The RCSB Protein Data Bank: views of structural biology for basic and applied research and education.

The RCSB Protein Data Bank (RCSB PDB, http://www.rcsb.org) provides access to 3D structures of biological macromolecules and is one of the leading resources in biology and biomedicine worldwide. Our efforts over the past 2 years focused on enabling a deeper understanding of structural biology and providing new structural views of biology that support both basic and applied research and education. Herein, we describe recently introduced data annotations including integration with external biological resources, such as gene and drug databases, new visualization tools and improved support for the mobile web. We also describe access to data files, web services and open access software components to enable software developers to more effectively mine the PDB archive and related annotations. Our efforts are aimed at expanding the role of 3D structure in understanding biology and medicine

ROLES OF CYCLOOXYGENASE-2 IN MICROGLIAL ACTIVATION AND DOPAMINERGIC CELL DEATH

Accumulating evidence suggests that inflammation plays an important role in the progression ofParkinson\u27s disease (PD). Among many inflammatory factors found in the PD brain, cyclooxygenase(COX), especially the inducible isoform, COX-2, is believed to be the critical enzyme in theinflammatory response. Induction of COX-2 is also found in an experimental model of PD producedby administration of 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To investigate whetherinhibition of COX-2 by valdecoxib or deficiency in COX-2 could prevent dopaminergic neuronaltoxicity and locomotor activity impairment, we injected MPTP into valdecoxib-treated C57BL/6N miceand COX-2 deficient mice, respectively. Both automated total distance and vertical activitymeasurements of the open-field test were significantly reduced in the vehicle-treated mice at two weekspost-MPTP injection. In contrast, valdecoxib treatment significantly attenuated these deficits.Similarly, COX-2 deficiency attenuated MPTP-induced loss of coordination on a rotarod assay.Valdecoxib or deficiency of COX-2 reduced microglial activation while preventing loss of tyrosinehydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc). The total number ofactivated microglia in the SNpc had a strong positive correlation with the level of COX-2 anddopaminergic neurodegeneration. The results of this study indicate that reducing the activity of COX-2can mitigate the progressive loss of dopaminergic neurons as well as the motor deficits caused byMPTP neurotoxicity, possibly by suppressing the activation of microglia in the SNpc

Pharmacological modulation and manipulation of cancer drug resistance

The aim of this project was to investigate pharmacological methods of overcoming resistance in cancer. Novel compounds, targeted therapies and non-steroidal anti-inflammatory drugs (NSAIDs) were examined for their potential to modulate and manipulate specific forms of drug resistance. Sixty one novel compounds were tested in combination with chemotherapeutic drugs in proliferation assays for their ability to overcome MRP1 and P-gp-mediated drug resistance. Two compounds were successful P-gp modulators in the DLKP-A cell line; the ditrifluoroacetyl resveratrol derivative, RBM15, and the macrocycle derivative, KG104. A panel of nine therapeutic agents were evaluated for their potential to down-regulate multidrug resistant protein expression and thus, overcome P-gp, MRP1 or BCRP-mediated drug resistance, at and below pharmacologically-relevant concentrations. Two of these agents (indomethacin and 17-AAG) partially down-regulated the expression of P-gp in the A549-Taxol cell line but did not overcome P-gp-mediated resistance when combined with docetaxel simultaneously or in pre-treated proliferation assays. Three agents (lapatinib, sulindac sulphide and 17-AAG) reduced the expression of MRP1 in the A549 cell line. Only sulindac sulphide overcame MRP1-mediated resistance in the combination proliferations assays; however, this was due to the inhibitory mechanism of sulindac sulphide and not due to the down-regulation of the MRP1 protein. Five agents (17-AAG, lapatinib, indomethacin, elacridar and gefitinib) down-regulated the expression of BCRP in the DLKP-SQ/mitox cell line. Lapatinib, gefitinib, elacridar and 17-AAG overcame BCRP-mediated resistance in both the combination and pre-treatment proliferation assays. The data indicates that the amount of down-regulation resulting from treatment with these drugs was insufficient to overcome drug resistance. Up-regulation of the three MDR transporter proteins was observed with a variety of agents tested. This suggests that, long-term treatment with such agents could lead to the development and amplification of multidrug resistance, and therefore, reduce the effectiveness of substrate chemotherapies in patients. Targeted therapies, including tyrosine kinase inhibitors (TKIs, such as lapatinib), are the latest significant development in the treatment of cancer. Lapatinib sensitised HER2-expressing cell lines to chemotherapeutic agents in the presence or absence of EGFR expression. This agent was also found to be a more active sensitiser in P-gp-expressing cell lines, while erlotinib was more active in BCRP-expressing cell lines. Gefitinib was the least active of three TKIs at modulating P-gp, MRP1 or BCRP. Following a 48 hours treatment, lapatinib up-regulated the expression and function of COX-2. It also stimulated COX-2 activity directly. This lapatinib-mediated COX-2 induction was independent of its TKI action on EGFR, and HER2 and could have serious therapeutic effects as COX-2 is known to increase cell growth, inhibit apoptosis, and enhance metastasis and angiogenesis. A COX-2-specific inhibitor, celecoxib, overcame P-gp, MRP1 and BCRP-mediated resistance. At pharmacologically relevant concentrations, celecoxib significantly overcame MRP1 and BCRP-mediated resistance. And to a much lesser extent celecoxib overcame P-gp mediated resistance above pharmacologically relevant concentrations. The combination of lapatinib with celecoxib could be of therapeutic benefit, as the combination of these agents could collectively inhibit COX-2, P-gp, MRP1, BCRP, HER2 and EGFR activity in tumours expressing multiple oncoproteins resistant pathways and enhanced signalling pathways. It is hoped that a novel treatment regimens, using these agents and TKI drugs with traditional chemotherapeutic agents, could improve current treatment strategies resulting in increased survival rates and decreased mortality

