To emphasize the importance of gatekeeper Phe605, two haspin mutants (F605Y and F605T) were also built. The results showed that the binding affinity of 5-iTU and haspin was highest in all wild type (WT) systems, relying on the strong halogen aromatic π interaction between 5-iTU and gatekeeper Phe605. Gatekeeper mutations, because of damage to this interaction, led to the rearrangement of water distributions at the binding site and the decrease of 5-iTU residence times. Additionally, compared with the smaller 5-fTU, 5-iTU dissociated from WT haspin with more difficulty through distinct unbinding pathways. These findings will provide crucial guidance for the design and development of novel haspin inhibitors and the rational modification of existing inhibitors.

Impact of 3-deazapurine nucleobases on RNA properties.

Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.

Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine C-Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.

A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.

[Chemical Approaches for RNAi Drug Development].

RNA interference (RNAi) is the standard method of suppressing gene expression because of its target specificity, potency, and ability to silence the expression of virtually any gene. Using 21-mer small interfering RNA (siRNA) is the general approach for inducing RNAi, as siRNA can be easily prepared using a DNA/RNA synthesizer. Synthetic siRNA can be chemically modified to increase the potency of RNAi activity and abrogate innate immune stimulation. However, designing chemically modified siRNA requires substantial experimentation. A practical method for understanding the interaction of siRNA and RNAi-related proteins and how modifications affect RNA-protein interactions is therefore needed. Plasmid DNA (pDNA) expressing short hairpin RNA (shRNA) can also be used to induce RNAi. pDNA produces numerous shRNAs that induce RNAi with potent and longterm RNAi activity, even if only one pDNA molecule is delivered to the nucleus. However, this approach has some drawbacks with regard to its therapeutic application, such as a low pDNA transfection efficiency due to its huge molecular size and innate immune responses induced by extra genes, such as CpG motifs. To overcome these issues with RNAi inducers (siRNA and pDNA), our group developed some chemical approaches using chemically modified oligonucleotides. This article focuses on our two original approaches. The first involves the groove modification of siRNA duplexes to understand siRNA-protein interactions using 7-bromo-7-deazaadenosine and 3-bromo-3-deazaadenosine as chemical probes, while the second involves the generation of RNAi medicine using chemically modified DNA, known as an intelligent shRNA expression device (iRed).

Novel Nucleoside Analogues as Effective Antiviral Agents for Zika Virus Infections.

Previously considered a neglected flavivirus, Zika virus has recently emerged as a public health concern due to its ability to spread rapidly and cause severe neurological disorders, such as microcephaly in newborn babies from infected mothers, and Guillain-Barré syndrome in adults. Despite extensive efforts towards the identification of effective therapies, specific antivirals are still not available. As part of ongoing medicinal chemistry studies to identify new antiviral agents, we screened against Zika virus replication in vitro in a targeted internal library of small-molecule agents, comprising both nucleoside and non-nucleoside agents. Among the compounds evaluated, novel aryloxyphosphoramidate prodrugs of the nucleosides 2'-C-methyl-adenosine, 2-CMA, and 7-deaza-2'C-methyl-adenosine, 7-DMA, were found to significantly inhibit the virus-induced cytopathic effect in multiple relevant cell lines. In addition, one of these prodrugs exhibits a synergistic antiviral effect against Zika virus when applied in combination with an indirect antiviral agent, a l-dideoxy bicyclic pyrimidine nucleoside analogue, which potently inhibits vaccinia and measles viruses in vitro by targeting a host pathway. Our findings provide a solid basis for further development of an antiviral therapy for Zika virus infections, possibly exploiting a dual approach combining two different agents, one targeting the viral polymerase (direct-acting antiviral), the second targeting a host-directed autophagy mechanism.

Combining tubercidin and cordycepin scaffolds results in highly active candidates to treat late-stage sleeping sickness.

