Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation and ssDNA binding kinetics

Orris, Benjamin; Huynh, Kevin W.; Ammirati, Mark; Han, Seungil; Bolaños, Ben; Carmody, Jason; Petroski, Matthew D.; Bosbach, Benedikt; Shields, David J.; Stivers, James T.

doi:10.1093/nar/gkac573

Cited by 8 publications

(22 citation statements)

References 46 publications

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“…3b-c ) led to a concentration-dependent transition away from the Tm 1 at 40-45°C to Tm 2 at 57-60°C, with the latter being the only observed species at 5 mM GTP. Despite variations in the content of folded apo-protein across individual experiments, the observed Tm’s are in good agreement with the known equilibrium between mono- and dimeric species for recombinant SAMHD1 61 , and with recently published thermal shift data for SAMHD1 by Orris et al 62 . While the latter study only quotes the first transition between 44-46°C, the figures clearly show two transitions for SAMHD1 in the absence of ligands, which we interpret as representing monomeric (Tm 1 ) and dimeric (Tm 2 ) species, respectively.…”

Section: Resultssupporting

confidence: 89%

“…3d-e, dGTPαS concentration-dependently stabilised SAMHD1 beyond the first two transitions and elevated its melting temperature to a third Tm3 close to 70°C, which likely represents the tetrameric species. The observation of such significant stabilisation is also in agreement with the study by Orris et al 62 , although they used elevated dGTPαS concentrations at 2 mM and observed a higher Tm3 of 74-76°C across different SAMHD1 forms. Our data clearly shows how dGTPαS is a more potent stabiliser of SAMHD1, as the first (monomer) transition is largely lost already at 0.25 mM concentration.…”

Section: Th6342 and Analogues Deterred Recombinant Samhd1 Oligomerisa...supporting

confidence: 92%

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Identification and evaluation of small-molecule inhibitors against the dNTPase SAMHD1viaa comprehensive screening funnel

Zhang

Paulin

Michel

et al. 2023

Preprint

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Sterile alpha motif and histidine-aspartic acid domain containing protein-1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase central to cellular nucleotide pool homeostasis. Recent literature has also demonstrated how SAMHD1 can detoxify chemotherapy metabolites thereby controlling their clinical responses. To further understand SAMHD1 biology and to investigate the potential of targeting this enzyme as a neoadjuvant to existing chemotherapies we set out to discover selective small molecule-based inhibitors of SAMHD1. Here we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for further characterisation of the screen output. The identified hit compound TH6342 and its analogues, accompanied by their inactive negative control analogue TH7126, demonstrated specific, low μM potency in inhibiting the hydrolysis of both natural substrates and nucleotide analogue therapeutics, shown using complementary enzyme-coupled and direct enzymatic activity assays. Their mode of inhibition was subsequently detailed by coupling kinetic studies with thermal shift assays, where TH6342 and analogues were shown to engage with pre-tetrameric SAMHD1 and deter the oligomerisation and allosteric activation of SAMHD1 without occupying nucleotide binding pockets. We further outline the development and application of multiple cellular assays for assessing cellular target engagement and associated functional effects, including CETSA and an in-cell dNTP hydrolase activity assay, which highlighted future optimisation strategies of this chemotype. In summary, with a novel mode of inhibition, TH6342 and analogues broaden the set of tool compounds available in deciphering SAMHD1 enzymology and functions, and furthermore, the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development.

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Section: Resultssupporting

confidence: 89%

Section: Th6342 and Analogues Deterred Recombinant Samhd1 Oligomerisa...supporting

confidence: 92%

Identification and evaluation of small-molecule inhibitors against the dNTPase SAMHD1viaa comprehensive screening funnel

Zhang

Paulin

Michel

et al. 2023

Preprint

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“…Previously, we suggested that high dNTP levels found in cycling cells promote tetramerisation and catalytic activity of an unstable phospho-T592 SAMHD1 [ 5 ] and proposed this as an explanation for why phosphorylated SAMHD1 was still active in vitro. Although there are many conflicting reports, similar observations were reported by Bhattacharya et al [ 34 ], and more recently by Orris et al [ 57 ], where they found that phosphomimetic mutations affect the kinetics of tetramer assembly and disassembly, without affecting tetramerization equilibrium and overall triphosphohydrolase activity. Here, using our panel of T592 stable and unstable tetramer mutants (Fig.…”

Section: Discussionsupporting

confidence: 70%

Attenuation of reverse transcriptase facilitates SAMHD1 restriction of HIV-1 in cycling cells

et al. 2023

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Background SAMHD1 is a deoxynucleotide triphosphohydrolase that restricts replication of HIV-1 in differentiated leucocytes. HIV-1 is not restricted in cycling cells and it has been proposed that this is due to phosphorylation of SAMHD1 at T592 in these cells inactivating the enzymatic activity. To distinguish between theories for how SAMHD1 restricts HIV-1 in differentiated but not cycling cells, we analysed the effects of substitutions at T592 on restriction and dNTP levels in both cycling and differentiated cells as well as tetramer stability and enzymatic activity in vitro. Results We first showed that HIV-1 restriction was not due to SAMHD1 nuclease activity. We then characterised a panel of SAMHD1 T592 mutants and divided them into three classes. We found that a subset of mutants lost their ability to restrict HIV-1 in differentiated cells which generally corresponded with a decrease in triphosphohydrolase activity and/or tetramer stability in vitro. Interestingly, no T592 mutants were able to restrict WT HIV-1 in cycling cells, despite not being regulated by phosphorylation and retaining their ability to hydrolyse dNTPs. Lowering dNTP levels by addition of hydroxyurea did not give rise to restriction. Compellingly however, HIV-1 RT mutants with reduced affinity for dNTPs were significantly restricted by wild-type and T592 mutant SAMHD1 in both cycling U937 cells and Jurkat T-cells. Restriction correlated with reverse transcription levels. Conclusions Altogether, we found that the amino acid at residue 592 has a strong effect on tetramer formation and, although this is not a simple “on/off” switch, this does correlate with the ability of SAMHD1 to restrict HIV-1 replication in differentiated cells. However, preventing phosphorylation of SAMHD1 and/or lowering dNTP levels by adding hydroxyurea was not enough to restore restriction in cycling cells. Nonetheless, lowering the affinity of HIV-1 RT for dNTPs, showed that restriction is mediated by dNTP levels and we were able to observe for the first time that SAMHD1 is active and capable of inhibiting HIV-1 replication in cycling cells, if the affinity of RT for dNTPs is reduced. This suggests that the very high affinity of HIV-1 RT for dNTPs prevents HIV-1 restriction by SAMHD1 in cycling cells.

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“…Inhibition of ssDNA and ssRNA binding also likely arises from 5a blocking the A1 site that is used to bind to guanine nucleotides within nucleic acid sequences. 23,37 ■ CONCLUSIONS Although several unsuccessful library screens have been reported with SAMHD1, 31,46,47 and a nucleotide-based inhibitor with a methylene bridge connecting the α-phosphate and 5′-carbon has been described, 35 a general strategy with the potential for discovery of biologically active inhibitors is needed. In this regard, we have described an efficient strategy that has led to the first bifunctional small molecule ligand for SAMHD1 that inhibits both its dNTPase and nucleic acid binding activities.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Deoxyguanosine-Linked Bifunctional Inhibitor of SAMHD1 dNTPase Activity and Nucleic Acid Binding

et al. 2023

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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 (dGpC3NH2) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC3NHCO-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.

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Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation and ssDNA binding kinetics

Cited by 8 publications

References 46 publications

Identification and evaluation of small-molecule inhibitors against the dNTPase SAMHD1viaa comprehensive screening funnel

Identification and evaluation of small-molecule inhibitors against the dNTPase SAMHD1viaa comprehensive screening funnel

Attenuation of reverse transcriptase facilitates SAMHD1 restriction of HIV-1 in cycling cells

Deoxyguanosine-Linked Bifunctional Inhibitor of SAMHD1 dNTPase Activity and Nucleic Acid Binding

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