COMPUTATIONAL DESIGN OF 3-PHOSPHOINOSITIDE DEPENDENT KINASE-1 INHIBITORS AS POTENTIAL ANTI-CANCER AGENTS

Computational drug design methods have great potential in drug discovery particularly in lead identification and lead optimization. 3-Phosphoinositide dependent kinase-1 (PDK1) is a protein kinase and a well validated anti-cancer target. Inhibitors of PDK1 have the potential to be developed as anti-cancer drugs. In this work, we have applied various novel computational drug design strategies to design and identify new PDK1 inhibitors with potential anti-cancer activity. We have pursued novel structure-based drug design strategies and identified a new binding mode for celecoxib and its derivatives binding with PDK1. This new binding mode provides a valuable basis for rational design of potent PDK1 inhibitors. In order to understand the structure-activity relationship of indolinone-based PDK1 inhibitors, we have carried out a combined molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) modeling study. The predictive ability of the developed 3D-QSAR models were validated using an external test set of compounds. An efficient strategy of the hierarchical virtual screening with increasing complexity was pursued to identify new hits against PDK1. Our approach uses a combination of ligand-based and structure-based virtual screening including shape-based filtering, rigid docking, and flexible docking. In addition, a more sophisticated molecular dynamics/molecular mechanics- Poisson-Boltzmann surface area (MD/MM-PBSA) analysis was used as the final filter in the virtual screening. Our screening strategy has led to the identification of a new PDK1 inhibitor. The anticancer activities of this compound have been confirmed by the anticancer activity assays of national cancer institute-developmental therapeutics program (NCI-DTP) using 60 cancer cell lines. The PDK1-inhibitor binding mode determined in this study may be valuable in future de novo drug design. The virtual screening approach tested and used in this study could also be applied to lead identification in other drug discovery efforts

Applications of the indole scaffold in medicinal chemistry: development of new antioxidants, COX inhibitors and antitubercular agents

Dissertação para obtenção do Grau de Doutor em Química, especialidade Química OrgânicaFundação para a Ciência e Tecnologia - SFRH/BD/46234/200

Molecular Docking and Synthesis of 1, 2, 4 - Triazin Analogue of Diclofenac as Potential Ligand for Chlorpromazine Induced Parkinson’s in Rat Model

