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SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in noncycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analog drugs important for treating a variety of cancers, including acute myeloid leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We found that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogs tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regard to nucleotide analogs, which can be used to improve current cancer and antiviral therapies.

The functions of RNA pseudoknots (PKs), which are minimal tertiary structural motifs and an integral part of several ribozymes and ribonucleoprotein complexes, are determined by their structure, stability, and dynamics. Therefore, it is important to elucidate the general principles governing their thermodynamics/folding mechanisms. Here, we combine laser temperature-jump experiments and coarse-grained simulations to determine the folding/unfolding pathways of VPK, a variant of the mouse mammary tumor virus (MMTV) PK involved in ribosomal frameshifting. Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at different stem/loop positions in the PK serve as local probes allowing us to monitor the order of assembly of VPK that has two constituent hairpins with different intrinsic stabilities. We show that at 50 mM KCl, the dominant folding pathway populates only the more stable hairpin intermediate; as the salt concentration is increased, a parallel folding pathway emerges involving the less stable hairpin as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. Our findings support the principle that the order of PK structure formation is determined by the relative stabilities of the hairpins, which can be altered by sequence variations or salt concentrations. The experimental results of salt effects on the partitioning between the two folding pathways are in remarkable agreement with simulations that were performed with no adjustable parameters. Our study not only unambiguously demonstrates that VPK folds by parallel pathways but also showcases the power of combining experiments and simulations for a more enriched description of RNA self-assembly.

Flaviviruses are arthropod-borne RNA viruses associated with significant human diseases globally. There are no effective direct-acting antivirals approved to treat these viral infections. Given its critical role in viral replication, the RNA-dependent RNA polymerase (RdRp) is a logical target for antiviral drug development. Remdesivir (formerly GS-5734), a 1′-cyano modified C-adenosine monophosphate prodrug, was the first US Food and Drug Administration (FDA) approved antiviral for coronavirus disease 2019 (COVID-19) and was also shown to inhibit flavivirus replication. GS-7682, a 4′-cyano modified C-adenosine prodrug, exhibits a broad-spectrum antiviral activity. Here, we determined the anti-flavivirus potency of both remdesivir and GS-7682 and characterized their active triphosphate forms, GS-443902 and GS-646939, respectively, against a panel of purified flavivirus RdRps. These include dengue, Japanese encephalitis, West Nile, yellow fever, and Zika. Enzyme kinetics demonstrate efficient RNA incorporation of GS-443902 and GS-646939. GS-646939 acts as an immediate chain terminator. Conversely, GS-443902 acts through a template-dependent inhibition mechanism by impeding the incorporation of the complementary UTP. Both mechanisms correlate with anti-flavivirus activity, although remdesivir is generally superior. The data demonstrate that immediate chain termination is not necessarily a preferred mechanism of action of nucleotide analogs. Template-dependent inhibition should also be considered, especially for viruses lacking intrinsic proofreading activities.

Background Hepatitis B virus (HBV) reactivation is one of the most common precipitating events associated with acute decompensation (AD) or acute-on-chronic liver failure (ACLF) in chronic hepatitis B (CHB)-related cirrhotic patients. However, whether their serum HBV deoxyribonucleic acid (DNA) levels are associated with ACLF incidence and short-term mortality rate is still ambiguous. Methods The ACLF incidences, 28-day and 90-day liver transplantation (LT)-free mortality rates, previous nucleoside/nucleotide analogues (NUCs) treatments and serum HBV DNA levels at admission (ad-levels) of 111 hospitalized patients with AD of CHB-related cirrhosis were analyzed. Results 43 (38.7%) patients developed ACLF. The 28-day and 90-day LT-free mortality rates of the ACLF cases were 15.4 and 40.9%, respectively. Though NUCs inhibited HBV replication effectively, there were no differences in the ACLF incidence between antiviral treatment-naïve patients and NUCs treatment-experienced patients with or without interruptions (37.5, 41.7 and 45.5%, respectively, P >0.05). The serum HBV DNA ad-level was similar between the patients with and without ACLF development (logarithms: 4.50 ± 1.96 vs 4.32 ± 1.99; ≥2000 IU/ml: 67.4% vs 67.6%; both P >0.05), so was between the ACLF patients died or survived in 28 or 90 days (logarithms: 4.31 ± 1.91 vs 5.54 ± 2.53, 4.81 ± 1.76 vs 4.84 ± 2.40, respectively, both P >0.05). Conclusion Serum HBV DNA ad-level and previous NUCs treatment are not associated with incidence of ACLF and short-term mortality rate in the hospitalized patients with AD of CHB-related cirrhosis.

