Journal: The Journal of Biological Chemistry

Article Title: Allosteric targeting with antiviral nucleotide analogs allows fine-tuning of SAMHD1 dNTPase activity

doi: 10.1016/j.jbc.2026.111214

Figure Lengend Snippet: Acyclovir-triphosphate (Ac-TP) and ganciclovir-triphosphate (Gc-TP) are allosteric site 1 (AS1) activators that differentially activate dNTP hydrolysis . A , rationale of the enzymatic assay. SAMHD1 is incubated with test compounds (100 μM, "X") and allosteric activators, substrates, or buffer (indicated by color ), allowing determination of where test compounds bind. Control compounds: GTP (AS1-activator), dGTP (AS1-/AS2-activator/substrate), and dATP (AS2-activator/substrate). B , SAMHD1 was incubated with different combinations of activators/substrates/buffer (indicated by color ) and test compounds ( left ) in the enzyme-coupled assay. SAMHD1 activity normalized to buffer + dGTP (100 μM) control. Bars indicate means of three independent experiments, where dots represent individual values of technical duplicates. Error bars indicate SD. C , SAMHD1 was incubated with varying concentrations of GTP, Ac-TP, or Gc-TP and 100 μM of the indicated dNTP in the enzyme-coupled assay. SAMHD1 activity was normalized to dATP (100 μM) + GTP (100 μM) control. Data points indicate the mean of two independent experiments; each performed with technical duplicates; error bars indicate SD. SAMHD1, SAM and HD domain–containing protein 1.

Article Snippet: Ac-TP (NU-877), Gc-TP (NU-275), dGTPαS (NU-424), mant-GTP (NU-206), and ara-CTP (NU-117) were obtained from Jena Bioscience. dATP (27-1850-04), dCTP (27-1850-04), and dTTP (27-1880-04) were obtained from GE Healthcare.

Techniques: Enzymatic Assay, Incubation, Control, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Allosteric targeting with antiviral nucleotide analogs allows fine-tuning of SAMHD1 dNTPase activity

doi: 10.1016/j.jbc.2026.111214

Figure Lengend Snippet: Acyclovir-triphosphate (Ac-TP) and ganciclovir-triphosphate (Gc-TP) bind to allosteric site 1 (AS1) and induce tetramerization . A , schematic of the AS1 competitive binding assay using the environmentally sensitive mant-GTP probe. As mant-GTP is displaced from AS1 by competitors, fluorescence signal decreases. B , SAMHD1 (2 μM) and mant-GTP (0.5 μM) were incubated with varying concentrations of AS1 competitors. Data points indicate means of three independent experiments; each performed with technical duplicates. No-compound controls for each competitor dilution series were considered as shared technical replicates (N = 6). Error bars indicate SD. p K i values ± SD were calculated from a competitive binding equation (see the section). ANOVA shows significant differences between p K i values for the three AS1 competitors ( p = 0.0007). Results of a post hoc Tukey’s multiple comparisons are indicated by asterisks , where ∗∗∗ and ∗∗ indicate adjusted p values ≤0.001 and ≤0.01, respectively. GTP versus Ac-TP: adjusted p = 0.0006; GTP versus Gc-TP: adjusted p = 0.0041; and Ac-TP versus Gc-TP: adjusted p = 0.1331. C , mass photometry quantification of SAMHD1 oligomeric states (monomer: M, dimer: D, and tetramer: T) after incubation with AS2 activator dATP (250 μM) and increasing concentrations of either GTP, Ac-TP, or Gc-TP between 0 and 1200 μM. Data shown are representative of three independent experiments. D , quantification of relative abundance of monomer, dimer, and tetramer oligomeric states as seen in C . Intensities are grouped by oligomeric state, and colors indicate different AS1 activators. Data points indicate means of three independent experiments; error bars indicate SD. mant-GTP, 2'/3′-O- N -methyl-anthraniloyl)-GTP; SAMHD1, SAM and HD domain–containing protein 1.