Aim: Parkinson’s disease (PD) is a common neurodegenerative disorder, which is characterized by slowly progressive loss of dopaminergic neurons associated with substantial. The present study was aimed for molecular docking and synthesis of 1, 2, 4–Triazin analogue of diclofenac as potential ligand for chlorpromazine induced Parkinson’s in rat model. Materials and methods: Twenty four Albino Wistar male Rats weighing 250-270 gm were randomly assigned to four groups, each group contain 6 animals. Group I Vehicle group-Received 1% Gum acacia + distilled water p.o for 14 days, Group II Negative control group- Received Chlorpromazine 3mg/kg, i.p. (dissolved with 1% gum acacia in distilled water suspension), Group III Standard group - Received Chlorpromazine (3mg/kg/day), i.p. + Diclofenac (10mg/kg/day) p. o., Group IV treatment group- Received Chlorpromazine (3mg/kg/day) i.p. + 1,2,4-Triazin derivative of Diclofenac at dose of 30mg/kg, p. o. Parkinson’s disease (PD) was induced by intra peritoneal injection of chlorpromazine 3mg/kg, i. p (dissolved with 1% gum acacia in distilled water suspension) daily for a period of 14 days. The 1, 2, 4-Trianine analogue of diclofenac was synthesised and evaluated against chlorpromazine induced PD by monitoring in vivo behavioural paramerters like muscle coordination, cognitive performance, catalepsy activity, Biochemical estimation of SGPT, SGOT, ALP, Total bilirubin, urea, creatinie and brain antioxidant levels, Acetylcholine, dopamine. Changes were confirmed by Histopathological studies. Results:Molecular docking of 1, 2, 4- Triazin derivative of diclofenac, binding scores of designed ligand was 1-10 scores with D3 protein, DDC, AA2AR, MAPK and MAO-B enzymes ranging from -6.35 to -5.64 Kcal/mol, -6.86 to -5.81 Kcal/mol, -6.11 to -5.02, -8.67 to -4.63 Kcal/mol, and -10.25 to -6.76 Kcal/mol respectively. Treatment group shows significant increase the body weight, feed intake, Locomotion action, muscle coordination, cognitive performance and dopamine level, also decrease in muscle rigidity, oxidative stress and cholinergic over activity. Conclusion: 1, 2, 4 -Triazin derivative of diclofenac shows significantly anti Parkinson’s activity against chlorpromazine induced PD rats with milder GI toxicity as compared to the diclofenac treatment. Further clinical data are required to explore this Analogue of diclofenac as Potential Ligand for improving the status of PD patients