Aptamers are short single-strand oligonucleotides that can form secondary and tertiary structures, fitting targets with high affinity and specificity. They are so-called “chemical antibodies” and can target specific biomarkers in both diagnostic and therapeutic applications. Systematic evolution of ligands by exponential enrichment (SELEX) is usually used for the enrichment and selection of aptamers, and the targets could be metal ions, small molecules, nucleotides, proteins, cells, or even tissues or organs. Due to the high specificity and distinctive binding affinity of aptamers, aptamer–drug conjugates (ApDCs) have demonstrated their potential role in drug delivery for cancer-targeting therapies. Compared with antibodies which are produced by a cell-based bioreactor, aptamers are chemically synthesized molecules that can be easily conjugated to drugs and modified; however, the conventional ApDCs conjugate the aptamer with an active drug using a linker which may add more concerns to the stability of the ApDC, the drug-releasing efficiency, and the drug-loading capacity. The function of aptamer in conventional ApDC is just as a targeting moiety which could not fully perform the advantages of aptamers. To address these drawbacks, scientists have started using active nucleotide analogs as the cargoes of ApDCs, such as clofarabine, ara-guanosine, gemcitabine, and floxuridine, to replace all or part of the natural nucleotides in aptamer sequences. In turn, these new types of ApDCs, aptamer nucleotide analog drug conjugates, show the strength for targeting efficacy but avoid the complex drug linker designation and improve the synthetic efficiency. More importantly, these classic nucleotide analog drugs have been used for many years, and aptamer nucleotide analog drug conjugates would not increase any unknown druggability risk but improve the target tumor accumulation. In this review, we mainly summarized aptamer-conjugated nucleotide analog drugs in cancer-targeting therapies.

Tissue-nonspecific alkaline phosphatase (TNAP) is known to be involved in the degradation of extracellular ATP via the hydrolysis of pyrophosphate (PPi). We investigated, using three different computational methods, namely molecular docking, thermodynamic integration (TI) and conventional molecular dynamics (MD), whether TNAP may also be involved in the utilization of β,γ-modified ATP analogues. For that, we analyzed the interaction of bisphosphonates with this enzyme and evaluated the obtained structures using in silico studies. Complexes formed between pyrophosphate, hypophosphate, imidodiphosphate, methylenediphosphonic acid monothiopyrophosphate, alendronate, pamidronate and zoledronate with TNAP were generated and analyzed based on ligand docking, molecular dynamics and thermodynamic integration. The obtained results indicate that all selected ligands show high affinity toward this enzyme. The forming complexes are stabilized through hydrogen bonds, electrostatic interactions and van der Waals forces. Short- and middle-term molecular dynamics simulations yielded very similar affinity results and confirmed the stability of the protein and its complexes. The results suggest that certain effectors may have a significant impact on the enzyme, changing its properties.

With the recent global spread of new SARS-CoV-2 variants, there remains an urgent need to develop effective and variant-resistant oral drugs. Recently, we reported in vitro results validating the use of combination drugs targeting both the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and proofreading exonuclease (ExoN) as potential COVID-19 therapeutics. For the nucleotide analogues to be efficient SARS-CoV-2 inhibitors, two properties are required: efficient incorporation by RdRp and substantial resistance to excision by ExoN. Here, we have selected and evaluated nucleotide analogues with a variety of structural features for resistance to ExoN removal when they are attached at the 3′ RNA terminus. We found that dideoxynucleotides and other nucleotides lacking both 2′- and 3′-OH groups were most resistant to ExoN excision, whereas those possessing both 2′- and 3′-OH groups were efficiently removed. We also found that the 3′-OH group in the nucleotide analogues was more critical than the 2′-OH for excision by ExoN. Since the functionally important sequences in Nsp14/10 are highly conserved among all SARS-CoV-2 variants, these identified structural features of nucleotide analogues offer invaluable insights for designing effective RdRp inhibitors that can be simultaneously efficiently incorporated by the RdRp and substantially resist ExoN excision. Such newly developed RdRp terminators would be good candidates to evaluate their ability to inhibit SARS-CoV-2 in cell culture and animal models, perhaps combined with additional exonuclease inhibitors to increase their overall effectiveness.