Article Snippet: Ac-TP (NU-877), Gc-TP (NU-275), dGTPαS (NU-424), mant-GTP (NU-206), and ara-CTP (NU-117) were obtained from Jena Bioscience. dATP (27-1850-04), dCTP (27-1850-04), and dTTP (27-1880-04) were obtained from GE Healthcare.

Techniques: Competitive Binding Assay, Fluorescence, Incubation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Allosteric targeting with antiviral nucleotide analogs allows fine-tuning of SAMHD1 dNTPase activity

doi: 10.1016/j.jbc.2026.111214

Figure Lengend Snippet: SAMHD1 is an allosterically activated dNTP hydrolase . A , schematic representation of the current model of SAMHD1 allosteric activation and oligomerization. Inactive SAMHD1 monomers dimerize upon binding of (d)GTP to allosteric site 1 (AS1). Upon binding of any dNTP to allosteric site 2 (AS2), the catalytically active homotetramer is formed. B , the guanine nucleotide analogs acyclovir-triphosphate (Ac-TP) and ganciclovir-triphosphate (Gc-TP) closely resemble SAMHD1 AS1 activator GTP. SAMHD1, SAM and HD domain–containing protein 1.

Article Snippet: Ac-TP (NU-877), Gc-TP (NU-275), dGTPαS (NU-424), mant-GTP (NU-206), and ara-CTP (NU-117) were obtained from Jena Bioscience. dATP (27-1850-04), dCTP (27-1850-04), and dTTP (27-1880-04) were obtained from GE Healthcare.

Techniques: Activation Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Allosteric targeting with antiviral nucleotide analogs allows fine-tuning of SAMHD1 dNTPase activity

doi: 10.1016/j.jbc.2026.111214

Figure Lengend Snippet: Acyclovir-triphosphate (Ac-TP) and ganciclovir-triphosphate (Gc-TP) modulate SAMHD1 activity by changing the kinetic profile . A , an overlay of five NMR spectra, showing peaks for SAMHD1 substrate and activator dATP, as well as reaction product dA. Different measurement timepoints (2, 5, 10, 15, and 20 min after reaction start) show reaction progress over time. B , Michaelis–Menten (GTP) or allosteric sigmoidal (Ac-TP/Gc-TP) equation parameters fitted to experimental data by least squares regression for the depletion of 500 μM dATP in the presence of 250 μM GTP, Ac-TP, or Gc-TP AS1 activator by SAMHD1 (1 μM). Data are shown representative of three independent experiments. NMR spectra were recorded in 30 s intervals for GTP- and Ac-TP-activated SAMHD1 and 5- or 7-min intervals for Gc-TP-activated SAMHD1. Estimated Michaelis–Menten equation parameters for GTP-activated dATP depletion (numbers in brackets indicate SD for three experimental replicates): k cat : 0.88 s -1 (±0.54 s -1 ), K M : 140 μM (±84 μM), k cat / K M : 0.006 μM -1 s -1 (±0.001 μM -1 s -1 ). Estimated allosteric sigmoidal equation parameters for Ac-TP-activated dATP depletion (numbers in brackets indicate SD for three experimental replicates): k cat : 0.99 s -1 (±0.58 s -1 ), K half : 244 μM (±46 μM), h: 2.1 (±0.2), k cat / K half : 0.004 μM -1 s -1 (±0.002 μM -1 s -1 ). Estimated allosteric sigmoidal equation parameters for Gc-TP-activated dATP depletion (numbers in brackets indicate SD for three replicates): k cat : 0.03 s -1 (±0.01 s -1 ), K half : 211 μM (±25 μM), h: 3.1 (±0.6), k cat / K half : 0.13 nM -1 s -1 (±0.08 nM -1 s -1 ). SAMHD1, SAM and HD domain–containing protein 1.

Article Snippet: Ac-TP (NU-877), Gc-TP (NU-275), dGTPαS (NU-424), mant-GTP (NU-206), and ara-CTP (NU-117) were obtained from Jena Bioscience. dATP (27-1850-04), dCTP (27-1850-04), and dTTP (27-1880-04) were obtained from GE Healthcare.

