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

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

10.1101/296624 ◽

2018 ◽

Author(s):

Kirsten M. Knecht ◽

Olga Buzovetsky ◽

Constanze Schneider ◽

Dominique Thomas ◽

Vishok Srikanth ◽

...

Keyword(s):

Cancer Therapy ◽

Genome Stability ◽

Viral Infections ◽

Structural Basis ◽

Cancer Drug ◽

Allosteric Site ◽

Nucleotide Analogues ◽

Future Design ◽

Nucleotide Analogue ◽

Allosteric Sites

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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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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Allosteric inhibition of the SARS-CoV-2 main protease – insights from mass spectrometry-based assays

10.1101/2020.07.29.226761 ◽

2020 ◽

Cited By ~ 1

Author(s):

Tarick J. El-Baba ◽

Corinne A. Lutomski ◽

Anastassia L. Kantsadi ◽

Tika R. Malla ◽

Tobias John ◽

...

Keyword(s):

Mass Spectrometry ◽

Native Mass Spectrometry ◽

Turnover Rates ◽

Kinetic Assay ◽

Antiviral Therapies ◽

Enzyme Substrate ◽

Main Protease ◽

Starting Point ◽

Catalytically Active ◽

Substrate Processing

AbstractFollowing translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease Mpro cleaves at eleven sites to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease (COVID-19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of Mpro. Analysis of the monomer-dimer equilibria reveals a dissociation constant of Kd = 0.14 ± 0.03 μM, revealing MPro has a strong preference to dimerize in solution. Developing an MS-based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, which are effectively “flash-frozen” as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x-ray crystal structures of these enzyme-small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.

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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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Discovery of cryptic allosteric sites using reversed allosteric communication by a combined computational and experimental strategy

Chemical Science ◽

10.1039/d0sc05131d ◽

2021 ◽

Vol 12 (1) ◽

pp. 464-476

Author(s):

Duan Ni ◽

Jiacheng Wei ◽

Xinheng He ◽

Ashfaq Ur Rehman ◽

Xinyi Li ◽

...

Keyword(s):

Md Simulations ◽

Allosteric Site ◽

Experimental Strategy ◽

Allosteric Communication ◽

Allosteric Sites

Using reversed allosteric communication, we performed MD simulations, MSMs, and mutagenesis experiments, to discover allosteric sites. It reproduced the known allosteric site for MDL-801 on Sirt6 and uncovered a novel cryptic allosteric Pocket X.

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The missing link: allostery and catalysis in the anti-viral protein SAMHD1

Biochemical Society Transactions ◽

10.1042/bst20180348 ◽

2019 ◽

Vol 47 (4) ◽

pp. 1013-1027 ◽

Cited By ~ 2

Author(s):

Elizabeth R. Morris ◽

Ian A. Taylor

Keyword(s):

Allosteric Regulation ◽

Active Form ◽

Inflammatory Disorder ◽

Dna Viruses ◽

Allosteric Coupling ◽

Allosteric Sites ◽

Catalytically Active ◽

Important Regulator ◽

Hydrolysis Of ◽

Vertebrate Protein

Abstract Vertebrate protein SAMHD1 (sterile-α-motif and HD domain containing protein 1) regulates the cellular dNTP (2′-deoxynucleoside-5′-triphosphate) pool by catalysing the hydrolysis of dNTP into 2′-deoxynucleoside and triphosphate products. As an important regulator of cell proliferation and a key player in dNTP homeostasis, mutations to SAMHD1 are implicated in hypermutated cancers, and germline mutations are associated with Chronic Lymphocytic Leukaemia and the inflammatory disorder Aicardi–Goutières Syndrome. By limiting the supply of dNTPs for viral DNA synthesis, SAMHD1 also restricts the replication of several retroviruses, such as HIV-1, and some DNA viruses in dendritic and myeloid lineage cells and resting T-cells. SAMHD1 activity is regulated throughout the cell cycle, both at the level of protein expression and post-translationally, through phosphorylation. In addition, allosteric regulation further fine-tunes the catalytic activity of SAMHD1, with a nucleotide-activated homotetramer as the catalytically active form of the protein. In cells, GTP and dATP are the likely physiological activators of two adjacent allosteric sites, AL1 (GTP) and AL2 (dATP), that bridge monomer–monomer interfaces to stabilise the protein homotetramer. This review summarises the extensive X-ray crystallographic, biophysical and molecular dynamics experiments that have elucidated important features of allosteric regulation in SAMHD1. We present a comprehensive mechanism detailing the structural and protein dynamics components of the allosteric coupling between nucleotide-induced tetramerization and the catalysis of dNTP hydrolysis by SAMHD1.

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Unraveling allosteric landscapes of allosterome with ASD

Nucleic Acids Research ◽

10.1093/nar/gkz958 ◽

2019 ◽

Cited By ~ 1

Author(s):

Xinyi Liu ◽

Shaoyong Lu ◽

Kun Song ◽

Qiancheng Shen ◽

Duan Ni ◽

...