African trypanosomiasis is a disease caused by Trypanosoma brucei parasites with limited treatment options. Trypanosoma is unable to synthesize purines de novo and relies solely on their uptake and interconversion from the host, constituting purine nucleoside analogues a potential source of antitrypanosomal agents. Here we combine structural elements from known trypanocidal nucleoside analogues to develop a series of 3'-deoxy-7-deazaadenosine nucleosides, and investigate their effects against African trypanosomes. 3'-Deoxytubercidin is a highly potent trypanocide in vitro and displays curative activity in animal models of acute and CNS-stage disease, even at low doses and oral administration. Whole-genome RNAi screening reveals that the P2 nucleoside transporter and adenosine kinase are involved in the uptake and activation, respectively, of this analogue. This is confirmed by P1 and P2 transporter assays and nucleotide pool analysis. 3'-Deoxytubercidin is a promising lead to treat late-stage sleeping sickness.

A promiscuous kinase inhibitor delineates the conspicuous structural features of protein kinase CK2a1.

Protein kinase CK2a1 is a serine/threonine kinase that plays a crucial role in the growth, proliferation and survival of cells and is a well known target for tumour and glomerulonephritis therapies. Here, the crystal structure of the kinase domain of CK2a1 complexed with 5-iodotubercidin (5IOD), an ATP-mimetic inhibitor, was determined at 1.78 Šresolution. The structure shows distinct structural features and, in combination with a comparison of the crystal structures of five off-target kinases complexed with 5IOD, provides valuable information for the development of highly selective inhibitors.

Triazine-Modified 7-Deaza-2'-deoxyadenosines: Better Suited for Bioorthogonal Labeling of DNA by PCR than 2'-Deoxyuridines.

6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels-Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate and to the 5-position of 2'-deoxyuridine triphosphate. Both modified nucleotide triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA polymerase when attached to the 7-position of 7-deaza-2'-deoxyadenosine in comparison to the attachment at the 5-position of 2'-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.

Uncoupling of nucleo-cytoplasmic RNA export and localization during stress.

Eukaryotic cells contain sub-cellular compartments that are not membrane bound. Some structures are always present, such as nuclear speckles that contain RNA-binding proteins (RBPs) and poly(A)+ RNAs. Others, like cytoplasmic stress granules (SGs) that harbor mRNAs and RBPs, are induced upon stress. When we examined the formation and composition of nuclear speckles during stress induction with tubercidin, an adenosine analogue previously shown to affect nuclear speckle composition, we unexpectedly found that it also led to the formation of SGs and to the inhibition of several crucial steps of RNA metabolism in cells, thereby serving as a potent inhibitor of the gene expression pathway. Although transcription and splicing persisted under this stress, RBPs and mRNAs were mislocalized in the nucleus and cytoplasm. Specifically, lncRNA and RBP localization to nuclear speckles was disrupted, exon junction complex (EJC) recruitment to mRNA was reduced, mRNA export was obstructed, and cytoplasmic poly(A)+ RNAs localized in SGs. Furthermore, nuclear proteins that participate in mRNA export, such as nucleoporins and mRNA export adaptors, were mislocalized to SGs. This study reveals structural aspects of granule assembly in cells, and describes how the flow of RNA from the nucleus to the cytoplasm is severed under stress.

Revisiting tubercidin against kinetoplastid parasites: Aromatic substitutions at position 7 improve activity and reduce toxicity.

The nucleoside antibiotic tubercidin displays strong activity against different target organisms, but it is notoriously toxic to mammalian cells. The effects of tubercidin against T. brucei parasites inspired us to synthesize several C7 substituted analogs for in vitro evaluation in order to find suitable hit compounds. C7 Deazaadenosines substituted with electron-poor phenyl groups were found to have micromolar activity against T. brucei in vitro. Replacement of the phenyl for a pyridine ring gave compound 13, with submicromolar potency and much-attenuated cytotoxicity compared to tubercidin. The veterinary pathogen T. congolense was equally affected by 13in vitro. Transporter studies in T. b. brucei indicated that 13 is taken up efficiently by both the P1 and P2 adenosine transporters, making the occurrence of transporter-related resistance and cross-resistance with diamidine drugs such as diminazene aceturate and pentamidine as well as with melaminophenyl arsenicals unlikely. Evaluation of the in vitro metabolic stability of analog 13 indicated that this analog was significantly metabolized in mouse microsomal fractions, precluding further in vivo evaluation in mouse models of HAT.