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Allosteric targeting with antiviral nucleotide analogs allows fine-tuning of SAMHD1 dNTPase activity

doi: 10.1016/j.jbc.2026.111214

Figure Lengend Snippet: Combination of GTP and acyclovir-triphosphate (Ac-TP)/ganciclovir-triphosphate (Gc-TP) allosteric activators promotes SAMHD1 activity . A , a schematic representation of the experiment flow. SAMHD1 was preincubated with varying concentrations of GTP (0–50 μM, indicated by color gradient in B – D ), followed by the simultaneous addition of varying concentrations of AS1-activators GTP ( B ), Ac-TP ( C ), or Gc-TP ( D ) (0–50 μM) and a fixed concentration of AS2-activator and substrate dATP (50 μM). B – D , dATP substrate turnover was quantified using the enzyme-coupled activity assay, and activity was normalized to GTP (50 μM, preincubation) + GTP (50 μM, added) control. Select experimental conditions are highlighted as bar charts for easier comparison. Left , data points indicate means of three independent experiments; each performed with technical duplicates; error bars indicate SD. Right , bars indicate means of three independent experiments, where dots represent individual values of technical duplicates. Error bars indicate SD. SAMHD1, SAM and HD domain–containing protein 1.

Article Snippet: Ac-TP (NU-877), Gc-TP (NU-275), dGTPαS (NU-424), mant-GTP (NU-206), and ara-CTP (NU-117) were obtained from Jena Bioscience. dATP (27-1850-04), dCTP (27-1850-04), and dTTP (27-1880-04) were obtained from GE Healthcare.

Techniques: Activity Assay, Concentration Assay, Control, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Allosteric targeting with antiviral nucleotide analogs allows fine-tuning of SAMHD1 dNTPase activity

doi: 10.1016/j.jbc.2026.111214

Figure Lengend Snippet: Antiviral nucleotide analogs fine-tune SAMHD1 dNTPase activity . A , in the presence of guanine nucleotide analogs, Ac-TP and Gc-TP, SAMHD1 forms homotetramers that are catalytically competent but display reduced catalytic activity with distinct kinetic profiles compared with those containing the endogenous activator GTP. B , due to the cooperativity between Ac-TP/Gc-TP and GTP in allosterically activating SAMHD1, we suggest the existence of mixed SAMHD1 tetramers where a mixture of GTP- and analog-bound monomers make up a homotetramer. The resulting mixed tetramers can display enzymatic activity comparable with GTP-only-activated SAMHD1. Ac-TP, acyclovir-triphosphate; Gc-TP, ganciclovir-triphosphate; SAMHD1, SAM and HD domain–containing protein 1.

Article Snippet: Ac-TP (NU-877), Gc-TP (NU-275), dGTPαS (NU-424), mant-GTP (NU-206), and ara-CTP (NU-117) were obtained from Jena Bioscience. dATP (27-1850-04), dCTP (27-1850-04), and dTTP (27-1880-04) were obtained from GE Healthcare.

Techniques: Activity Assay

Journal: Annals of Hematology

Article Title: BCOR mutations define a therapeutic vulnerability to DHODH Inhibition in acute myeloid leukemia