Clinical studies on the role of eicosanoids in the asthmatic airway inflammation

The underlying mechanisms in the asthmatic airway inflammation involve the interaction between different cells and mediators that consequently result in different clinical phenotypes. The aim of this thesis was to investigate the impact of inflammatory mediators, with emphasis on eicosanoids, on the inflammatory and functional airway responses under basal and triggered conditions in subjects with asthma, in particular ASA/NSAID - intolerant and allergic phenotypes. In the studies included in this thesis, we investigated the possibility of finding new phenotype - specific biomarkers of asthma in connection with mechanistic pathwa ys of eico sanoid biosynthesis. The studies were possible because of careful and extensive characterizations of the patients. Eleven aspirin - sensitive asthmatics had, in comparison with ten aspirin - tolerant asthmatics, higher exhaled nitric oxide levels and higher baseline levels of CysLTs in saliva, sputum, blood ex vivo a nd urine. Levels of urinary LTE 4 and 9α,1 1β - prostaglandin F 2 increased after aspirin provocation whereas leukotriene levels in saliva and ex vivo stimulated blood did not increase. These findings support a selective CysLT - overproduction in this distinct clinical syndrome. CysLTs in saliva should be explored as a new and clinically convenient biomarker of AIA and other diseases associated with increased production of leukotrienes. In an explorative study, the capacity of eosinophils to produce 15 - LO pathway products and their ex vivo responsiveness to COX inhibiti on was studied in the peripheral blood drawn from healthy volunteers and three asthma groups. In the absence or presence of lysine - aspirin, eosinophils were stimulated with arachidonic acid and calcium ionophore to trigger the 15 - lipoxygenase - 1 (15 - LO) and 5 - lipoxygenase (5 - LO) pathways, respectively. The results displayed an increased release of the recently di scovered lipid mediator eoxin C 4 (EXC 4 ) as well as the main indicator of 15 - LO activity, 15 - HETE, in activated eosinophils from severe and aspirin - i ntolerant asthmatics. Eosinophils from AIA su bjects also showed elevated EXC 4 and LTC 4 formation after cellular activation in the presence of lysine - aspirin. This higher biosynthetic activity of 15 - LO pathway in AIA is in part due to increased numbers of eosinophils, but the data also support enhanced eosinophil function, possibly involving transcellular interactions with platelets. The findings support contribution of 15 - LO pathway in the pathophysiology of severe and aspirin - intolerant asthma. This the sis also aimed at evaluating the role of COX - 1 and COX - 2 in the biosynthesis of the p ro - inflammatory prostaglandin D 2 (PGD 2 ) and br onchoprotective prostaglandin E 2 (PGE 2 ) under basal conditions and during heightened airway inflammation and responses after inhaled allergen provocation. Eighteen subjects with asthma and six healthy controls participated in a cross - over study where a selective COX - 2 inhhibitor, celecoxib 200 mg, or placebo were given b.i.d. on 3 consecutive days following 2 untreated baseline days. Celecoxib treatment inhibited urinary excretion of the tetranor metabolite of PGE 2 , PGEM, by 50% or more in asthmatic subjects and healthy controls, whereas there was no significant change in the excretion of the tetranor metabolite of PGD 2 , PGDM. I n addition, celecoxib did not cause any significant changes in FEV 1 or F E NO. In comparison with the healthy controls, the subjects with asthma had higher baseline levels of urinary PGDM but not of PGEM. These findings indicate that biosynthes is of PGD 2 is catalysed predominantly by COX - 1 and that COX - 2 contributes substanti ally to the biosynthesis of PGE 2 . The asymmetric impact of COX - 2 inhibition on prostanoid formation raises the possibility of long - term adverse consequences of COX - 2 inhibition on airway homeostasis by the decreased formation of PGE 2 and maintained produc tion of increased levels of PGD 2 in asthmatics. Therefore, the effect of selective COX - 2 inhibition on induced asthmatic airway obstruction and inflammation was investigated in 16 subject s with mild atopic asthma who underwent rising dose inhalation challenges with allergen and methacholine (MCh) to determine the provocative dose causing a 20% drop in FEV 1 (PD 20 ) during a control study period and following 10 - 13 days of treatment with etor icoxib (90 mg once daily). Study periods were randomized with at least 2 weeks washout between and induced sputum cells and exhaled nitric oxide levels (F E NO) were used to assess airway inflammation. Blood assays for COX - 1 and COX - 2 activity to determine biochemical efficacy were performed and urinary excretion of lipid mediators was measured by mass - spectrometry. The intervention with COX - 2 inhibitor in pro voked asthma was not found to have any negative effects on allergen - induced airflow obstruction and sputum eosinophils, basal lung function or methacholine responsiveness. The study suggests that short - term use of COX - 2 inhibitors is safe in asthmatics. In summa ry: 1) The higher baseline LTE 4 levels found in three body matrices lends further support to CysLT - overproduction in AIA and the higher salivary levels should be explored as a new and clinically convenient biomarker of AIA and other diseases with inc reased CysLT - production. 2) The increased rel ease of the 15 - LO products, EXC 4 , and 15 - HETE, in activated eosinophils from severe asthma and AIA patients, and the elevated EXC 4 and LTC 4 formation in activated eosinophils from AIA subjects in the presence of ASA support a pathophysiological role of the 15 - LO pathway in AIA and severe asthma. 3 ) Basal biosynthesis of PGD 2 is increased in subjects with asthma and its formation is catalysed predominantly by COX - 1. By contrast, COX - 2 contributes substanti ally to the biosynthesis of PGE 2 . 4) COX - 2 inhibition in provoked asthma is found to have no negative effects on allergen - induced airflow obstruction and sputum eosinophils, basal lung function or MCh responsiveness suggesting that short - term use of COX - 2 inhibito rs is safe in asthmatic

From a Molecule to a Drug: Chemical Features Enhancing Pharmacological Potential

This book collects contributions published in the Special Issue “From a Molecule to a Drug: Chemical Features Enhancing Pharmacological Potential” and dealing with successful stories of drug improvement or design using classic protocols, quantum mechanical mechanistic investigation, or hybrid approaches such as QM/MM or QM/ML (machine learning). In the last two decades, computer-aided modeling has strongly supported scientists’ intuition to design functional molecules. High-throughput screening protocols, mainly based on classical mechanics’ atomistic potentials, are largely employed in biology and medicinal chemistry studies with the aim of simulating drug-likeness and bioactivity in terms of efficient binding to the target receptors. The advantages of this approach are quick outcomes, the possibility of repurposing commercially available drugs, consolidated protocols, and the availability of large databases. On the other hand, these studies do not intrinsically provide reactivity information, which requires quantum mechanical methodologies that are only applicable to significantly smaller and simplified systems at present. These latter studies focus on the drug itself, considering the chemical properties related to its structural features and motifs. Overall, such simulations provide necessary insights for a better understanding of the chemistry principles that rule the diseases at the molecular level, as well as possible mechanisms for restoring the physiological equilibrium