Background There are inadequate data and no histological evidence regarding the effects of antiviral treatment for hepatitis B e-antigen (HBeAg)-negative chronic hepatitis B (CHB) patients with normal or mildly elevated alanine aminotransferase (ALT). This study investigated the effects of antiviral treatment on these patients. Methods We retrospectively analysed the outcomes of antiviral treatment for HBeAg-negative CHB patients with normal or mildly elevated ALT who were treated with nucleoside/nucleotide analogues (NAs) for up to 96 weeks. Results A total of 128 patients were enrolled; 74 patients had normal ALT and 54 patients had mildly elevated ALT. The total cumulative rates of viral suppression were 64.06%, 81.97%, and 96.39%, at weeks 24, 48, and 96, respectively. The cumulative rates of viral suppression for the normal and mildly elevated ALT groups were 67.85% and 58.97%, 86.39% and 76.31%, and 93.13% and 97.04% at weeks 24, 48, and 96, respectively. The serum HBV DNA levels at week 12 and hepatitis B surface antigen (HBsAg) levels at week 24 were significant predictors of the 96-week virological response. Of the 128 patients, 54 with normal ALT and 33 with mildly elevated ALT underwent FibroScan at baseline. Significant fibrosis (F ≥ 2) was found in 44.4% (n = 24) and 51.5% (n = 17) of the patients in the normal ALT group and mildly elevated ALT group, respectively. Compared with the values at baseline, liver stiffness values significantly decreased at week 48 (8.12 kPa vs. 6.57 kPa; p  < 0.001) and week 96 (8.87 kPa vs. 6.43 kPa; p  < 0.001), respectively. Conclusions HBeAg-negative CHB patients with normal ALT could benefit from antiviral therapy with NAs, similar to patients with mildly elevated ALT. Antiviral treatment is strongly recommended for HBeAg-negative CHB patients with normal ALT. Additionally, significant liver fibrosis is not rare in HBeAg-negative CHB patients with ALT less than two-times the upper limit of normal, and FibroScan should be performed regularly for these patients.

No information is available regarding the influence of besifovir (BSV), a new nucleotide analogue, on the occurrence of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). This study evaluated the reduced risk of HCC in patients undergoing BSV treatment. A total of 188 patients with CHB were treated with BSV for up to 8 years. We prospectively assessed the incidence of HCC compared with the risk from prediction models. During the follow-up, 5 patients developed HCC: 1 of 139 patients with non-cirrhotic CHB, and 4 of 49 patients with liver cirrhosis. We compared the HCC incidence in non-cirrhotic and cirrhotic patients with the predicted number derived from the REACH-B (risk estimation for HCC in CHB) model and GAG-HCC (guide with age, gender, HBV DNA, core promotor mutation, and cirrhosis) model, respectively. The standardized incidence ratio (SIR) was 0.128 (p = 0.039) at 7 years in non-cirrhotic CHB patients, and the SIR was 0.371 (p = 0.047) at 7.5 years in cirrhotic patients, suggesting a significantly decreased HCC incidence in both groups. HCC prediction was available for BSV-treated patients using existing models. In conclusion, BSV decreased the risk of HCC in patients with CHB, and prediction models were applicable. Clinical trial registry website and trial number: ClinicalTrials.gov no: NCT01937806.

A cohort of patients with severe Covid-19 received treatment with remdesivir under a compassionate-use protocol. Improvement in oxygen-support status was observed in 68% of patients, and overall mortality was 13% over a median follow-up of 18 days.