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The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish in cryo-EM and biochemical studies that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ∼20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom ∼69 000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC 3 NH 2 ) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC 3 NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a ) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1- 5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA, and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ~20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity., Competing Interests: Conflict of interest statement M.W.S, Y.X., K.W.H, S.H., B.B., and D.S. are employees of Pfizer Inc. and may hold shares in the company.

SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), localizing the enzyme to stalled replication forks (RFs) to promote their restart. Although phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (∼15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial dynamics in the C-terminal domain (which contains T592), which could be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Previous short-hairpin RNA knockdown studies have established that depletion of human uracil DNA glycosylase (hUNG) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here, we selectively inhibit the catalytic activity of hUNG by lentiviral transduction of uracil DNA glycosylase inhibitor protein into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small-molecule targeting of hUNG. In total, 6 of 11 colorectal lines showed 6- to 70-fold increases in FdU potency upon hUNG inhibition ("responsive"). This hUNG-dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU in vitro. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates deoxyuridine triphosphate levels, was only incrementally enhanced upon hUNG inhibition (<40%), suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than deoxyuridine. The importance of FU/A and FU/G lesions in the toxicity of FdU is supported by the observation that dT supplementation completely rescued the toxic effects of U/A lesions resulting from RTX, but dT only increased the IC 50 for FdU, which forms both FU/A and FU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested in which the persistence of FU/A and FU/G base pairs in the absence of hUNG activity elicits an apoptotic DNA damage response in both responsive and nonresponsive colorectal lines. SIGNIFICANCE STATEMENT: The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here, this study shows that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme human uracil DNA glycosylase. The hUNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small-molecule hUNG inhibitor., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Many human DNA repair proteins have disordered domains at their N- or C-termini with poorly defined biological functions. We recently reported that the partially structured N-terminal domain (NTD) of human uracil DNA glycosylase 2 (hUNG2), functions to enhance DNA translocation in crowded environments and also targets the enzyme to single-stranded/double-stranded DNA junctions. To understand the structural basis for these effects we now report high-resolution heteronuclear NMR studies of the isolated NTD in the presence and absence of an inert macromolecular crowding agent (PEG8K). Compared to dilute buffer, we find that crowding reduces the degrees of freedom for the structural ensemble, increases the order of a PCNA binding motif and dramatically promotes binding of the NTD for DNA through a conformational selection mechanism. These findings shed new light on the function of this disordered domain in the context of the crowded nuclear environment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Severe urinary retention is not a common condition, but may occur following some pelvic surgeries or other medical conditions. Electrical stimulation of the bladder has been examined as a means of managing this difficult problem. We conducted preliminary investigations in cats to prove the hypothesis that pelvic-plexus (bladder-neck) stimulation would produce greater micturition response with reduced side effects, such as animal movement or discomfort, than bladder-wall stimulation with electrodes implanted higher on the bladder wall. We used model microstimulators that mimic the look and function of commercial microstimulators, but that we constructed. We instrumented four female cats during a survival surgery. Animals recovered well and studies were conducted over a 1-month period in the conscious animal and under anesthesia. We performed a variety of studies with different stimulation parameters and electrode locations to evaluate our hypothesis. In the active animal, we supplied only low currents, but two animals responded to stimulation with bladder contractions and voiding. Following anesthesia, higher stimulating currents resulted in greater bladder contractions during stimulation in two of the three animals. In two cases, pelvic-plexus (bladder-neck) stimulation induced greater micturition responses than direct bladder-wall stimulation. In conclusion, we learned from these preliminary observations that stimulation at the pelvic plexus (bladder neck) may induce a better micturition response than stimulation higher on the bladder-wall. Newly available commercial microstimulators should be further studied for the treatment of urinary retention.

Background: Ovarian cancer is a major cause of cancer deaths in women and usually presents with diffuse abdominal disease. Lymph node metastases are common, but axillary lymph nodes are rarely involved., Case: A 63-year-old woman, initially presenting with abdominal symptoms and massive ascites, underwent optimal cytoreduction followed by intravenous chemotherapy with paclitaxel and carboplatin. The patient subsequently underwent a second-look surgery revealing only microscopically positive disease and then received intraperitoneal chemotherapy with cisplatin. At a follow-up visit, she was found to have bilateral axillary lymph node enlargement. Mammography revealed no lesions in either breast. Fine needle aspiration dissection confirmed the diagnosis of metastatic, recurrent ovarian adenocarcinoma and subsequently axillary lymph node dissection was undertaken., Conclusion: Ovarian carcinoma, which usually follows typical patterns of metastatic involvement, can appear in unusual areas.

The Expected Treatment Outcome Scales was developed to gather information on clients who abuse drugs or alcohol, to assess their severity of illness, and to evaluate the effectiveness of drug treatment in nonrandomized clinical studies. The scale is based upon a multiattribute value model reflecting the opinions of an expert panel. The experts identified 25 variables, or predictors of relapse, from which 48 questions were constructed. Answers to the questions are individually scored. These scores are summed to produce an overall Expected Treatment Outcome score. This paper focuses on the development and preliminary validation of the Expected Treatment Outcome Scale. Results of our analysis show a correlation of .89 between the experts' average ratings of hypothetical clients and scores based on our scale. This finding suggests that the Expected Treatment Outcome Scale has face validity and accurately simulates the experts' judgments regarding treatment outcome. Further research is necessary to assess the reliability as well as the concurrent and predictive validity of our instrument.