Publications by authors named "Vigneshwaran Namasivayam"

53 Publications

Structural Insights to Human Immunodeficiency Virus (HIV-1) Targets and Their Inhibition.

Adv Exp Med Biol 2021 ;1322:63-95

Pharmaceutical Chemistry, Pharmaceutical Institute, University of Bonn, Bonn, Germany.

Human immunodeficiency virus (HIV) is a deadly virus that attacks the body's immune system, subsequently leading to AIDS (acquired immunodeficiency syndrome) and ultimately death. Currently, there is no vaccine or effective cure for this infection; however, antiretrovirals that act at various phases of the virus life cycle have been useful to control the viral load in patients. One of the major problems with antiretroviral therapies involves drug resistance. The three-dimensional structure from crystallography studies are instrumental in understanding the structural basis of drug binding to various targets. This chapter provides key insights into different targets and drugs used in the treatment from a structural perspective. Specifically, an insight into the binding characteristics of drugs at the active and allosteric sites of different targets and the importance of targeting allosteric sites for design of new-generation antiretrovirals to overcome complex and resistant forms of the virus has been reviewed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-981-16-0267-2_3DOI Listing
July 2021

Feature-Based Molecular Networking for the Targeted Identification of G-Inhibiting FR900359 Derivatives.

J Nat Prod 2021 Jul 1;84(7):1941-1953. Epub 2021 Jul 1.

Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, D-53115 Bonn, Germany.

Both the soil bacterium and the bacterial endosymbiont Burkholderia crenata of the plant are producers of FR900359 (FR). This cyclic depsipeptide is a potent and selective G protein inhibitor used extensively to investigate the intracellular signaling of G protein coupled receptors (GPCRs). In this study, the metabolomes of both FR producers were investigated and compared using feature-based molecular networking (FBMN). As a result, 30 previously unknown FR derivatives were identified, one-third being unique to . Guided by MS, a novel FR derivative, FR-6 (compound ), was isolated, and its structure unambiguously established. In a whole-cell biosensing assay based on detection of dynamic mass redistribution (DMR) as readout for G inhibition, FR-6 suppressed G signaling with micromolar potency (pIC = 5.56). This functional activity was confirmed in radioligand binding assays (p = 7.50). This work demonstrates the power of molecular networking, guiding the way to a novel G-inhibiting FR derivative and underlining the potency of FR as a G inhibitor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jnatprod.1c00194DOI Listing
July 2021

Scaffold fragmentation and substructure hopping reveal potential, robustness, and limits of computer-aided pattern analysis ([email protected]).

Comput Struct Biotechnol J 2021 10;19:3269-3283. Epub 2021 May 10.

Department of Pharmaceutical and Cellbiological Chemistry, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany.

Computer-aided pattern analysis ([email protected]) was recently presented as a powerful tool to predict multitarget ABC transporter inhibitors. The backbone of this computational methodology was the statistical analysis of frequently occurring molecular features amongst a fixed set of reported small-molecules that had been evaluated toward ABCB1, ABCC1, and ABCG2. As a result, negative and positive patterns were elucidated, and secondary positive substructures could be suggested that complemented the multitarget fingerprints. Elevating [email protected] to a non-statistical and exploratory level, the concluded secondary positive patterns were extended with potential positive substructures to improve [email protected]'s prediction capabilities and to explore its robustness. A small-set compound library of known ABCC1 inhibitors with a known hit rate for triple ABCB1, ABCC1, and ABCG2 inhibition was taken to virtually screen for the extended positive patterns. In total, 846 potential broad-spectrum ABCB1, ABCC1, and ABCG2 inhibitors resulted, from which 10 have been purchased and biologically evaluated. Our approach revealed 4 novel multitarget ABCB1, ABCC1, and ABCG2 inhibitors with a biological hit rate of 40%, but with a slightly lower inhibitory power than derived from the original [email protected] This is the very first report about discovering novel broad-spectrum inhibitors against the most prominent ABC transporters by improving [email protected]
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.csbj.2021.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8193046PMC
May 2021

Adenosine AR/AR Antagonists Enabling Additional HR Antagonism for the Treatment of Parkinson's Disease.

J Med Chem 2021 Jun 9;64(12):8246-8262. Epub 2021 Jun 9.

Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Duesseldorf, Universitaets street 1, 40225 Duesseldorf, Germany.