Keyword(s):

Drug Discovery ◽

Protein Function ◽

Target Identification ◽

Allosteric Regulation ◽

Biological Research ◽

Missense Mutations ◽

Allosteric Site ◽

Comprehensive Information ◽

Allosteric Sites ◽

Human Proteins

Abstract Allosteric regulation is one of the most direct and efficient ways to fine-tune protein function; it is induced by the binding of a ligand at an allosteric site that is topographically distinct from an orthosteric site. The Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) was developed ten years ago to provide comprehensive information related to allosteric regulation. In recent years, allosteric regulation has received great attention in biological research, bioengineering, and drug discovery, leading to the emergence of entire allosteric landscapes as allosteromes. To facilitate research from the perspective of the allosterome, in ASD 2019, novel features were curated as follows: (i) >10 000 potential allosteric sites of human proteins were deposited for allosteric drug discovery; (ii) 7 human allosterome maps, including protease and ion channel maps, were built to reveal allosteric evolution within families; (iii) 1312 somatic missense mutations at allosteric sites were collected from patient samples from 33 cancer types and (iv) 1493 pharmacophores extracted from allosteric sites were provided for modulator screening. Over the past ten years, the ASD has become a central resource for studying allosteric regulation and will play more important roles in both target identification and allosteric drug discovery in the future.

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Addressing the selectivity and toxicity of antiviral nucleosides

Antiviral Chemistry and Chemotherapy ◽

10.1177/2040206618758524 ◽

2018 ◽

Vol 26 ◽

pp. 204020661875852 ◽

Cited By ~ 20

Author(s):

Joy Y Feng

Keyword(s):

New Drugs ◽

Attrition Rate ◽

Emerging Viruses ◽

Herpes Simplex Virus 1 ◽

Nucleotide Analogs ◽

Antiviral Therapies ◽

Animal Toxicity ◽

Simplex Virus ◽

Antiviral Nucleosides ◽

High Attrition Rate

Nucleoside and nucleotide analogs have played significant roles in antiviral therapies and are valued for their impressive potency and high barrier to resistance. They have been approved for treatment of herpes simplex virus-1, HIV, HBV, HCV, and influenza, and new drugs are being developed for the treatment of RSV, Ebola, coronavirus MERS, and other emerging viruses. However, this class of compounds has also experienced a high attrition rate in clinical trials due to toxicity. In this review, we discuss the utility of different biochemical and cell-based assays and provide recommendations for assessing toxicity liability before entering animal toxicity studies.

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PASSer: Prediction of Allosteric Sites Server

10.26434/chemrxiv.13360535.v1 ◽

2020 ◽

Author(s):

Hao Tian ◽

Xi Jiang ◽

Peng Tao

Keyword(s):

Drug Discovery ◽

Prior Information ◽

Gradient Boosting ◽

Command Line ◽

Allosteric Site ◽

Command Line Interface ◽

And Topology ◽

Extreme Gradient Boosting ◽

Allosteric Sites ◽

Allosteric Mechanisms

Allostery is considered important in regulating protein's activity. Drug development depends on the understanding of allosteric mechanisms, especially the identification of allosteric sites, which is prerequisite in drug discovery and design. Many computational methods have been developed for allosteric site prediction using pocket features and dynamics information. Here, we provide a novel ensembled model, consisting of eXtreme gradient boosting (XGBoost) and graph convolutional neural network (GCNN) to predict allosteric sites. Our model can learn both physical properties and topology structure without any prior information and exhibited good performance under several indicators. Prediction results have shown that 84.9% of allosteric pockets in the testing proteins appeared in the top 3 positions. The PASSer: Protein Allosteric Sites Server (https://passer.smu.edu), along with a command line interface (CLI, https://github.com/smutaogroup/passerCLI) provide insights for further analysis in drug discovery.

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Structural basis for small molecule targeting of Doublecortin Like Kinase 1 DCLK1

10.21203/rs.3.rs-133153/v1 ◽

2021 ◽

Author(s):

Onisha Patel ◽

Michael Roy ◽

Ashleigh Kropp ◽

Weiwen Dai ◽

Isabelle Lucet

Keyword(s):

Kinase Inhibitor ◽

Critical Role ◽

Structural Data ◽

Kinase Domain ◽

Microtubule Assembly ◽

Systematic Evaluation ◽

Structural Basis ◽

Allosteric Site ◽

Functional Protein ◽

Wide Range

Abstract Doublecortin-like kinase 1 (DCLK1) is a bi-functional protein classified as a Microtubule-Associated Protein (MAP) and as a serine/threonine kinase that plays a critical role in regulating microtubule assembly. This understudied kinase is upregulated or mutated in a wide range of cancers. Knockdown studies have shown that DCLK1 is functionally important for tumour growth. However, the presence of tissue and development specific spliced DCLK1 isoforms and the lack of systematic evaluation of their biological function have challenged the development of effective strategies to understand the role of DCLK1 in oncogenesis. Recently, DCLK1-IN-1 was reported as a potent and selective DCLK1 kinase inhibitor, a powerful new tool to dissect DCLK1 biological functions. Here, we report the crystal structures of DCLK1 kinase domain in complex with two DCLK1-IN-1 precursors and DCLK-IN-1. Combined, our structural data analysis illuminates and rationalises the structure-activity relationship that informed development of DCLK1-IN-1 and provides the basis for DCLK1-IN-1 increased selectivity. We show that DCLK1-IN-1 induces a drastic conformational change of the N-lobe, which uncovered a new allosteric site. In addition, we demonstrate that DCLK1-IN-1 binds DCLK1 long isoforms with high affinity but does not prevent DCLK1 MAP function. Together, our work outlines the need for in-depth studies to rationally design of isoform-specific modulators and provides an invaluable structural platform to further the design of selective DCLK1 therapeutic agents.

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