Investigating phosphate structural replacements through computational and experimental approaches

Bioisosteric replacements are used in drug design during lead generation and optimization processes with the aim to replace one functional group of a known molecule by another while retaining biological activity. The reason to use bioisosteric replacements are typically to optimize bioavailability or reducing toxicity. Phosphate groups represent a paradigm to study bioisosteric replacements. Protein-phosphate interaction plays a critical role during molecular recognition processes, and for example kinases represent one of the largest families of drug targets. However, some challenges exclude phosphate as a promising lead-like building block: i) charged phosphates do not cross molecular membranes; ii) some widely expressed proteins such as phosphatases easily hydrolyze phosphoric acid esters, which lead phosphate-containing ligands to lose their binding affinities before reaching their biological targets; iii) introduction of phosphate groups to parent scaffold is not easy. In the first part of the thesis work, I designed and implemented a computational protocol to mine information about phosphate structural replacements deposited in the Protein Data Bank. I constructed 116, 314, 271, and 42 sets of superimposed proteins where each set contains a reference protein to either POP, AMP, ADP, or ATP as well as a certain number of non-nucleotide ligands. 929 of such ligands are under study. The chemotypes that came out as structural replacements are diverse, ranging from common phosphate isosteres such as carboxyl, amide and squaramide to more surprising moieties such as benzoxaborole and aromatic ring systems. I exemplified some novel examples and interpreted the mechanism behind them. Local structural replacements are circumstance dependent: one chemical group valid in certain set-up cannot necessarily guarantee the success of another. The data from the study is available at http://86.50.168.121/phosphates_LSR.php. In the second part, I synthesized fifteen compounds retaining the adenosine moieties and bearing bioisosteric replacements of the phosphate at the ribose 5'-oxygen to test their stability toward human macro domain protein 1. These compounds are composed of either a squaryldiamide or an amide group as the bioisosteric replacement and/or as a linker. To these groups a variety of substituents were attached: phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biological evaluation using differential scanning fluorimetry showed that four compounds stabilized human MDO1 at levels comparable to ADP and one at level comparable to AMP. Virtual screening was also run to identify MDO1 binding ligands. Among 20,000 FIMM database lead-like molecules, 39 compounds were selected for testing and eleven compounds found active based on ADPr and Poly-ADPr competition binding assay. The assay is however not well validated and a second confirmatory assay was conducted using calorimetry. To the best of my knowledge, this is the first report of non-endogenous ligands of the human MDO1. Altogether, this thesis highlights the versatility of molecular recognition processes that accompanies chemical replacements in compounds; this in turns shows the limits of the concepts of molecular similarity and classical bioisosterism that are based on the conservation of molecular interactions.Bioisosteeristä korvausta käytetään lääkeainekehityksessä johtolankamolekyylien tuottamisessa ja optimoinnissa. Tarkoitus on vaihtaa molekyylin funktionaalinen ryhmä toiseksi biologisen aktiivisuuden muuttumatta. Yleensä tavoitteena on parantaa biologista hyötyosuutta tai vähentää toksisuutta. Fosfaattiryhmää on tässä työssä käytetty esimerkkiryhmänä bioisosteerisiä korvauksia tutkittaessa. Väitöskirjatyön ensimmäisessä osassa suunnittelin ja toteutin tiedonlouhintaprotokollan etsiäkseni Protein Data Bank -tietokannasta korvaavia rakenteita fosfaattiryhmälle. Kokosin 116, 314, 271 ja 42 proteiiniryhmää, joissa kussakin on vertailumolekyylinä fosfaattiryhmän sisältävä POP, AMP, ADP tai ATP, ja lisäksi ei-nukleotidisiä ligandeja. Yhteensä 929 ei-nukleotidistä ligandia tutkittiin. Niistä löydettiin monipuolisesti fosfaattiryhmän korvaavia rakenteita, muun muassa yleisesti tunnettuja fosfaatin bioisosteerejä kuten karboksyyli, amidi ja squaramidi, mutta myös erikoisempia ryhmiä kuten bentsoksaboroli ja aromaattisia rengasrakenteita. Työssäni esittelen muutamia uusia rakenteita ja tulkitsen niiden vaikutusmekanismeja. Rakenteiden korvaaminen riippuu tilanteesta; yhteen tapaukseen sopiva korvaava ryhmä ei välttämättä toimi toisessa. Työn toisessa osassa syntetisoin 15 adenosiiniyhdistettä, joiden riboosiosan 5'-hapessa oleva fosfaattiryhmä on korvattu vaihtelevalla bioisosteerisellä ryhmällä. Bioisosteerisenä ryhmänä tai linkkerinä oli joko squaramidi- tai amidiryhmä. Yhdisteiden vakaus testattiin ihmisen MDO1-makrodomeeniproteiinin kanssa.Julkaisussa virheellinen verkkoaineiston ISBN 978-951-51-0045-0