Adenosine A/A receptors (AR/AR) represent targets in nondopaminergic treatment of motor disorders such as Parkinson's disease (PD). As an innovative strategy, multitargeting ligands (MTLs) were developed to achieve comprehensive PD therapies simultaneously addressing comorbid symptoms such as sleep disruption. Recognizing the wake-promoting capacity of histamine H receptor (HR) antagonists in combination with the "caffeine-like effects" of AR/AR antagonists, we designed AR/AR/HR MTLs, where a piperidino-/pyrrolidino(propyloxy)phenyl HR pharmacophore was introduced with overlap into an adenosine antagonist arylindenopyrimidine core. These MTLs showed distinct receptor binding profiles with overall nanomolar HR affinities ( < 55 nM). Compound (, (AR) = 11.5 nM, (AR) = 7.25 nM) and (, (AR) = 11.2 nM, (AR) = 4.01 nM) were evaluated . l-DOPA-induced dyskinesia was improved after administration of compound (1 mg kg, i.p. rats). Compound (2 mg kg, p.o. mice) increased wakefulness representing novel pharmacological tools for PD therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c00914DOI Listing
June 2021

An Overview of Spike Surface Glycoprotein in Severe Acute Respiratory Syndrome-Coronavirus.

Front Mol Biosci 2021 16;8:637550. Epub 2021 Mar 16.

Faculty of Pharmacy, Karpagam Academy of Higher Education, Tamil Nadu, India.

The novel coronavirus originated in December 2019 in Hubei, China. This contagious disease named as COVID-19 resulted in a massive expansion within 6 months by spreading to more than 213 countries. Despite the availability of antiviral drugs for the treatment of various viral infections, it was concluded by the WHO that there is no medicine to treat novel CoV, SARS-CoV-2. It has been confirmed that SARS-COV-2 is the most highly virulent human coronavirus and occupies the third position following SARS and MERS with the highest mortality rate. The genetic assembly of SARS-CoV-2 is segmented into structural and non-structural proteins, of which two-thirds of the viral genome encodes non-structural proteins and the remaining genome encodes structural proteins. The most predominant structural proteins that make up SARS-CoV-2 include spike surface glycoproteins (S), membrane proteins (M), envelope proteins (E), and nucleocapsid proteins (N). This review will focus on one of the four major structural proteins in the CoV assembly, the spike, which is involved in host cell recognition and the fusion process. The monomer disintegrates into S1 and S2 subunits with the S1 domain necessitating binding of the virus to its host cell receptor and the S2 domain mediating the viral fusion. On viral infection by the host, the S protein is further cleaved by the protease enzyme to two major subdomains S1/S2. Spike is proven to be an interesting target for developing vaccines and in particular, the RBD-single chain dimer has shown initial success. The availability of small molecules and peptidic inhibitors for host cell receptors is briefly discussed. The development of new molecules and therapeutic druggable targets for SARS-CoV-2 is of global importance. Attacking the virus employing multiple targets and strategies is the best way to inhibit the virus. This article will appeal to researchers in understanding the structural and biological aspects of the S protein in the field of drug design and discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmolb.2021.637550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058706PMC
March 2021

[email protected]: Computer-Aided Pattern Analysis to Predict Multitarget ABC Transporter Inhibitors.

J Med Chem 2021 03 16;64(6):3350-3366. Epub 2021 Mar 16.

Department of Pharmaceutical and Cellbiological Chemistry, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany.

Based on literature reports of the last two decades, a computer-aided pattern analysis ([email protected]) was implemented for the discovery of novel multitarget ABCB1 (P-gp), ABCC1 (MRP1), and ABCG2 (BCRP) inhibitors. [email protected] included basic scaffold identification, substructure search and statistical distribution, as well as novel scaffold extraction to screen a large virtual compound library. Over 45,000 putative and novel broad-spectrum ABC transporter inhibitors were identified, from which 23 were purchased for biological evaluation. Our investigations revealed five novel lead molecules as triple ABCB1, ABCC1, and ABCG2 inhibitors. [email protected] is the very first successful computational approach for the discovery of promiscuous ABC transporter inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c02199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041314PMC
March 2021

Targeting the Main Protease of SARS-CoV-2: From the Establishment of High Throughput Screening to the Design of Tailored Inhibitors.

Angew Chem Int Ed Engl 2021 04 24;60(18):10423-10429. Epub 2021 Mar 24.

Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany), E-mails.