Reversible versus irreversible inhibition modes of ERK2: a comparative analysis for ERK2 protein kinase in cancer therapy.

AIM: Irreversible covalent drug inhibition is an emerging paradigm; however, critical gaps in unraveling the efficacy of molecular determinants still persist. METHODOLOGY: We compare two ERK2 inhibitors with different binding modes. A 5-7-Oxozeaenol is selective inhibitor which irreversibly binds ERK2 by the formation of covalent bond with Cys166 while 5-iodotubercidin binds noncovalently. Result & discussion: Covalent inhibition showed greater protein stability, favorable binding energetics (irreversible inhibition binding free energy [ΔGbind] = -40.4354 kcal/mol and reversible inhibition ΔGbind = -26.2515 kcal/mol); higher correlation in residual movement and multiple van der Waals interactions as evident from residue interaction analysis. CONCLUSION: This investigation of the different inhibition modes of ERK2 would assist toward the design of more potent and highly site-specific covalent inhibitors in cancer therapy.

Family-wide analysis of aminoacyl-sulfamoyl-3-deazaadenosine analogues as inhibitors of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that precisely attach an amino acid to its cognate tRNA. This process, which is essential for protein translation, is considered a viable target for the development of novel antimicrobial agents, provided species selective inhibitors can be identified. Aminoacyl-sulfamoyl adenosines (aaSAs) are potent orthologue specific aaRS inhibitors that demonstrate nanomolar affinities in vitro but have limited uptake. Following up on our previous work on substitution of the base moiety, we evaluated the effect of the N3-position of the adenine by synthesizing the corresponding 3-deazaadenosine analogues (aaS3DAs). A typical organism has 20 different aaRS, which can be split into two distinct structural classes. We therefore coupled six different amino acids, equally targeting the two enzyme classes, via the sulfamate bridge to 3-deazaadenosine. Upon evaluation of the inhibitory potency of the obtained analogues, a clear class bias was noticed, with loss of activity for the aaS3DA analogues targeting class II enzymes when compared to the equivalent aaSA. Evaluation of the available crystallographic structures point to the presence of a conserved water molecule which could have importance for base recognition within class II enzymes, a property that can be explored in future drug design efforts.

Slowly on, Slowly off: Bisubstrate-Analogue Conjugates of 5-Iodotubercidin and Histone H3 Peptide Targeting Protein Kinase Haspin.

The atypical protein kinase haspin is a key player in mitosis by catalysing the phosphorylation of Thr3 in histone H3, and thus ensuring the normal function of the chromosomal passenger complex. Here, we report the development of bisubstrate-analogue inhibitors targeting haspin. The compounds were constructed by linking 5-iodotubercidin to the N terminus of histone H3 peptide. The new conjugates show high affinity (sub-nanomolar KD ) towards haspin as well as slow kinetics of association and dissociation (residence time of several hours). This reflects a unique binding mode and translated into improved selectivity. The latter was confirmed in a biochemical binding/displacement assay with a panel of ten protein kinases, in a thermal shift assay with off-targets of 5-iodotubercidin (adenosine kinase and the Cdc2-like kinase family) and in assay with spiked HeLa cell lysate.

Structural Basis for the KlenTaq DNA Polymerase Catalysed Incorporation of Alkene- versus Alkyne-Modified Nucleotides.

Efficient incorporation of modified nucleotides by DNA polymerases is essential for many cutting-edge biomolecular technologies. The present study compares the acceptance of either alkene- or alkyne-modified nucleotides by KlenTaq DNA polymerase and provides structural insights into how 7-deaza-adenosine and deoxyuridine with attached alkene-modifications are incorporated into the growing DNA strand. Thereby, we identified modified nucleotides that prove to be superior substrates for KlenTaq DNA polymerase compared with their natural analogues. The knowledge can be used to guide future design of functionalized nucleotide building blocks.

The 5-chlorouracil:7-deazaadenine base pair as an alternative to the dT:dA base pair.