Stress during cancer diagnostic workup

Receiving a cancer diagnosis is an extremely stressful life event. Cancer patients have been reported to experience an excessive risk of stress-related health outcomes including suicide. The time period between the first suspicion of a potential cancer and cancer diagnosis or start of primary cancer treatment is commonly defined as “cancer diagnostic workup”. This critical time window is especially stressful, mainly due to the uncertainty about the final diagnosis. Such diagnostic process and its health consequences may also apply to a larger group of population who are evaluated for but never receive a cancer diagnosis. Till now, less attention has been devoted to the health impact of a cancer diagnostic workup. This thesis aims to assess the risk of different stress-related health outcomes during a cancer diagnostic workup, taken into account various diagnostic processes, individuals with or without a final diagnosis of cancer, and different reasons of workup initiation. We also investiagated effect of a potential treatment in modulating the excessive risk of stress-related health outcomes following a cancer diagnosis. In study I, we examined the risk of iatrogenic and non-iatrogenic injuries during weeks before and after diagnosis among all patients with cancer. To eliminate impact of shared risk factors between cancer and injuries, we compared the risk from 16 weeks before to 16 weeks after cancer diagnosis, to the same period one year earlier, of the same patient. We found that cancer patients had an increased rate of both iatrogenic and non-iatrogenic injuries requiring inpatient care shortly before and after cancer diagnosis, compared to one year before. Our findings shed further light on the total burden of medical complications and call for prevention of intentional and unintentional injuries during the diagnostic process of a cancer. In study II, we assessed the risk of injuries that required inpatient care in relation to the diagnostic workup of cervical cancer and its precursor lesions, among all women that participated screening for cervical cancer. Women diagnosed with invasive cervical cancer and its precursors lesions were identified from the National Cervical Screening Register as exposed to a diagnostic workup. Women who had a normal result in Pap smear were classified as referece group. Inpatient care of either iatrogenic injuries or non-iatrogenic injuries was extremely rare during the diagnostic procedures of cervical cancer and its precursor lesions. Although with a small number of outcomes, we still found that women with invasive cervical cancer had an increased rate of non-iatrogenic injuries in relation to receiving a diagnosis of cervical cancer. In study III, we investigated the risk of psychiatric disorders and cardiovascular diseases during the diagnostic workup of potential breast cancer. All women diagnosed with a breast cancer, a benign tumor in breast, or unspecified lump in breast, were identified and considered as exposed to a diagnostic workup of potential breast cancer. We compared the risks of psychiatric disorders and cardiovascular diseases during the six weeks before diagnosis of the exposed women to the risks among the unexposed women. We found that women with benign tumor and breast cancer had an increased rate of psychiatric disorders and cardiovascular diseases while waiting for the final diagnosis. In study IV, we aimed to assess the use of low-dose aspirin and non-aspirin NSAIDs in relation to the risk of unnatural death due to suicide or accidents among patients after receiving a cancer diagnosis. Cancer patients with aspirin or non-aspirin NSAIDs dispensed after diagnosis were considered to be medicated. We compared the risk of unnatural death during on-medication period with the risk during off-medication period. We found that aspirin intake was associated with a lower risk of unnatural death after a cancer diagnosis. We did however not find a statistically significant association between use of non-aspirin NSAIDs and risk of unnatural death. In conclusion, a cancer diagnostic workup is associated with increased risks of a spectrum of stress-related health outcomes. Such increased risks of health outcomes, including injuries, psychiatric disorders and cardiovascular diseases, are noted for a large group of individuals being evaluated for a potential cancer, regardless of the final diagnosis. It represents therefore a great disease burden for the society. Interventions, such as low-dose aspirin intake, might be effective in reducing such risks (e.g. risk of unnatural death), after cancer diagnosis