doi: 10.1007/s00277-026-06773-z

Figure Lengend Snippet: BCOR deficiency sensitizes cells to DHODH Inhibitors. ( A ) Heatmaps representing survival fractions of OCI-AML2, OCI-AML3 and RPE1 treated with increasing doses of TP-021 (BCOR inhibitor) and brequinar, for 6 days ( n = 3 per line). Survival fractions of OCI-AML2, OCI-AML3 and RPE1 treated with working dose of TP-021 (10 µM) compared to DMSO, with increasing doses of brequinar, for each cell line, for 6 days (two-way ANOVA, n = 3; p < 0.0001****; p < 0.001***). ( B ) Survival fractions of OCI-AML2, OCI-AML3 and RPE1; transfected with siRNA and ± treated with brequinar 1 µM (two-way ANOVA, n = 3; p < 0.0001****). ( C ) Cell proliferation assay of RPE1 transfected with siRNA, ± treated with brequinar 1 µM (two-way ANOVA, n = 2; p < 0.0001****) with ( D ) associated pictures taken at 48 h (basic analyzer – Incucyte, 10x magnification). (E) SKM-1 (BCOR-mutated AML) was treated with increasing doses of DHODH inhibitors for 6 days (two-way ANOVA, n = 3; p < 0.0001****). ( F ) OCI-AML3 BCOR_WT , OCI-AML3 BCOR_KO.03 , OCI-AML3 BCOR_KO.17 and OCI-AML3 BCOR_KO.24 were treated for 6 days with increasing doses of 5 different DHODH inhibitors (two-way ANOVA, n = 3; p < 0.0001****, p < 0.001***, p < 0.01**, p < 0.05*, n.s = non-significant)

Article Snippet: For pharmacologic studies, cells were treated with brequinar (Cayman Chemical), TP-021, leflunomide, teriflunomide, AG-636, farudodstat, sparfosic acid (MedChemExpress), or 6-azauridine (Thermo Fisher).

Techniques: Transfection, Proliferation Assay

Journal: NAR Molecular Medicine

Article Title: Inhibitory effects of molnupiravir on Crimean–Congo hemorrhagic fever virus polymerase

doi: 10.1093/narmme/ugaf041

Figure Lengend Snippet: CCHFV and LASV L protein RNA synthesis patterns succeeding a single NHC-monophosphate. ( A ) RNA primer/template that supports incorporation of single CMP or NHC-monophosphate as a C-analog at position 6. RNA synthesis products catalyzed by CCHFV ( B ) and LASV ( C ) L protein. G indicates incorporation of [α- 32 P]-GTP at position 5. A 5′- 32 P-labeled 4-nt primer (4) serves as a size marker ( m ). The numbers on the side of the gel image indicate the size of the products in nucleotides. The asterisk indicates products that are likely the result of sequence-dependent template slippage events. ( D ) Graphical representation of the incorporation of CTP and NHC-TP by CCHFV L protein when 0 µM ATP is present in the reaction mixture. ( E ) Graphical representation of the incorporation of CTP and NHC-TP by LASV L protein when 0 µM ATP is present in the reaction mixture. Here, the signal at position 6 is presented as a percentage of the total signal generated between positions 5 and 7.

Article Snippet: NHC-TP was purchased from MedChemExpress (HY-135867A), and NTPs were purchased from GE Healthcare.

Techniques: Labeling, Marker, Sequencing, Generated

Journal: NAR Molecular Medicine

Article Title: Inhibitory effects of molnupiravir on Crimean–Congo hemorrhagic fever virus polymerase

doi: 10.1093/narmme/ugaf041

Figure Lengend Snippet: AlphaFold model prediction of CCHFV RdRp domain bound to dsRNA and incorporating NHC-TP. ( A ) The molecular surface (transparent beige) of the AlphaFold-predicted full-length CCHFV L-protein; the polymerase domain, including the subdomains thumb (green), palm (orange), and fingers (blue), is highlighted and individually displayed in ( B ). The gray region constitutes an undefined region. ( C ) A model of the RdRp domain in the complex with dsRNA substrate, primer strand (light blue), and template strand (green) shows the binding of NHC-TP as a substrate at the active site. ( D ) A close-up view of the active site, showing the potential interactions of NHC-TP with surrounding protein atoms and a Mg 2+ cofactor. Arg-2280 forms a potentially conserved interaction with the incoming NTP, analogous to that observed in SARS-CoV-2 RdRp structure . Gln-2275 is located within motif F , which appears to interact with the hydroxylamine on NHC-TP.

Article Snippet: NHC-TP was purchased from MedChemExpress (HY-135867A), and NTPs were purchased from GE Healthcare.

Techniques: Binding Assay