5-Chloro-2'-deoxyuridine as a possible component of a chemically modified genome has been discussed in terms of its influence on duplex stability and DNA polymerase incorporation properties. The search for its counterpart among different deoxyadenosine analogs (7-deaza-, 8-aza- and 8-aza-7-deaza-2'-deoxyadenosines) showed that the stable duplex formation as well as the synthesis of long constructs, more than 2 kb, were successful with the 5-chloro-2'-deoxyuridine and 7-deaza-2'-deoxyadenosine combination and with Taq DNA polymerase.

Groove modification of siRNA duplexes to elucidate siRNA-protein interactions using 7-bromo-7-deazaadenosine and 3-bromo-3-deazaadenosine as chemical probes.

Elucidation of dynamic interactions between RNA and proteins is essential for understanding the biological processes regulated by RNA, such as RNA interference (RNAi). In this study, the logical chemical probes, comprising 7-bromo-7-deazaadenosine (Br7C7A) and 3-bromo-3-deazaadenosine (Br3C3A), to investigate small interfering RNA (siRNA)-RNAi related protein interactions, were developed. The bromo substituents of Br7C7A and Br3C3A are expected to be located in the major and the minor grooves, respectively, and to act as a steric hindrance in each groove when these chemical probes are incorporated into siRNAs. A comprehensive investigation using siRNAs containing these chemical probes revealed that (i) Br3C3A(s) at the 5'-end of the passenger strand enhanced their RNAi activity, and (ii) the direction of RISC assembly is determined by the interaction between Argonaute2, which is the main component of RISC, and siRNA in the minor groove near the 5'-end of the passenger strand. Utilization of these chemical probes enables the investigation of the dynamic interactions between RNA and proteins.

South (S)- and North (N)-Methanocarba-7-Deazaadenosine Analogues as Inhibitors of Human Adenosine Kinase.

Adenosine kinase (AdK) inhibitors raise endogenous adenosine levels, particularly in disease states, and have potential for treatment of seizures, neurodegeneration, and inflammation. On the basis of the South (S) ribose conformation and molecular dynamics (MD) analysis of nucleoside inhibitors bound in AdK X-ray crystallographic structures, (S)- and North (N)-methanocarba (bicyclo[3.1.0]hexane) derivatives of known inhibitors were prepared and compared as human (h) AdK inhibitors. 5'-Hydroxy (34, MRS4202 (S); 55, MRS4380 (N)) and 5'-deoxy 38a (MRS4203 (S)) analogues, containing 7- and N(6)-NH phenyl groups in 7-deazaadenine, robustly inhibited AdK activity (IC50 ∼ 100 nM), while the 5'-hydroxy derivative 30 lacking the phenyl substituents was weak. Docking in the hAdK X-ray structure and MD simulation suggested a mode of binding similar to 5'-deoxy-5-iodotubercidin and other known inhibitors. Thus, a structure-based design approach for further potency enhancement is possible. The potent AdK inhibitors in this study are ready to be further tested in animal models of epilepsy.

2-Aryl-8-aza-3-deazaadenosine analogues of 5'-O-[N-(salicyl)sulfamoyl]adenosine: Nucleoside antibiotics that block siderophore biosynthesis in Mycobacterium tuberculosis.

A series of 5'-O-[N-(salicyl)sulfamoyl]-2-aryl-8-aza-3-deazaadenosines were designed to block mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) through inhibition of the essential adenylating enzyme MbtA. The synthesis of the 2-aryl-8-aza-3-deazaadenosine nucleosides featured sequential copper-free palladium-catalyzed Sonogashira coupling of a precursor 4-cyano-5-iodo-1,2,3-triazolonucleoside with terminal alkynes and a Minakawa-Matsuda annulation reaction. These modified nucleosides were shown to inhibit MbtA with apparent Ki values ranging from 6.1 to 25nM and to inhibit Mtb growth under iron-deficient conditions with minimum inhibitory concentrations ranging from 12.5 to >50µM.

Chemical Morphing of DNA Containing Four Noncanonical Bases.

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo.