The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

AbstractSAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in non-cycling 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 analogue 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 find 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 analogues 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 regards to nucleotide analogues, which can be used to improve current cancer and antiviral therapies.SignificanceNucleoside analogue drugs are widely used to treat a variety of cancers and viral infections. With an essential role in regulating the nucleotide pool in the cell by degrading cellular nucleotides, SAMHD1 has the potential to decrease the cellular concentration of frequently prescribed nucleotide analogues and thereby decrease their clinical efficacy in cancer therapy. To improve future nucleotide analogue treatments, it is important to understand SAMHD1 interactions with these drugs. Our work thoroughly examines the extent to which nucleotide analogues interact with the catalytic and allosteric sites of SAMHD1. This work contributes to the assessment of SAMHD1 as a potential therapeutic target for cancer therapy and the future design of SAMHD1 modulators that might improve the efficacy of existing therapies.

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The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805593115 ◽

2018 ◽

Vol 115 (43) ◽

pp. E10022-E10031 ◽

Cited By ~ 11

Author(s):

Kirsten M. Knecht ◽

Olga Buzovetsky ◽

Constanze Schneider ◽

Dominique Thomas ◽

Vishok Srikanth ◽

...

Keyword(s):

Genome Stability ◽

Structural Basis ◽

Cancer Drug ◽

Allosteric Site ◽

Nucleotide Analogs ◽

Antiviral Therapies ◽

Allosteric Sites ◽

Catalytically Active ◽

Deoxynucleoside Triphosphate ◽

Patient Responses

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.

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Rely on Each Other: DNA Binding Cooperativity Shapes p53 Functions in Tumor Suppression and Cancer Therapy

Cancers ◽

10.3390/cancers13102422 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2422

Author(s):

Oleg Timofeev ◽

Thorsten Stiewe

Keyword(s):

Cancer Therapy ◽

Dna Binding ◽

Cell Fate ◽

Tumor Suppression ◽

Quaternary Structure ◽

Three Dimensional ◽

Structural Basis ◽

Sequence Specificity ◽

Cell Fate Decisions ◽

Cancer Mutations

p53 is a tumor suppressor that is mutated in half of all cancers. The high clinical relevance has made p53 a model transcription factor for delineating general mechanisms of transcriptional regulation. p53 forms tetramers that bind DNA in a highly cooperative manner. The DNA binding cooperativity of p53 has been studied by structural and molecular biologists as well as clinical oncologists. These experiments have revealed the structural basis for cooperative DNA binding and its impact on sequence specificity and target gene spectrum. Cooperativity was found to be critical for the control of p53-mediated cell fate decisions and tumor suppression. Importantly, an estimated number of 34,000 cancer patients per year world-wide have mutations of the amino acids mediating cooperativity, and knock-in mouse models have confirmed such mutations to be tumorigenic. While p53 cancer mutations are classically subdivided into “contact” and “structural” mutations, “cooperativity” mutations form a mechanistically distinct third class that affect the quaternary structure but leave DNA contacting residues and the three-dimensional folding of the DNA-binding domain intact. In this review we discuss the concept of DNA binding cooperativity and highlight the unique nature of cooperativity mutations and their clinical implications for cancer therapy.

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Targeting Pin1 for Modulation of Cell Motility and Cancer Therapy

Biomedicines ◽

10.3390/biomedicines9040359 ◽

2021 ◽

Vol 9 (4) ◽

pp. 359

Author(s):

Hsiang-Hao Chuang ◽

Yen-Yi Zhen ◽

Yu-Chen Tsai ◽

Cheng-Hao Chuang ◽

Ming-Shyan Huang ◽

...

Keyword(s):

Cancer Therapy ◽

Tumor Suppressors ◽

Protein Conformation ◽

Genome Stability ◽

Recent Progress ◽

Multiple Roles ◽

Cancer Development ◽

Cancer Hallmarks ◽

Underlying Mechanisms ◽

And Function

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) specifically binds and isomerizes the phosphorylated serine/threonine-proline (pSer/Thr-Pro) motif, which leads to changes in protein conformation and function. Pin1 is widely overexpressed in cancers and plays an important role in tumorigenesis. Mounting evidence has revealed that targeting Pin1 is a potential therapeutic approach for various cancers by inhibiting cell proliferation, reducing metastasis, and maintaining genome stability. In this review, we summarize the underlying mechanisms of Pin1-mediated upregulation of oncogenes and downregulation of tumor suppressors in cancer development. Furthermore, we also discuss the multiple roles of Pin1 in cancer hallmarks and examine Pin1 as a desirable pharmaceutical target for cancer therapy. We also summarize the recent progress of Pin1-targeted small-molecule compounds for anticancer activity.

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DNA–protein crosslink proteases in genome stability

Communications Biology ◽

10.1038/s42003-020-01539-3 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Annamaria Ruggiano ◽

Kristijan Ramadan

Keyword(s):

Dna Replication ◽

Cancer Therapy ◽

Genome Stability ◽

Dna Lesions ◽

Protein Component ◽

Human Diseases ◽

Direct Link ◽

The Past ◽

Protein Crosslinks ◽

Bulky Dna Lesions

AbstractProteins covalently attached to DNA, also known as DNA–protein crosslinks (DPCs), are common and bulky DNA lesions that interfere with DNA replication, repair, transcription and recombination. Research in the past several years indicates that cells possess dedicated enzymes, known as DPC proteases, which digest the protein component of a DPC. Interestingly, DPC proteases also play a role in proteolysis beside DPC repair, such as in degrading excess histones during DNA replication or controlling DNA replication checkpoints. Here, we discuss the importance of DPC proteases in DNA replication, genome stability and their direct link to human diseases and cancer therapy.

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A Self-Assembling Amphiphilic Peptide Dendrimer-Based Drug Delivery System for Cancer Therapy

Pharmaceutics ◽

10.3390/pharmaceutics13071092 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1092

Author(s):

Dandan Zhu ◽

Huanle Zhang ◽

Yuanzheng Huang ◽

Baoping Lian ◽

Chi Ma ◽

...

Keyword(s):

Breast Cancer ◽

Drug Delivery ◽

Drug Resistance ◽

Cancer Therapy ◽

Self Assembly ◽

Cancer Drug ◽

Acquired Drug Resistance ◽

Self Assembling ◽

Amphiphilic Peptide ◽

Peptide Dendrimer

Despite being a mainstay of clinical cancer treatment, chemotherapy is limited by its severe side effects and inherent or acquired drug resistance. Nanotechnology-based drug-delivery systems are widely expected to bring new hope for cancer therapy. These systems exploit the ability of nanomaterials to accumulate and deliver anticancer drugs at the tumor site via the enhanced permeability and retention effect. Here, we established a novel drug-delivery nanosystem based on amphiphilic peptide dendrimers (AmPDs) composed of a hydrophobic alkyl chain and a hydrophilic polylysine dendron with different generations (AmPD KK2 and AmPD KK2K4). These AmPDs assembled into nanoassemblies for efficient encapsulation of the anti-cancer drug doxorubicin (DOX). The AmPDs/DOX nanoformulations improved the intracellular uptake and accumulation of DOX in drug-resistant breast cancer cells and increased permeation in 3D multicellular tumor spheroids in comparison with free DOX. Thus, they exerted effective anticancer activity while circumventing drug resistance in 2D and 3D breast cancer models. Interestingly, AmPD KK2 bearing a smaller peptide dendron encapsulated DOX to form more stable nanoparticles than AmPD KK2K4 bearing a larger peptide dendron, resulting in better cellular uptake, penetration, and anti-proliferative activity. This may be because AmPD KK2 maintains a better balance between hydrophobicity and hydrophilicity to achieve optimal self-assembly, thereby facilitating more stable drug encapsulation and efficient drug release. Together, our study provides a promising perspective on the design of the safe and efficient cancer drug-delivery nanosystems based on the self-assembling amphiphilic peptide dendrimer.

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Viral hepatitis and liver cancer

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0274 ◽

2017 ◽

Vol 372 (1732) ◽

pp. 20160274 ◽

Cited By ~ 70

Author(s):

Marc Ringehan ◽

Jane A. McKeating ◽

Ulrike Protzer

Keyword(s):

Liver Cirrhosis ◽

Liver Cancer ◽

Hepatitis B ◽

Viral Infections ◽

Current Knowledge ◽

Primary Liver Cancer ◽

Oncogenic Viruses ◽

Chronic Infections ◽

Future Design ◽

End Stage

Hepatitis B and C viruses are a global health problem causing acute and chronic infections that can lead to liver cirrhosis and hepatocellular carcinoma (HCC). These infections are the leading cause for HCC worldwide and are associated with significant mortality, accounting for more than 1.3 million deaths per year. Owing to its high incidence and resistance to treatment, liver cancer is the second leading cause of cancer-related death worldwide, with HCC representing approximately 90% of all primary liver cancer cases. The majority of viral-associated HCC cases develop in subjects with liver cirrhosis; however, hepatitis B virus infection can promote HCC development without prior end-stage liver disease. Thus, understanding the role of hepatitis B and C viral infections in HCC development is essential for the future design of treatments and therapies for this cancer. In this review, we summarize the current knowledge on hepatitis B and C virus hepatocarcinogenesis and highlight direct and indirect risk factors. This article is part of the themed issue ‘Human oncogenic viruses’.

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Structural basis for subtype-specific inhibition of the P2X7 receptor

eLife ◽

10.7554/elife.22153 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 94

Author(s):

Akira Karasawa ◽

Toshimitsu Kawate

Keyword(s):

P2x7 Receptor ◽

Hydrophobic Interactions ◽

Binding Pocket ◽

Drug Binding ◽

Structural Basis ◽

Atp Binding ◽

Allosteric Site ◽

Channel Opening ◽

A Site ◽

Novel Drug

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

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Structural basis for SARM1 inhibition and activation under energetic stress

eLife ◽

10.7554/elife.62021 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Michael Sporny ◽

Julia Guez-Haddad ◽

Tami Khazma ◽

Avraham Yaron ◽

Moshe Dessau ◽

...

Keyword(s):

Cell Death ◽

Axonal Degeneration ◽

Structural Basis ◽

Allosteric Site ◽

Inactive Conformation ◽

Catalytic Domains ◽

Energetic Stress ◽

Catalytic Sites ◽

Peripheral Ring ◽

Cellular Metabolite

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1’s own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.

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Stereoselectivity of ectonucleotidases on vascular endothelial cells

Biochemical Journal ◽

10.1042/bj2140975 ◽

1983 ◽

Vol 214 (3) ◽

pp. 975-981 ◽

Cited By ~ 70

Author(s):

N J Cusack ◽

J D Pearson ◽

J L Gordon

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Thiol Group ◽

Side Chain ◽

Vascular Endothelial ◽

Nucleotide Analogues ◽

Aortic Endothelial Cells ◽

Nucleotide Analogue ◽

High Degree ◽

Aortic Endothelial

We have investigated the stereoselectivity of ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5′-nucleotidase, EC 3.1.3.5) on pig aortic endothelial cells using two classes of nucleotide analogue. In experiments with nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety, the rate of catabolism of 100 microM-L-ATP was one-fifth that of D-ATP, the rate of catabolism of 100 microM-L-ADP was one-fifteenth that of D-ADP and there was no detectable catabolism of 100 microM-L-AMP. Each of the L-enantiomers inhibited, apparently competitively, the catabolism of the corresponding D-enantiomer; Ki values were approx. 0.6 mM, 1.0 mM and 3.9 mM for L-ATP, L-ADP and L-AMP respectively. Experiments with adenosine 5′-[beta, gamma-imido]triphosphate and with D- and L-enantiomers of adenosine 5′-[beta, gamma-methylene]triphosphate revealed modest ectopyrophosphatase activity, undetectable in experiments with natural nucleotides, which was also stereoselective. Use of phosphorothioate nucleotide analogues demonstrated that ATP catabolism was virtually stereospecific with respect to the geometry of the thiol group substituted on the beta-phosphate: the Rp isomer was degraded, whereas there was little or no breakdown of the Sp isomer. ADP catabolism was also stereospecific with respect to the geometry of the thiol group substituted on the alpha-phosphate: the Sp isomer but not the Rp isomer was degraded. The geometry of thiol-group substitution on the alpha-phosphate had no effect on ATP catabolism to ADP. There was no detectable catabolism of analogues with thiol-group substitution on the terminal phosphate. Each of the phosphorothioate analogues that was catabolized broke down at a rate similar to that of the natural nucleotide from which it was derived. These results demonstrate that the ectonucleotidases on pig aortic endothelial cells exhibit a high degree of stereoselectivity, characteristic for each enzyme, both with respect to the ribofuranosyl moiety and to the phosphate side chain.

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