The main protease of SARS-CoV-2 (M ), the causative agent of COVID-19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed M . Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in-depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of M and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards M inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (k /K =37 500 m  s , K =24.0 nm) and pyridyl ester 17 (k /K =29 100 m  s , K =10.0 nm), promising drug candidates for further development have been discovered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.202016961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014119PMC
April 2021

Rational drug design of 6-substituted 4-anilino-2-phenylpyrimidines for exploration of novel ABCG2 binding site.

Eur J Med Chem 2021 Feb 3;212:113045. Epub 2020 Dec 3.

Pharmaceutical and Cellbiological Chemistry, Pharmaceutical Institute, Rheinische Friedrich-Wilhelms-University of Bonn, An der Immenburg 4, 53121, Bonn, Germany. Electronic address:

In the search for novel, highly potent, and nontoxic adjuvant chemotherapeutics to resolve the major issue of ABC transporter-mediated multidrug resistance (MDR), pyrimidines were discovered as a promising compound class of modern ABCG2 inhibitors. As ABCG2-mediated MDR is a major obstacle in leukemia, pancreatic carcinoma, and breast cancer chemotherapy, adjuvant chemotherapeutics are highly desired for future clinical oncology. Very recently, docking studies of one of the most potent reversers of ABCG2-mediated MDR were reported and revealed a putative second binding pocket of ABCG2. Based on this (sub)pocket, a series of 16 differently 6-substituted 4-anilino-2-phenylpyrimidines was designed and synthesized to explore the potential increase in inhibitory activity of these ABCG2 inhibitors. The compounds were assessed for their influence on the ABCG2-mediated pheophorbide A transport, as well as the ABCB1- and ABCC1-mediated transport of calcein AM. They were additionally evaluated in MDR reversal assays to determine their half-maximal reversal concentration (EC). The 6-substitution did not only show increased toxicity against ABCG2-overexpressing cells in combination with SN-38 but also a negative influence on cell viability in general. Nevertheless, several candidates had EC values in the low double-digit nanomolar concentration range, qualifying them as some of the most potent reversers of ABCG2-mediated MDR. In addition, five novel multitarget ABCB1, ABCC1, and ABCG2 inhibitors were discovered, four of them exerting their inhibitory power against the three stated transporters at least in the single-digit micromolar concentration range.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2020.113045DOI Listing
February 2021

Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359.

Nat Commun 2021 01 8;12(1):144. Epub 2021 Jan 8.

Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115, Bonn, Germany.

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-20418-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794379PMC
January 2021

Non-nucleoside reverse transcriptase inhibitors (NNRTIs): a brief overview of clinically approved drugs and combination regimens.

Curr Opin Pharmacol 2020 10 14;54:179-187. Epub 2020 Nov 14.

Pharamceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany. Electronic address:

The non-nucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of HIV-1 reverse transcriptase and are classified into generations depending on their discovery and resistance profiles. The NNRTIs are used in combination regimens with antiretroviral agents that target two or more enzymes in the viral life cycle. The combination regimens usually include a backbone of two nucleoside or nucleotide reverse transcriptase inhibitors and a third core agent among the NNRTIs or protease inhibitors. The combination regimens are maintained over long durations and consequently lead to long-term problems, including toxicity, drug-drug interactions, and increasing costs. This brief overview summarizes the pharmacokinetic profiles for NNRTIs and NNRTI-based combination regimens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.coph.2020.10.009DOI Listing
October 2020

Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors.

Front Pharmacol 2020 8;11:1294. Epub 2020 Sep 8.

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical & Medicinal Chemistry, University of Bonn, Bonn, Germany.

Nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) inhibitors have potential as novel drugs for the (immuno)therapy of cancer. They increase the extracellular concentration of immunostimulatory ATP and reduce the formation of AMP, which can be further hydrolyzed by ecto-5'-nucleotidase (CD73) to immunosuppressive, cancer-promoting adenosine. In the present study, we synthesized analogs and derivatives of the standard CD39 inhibitor ARL67156, a nucleotide analog which displays a competitive mechanism of inhibition. Structure-activity relationships were analyzed at the human enzyme with respect to substituents in the - and C8-position of the adenine core, and modifications of the triphosph(on)ate chain. Capillary electrophoresis coupled to laser-induced fluorescence detection employing a fluorescent-labeled ATP derivative was employed to determine the compounds' potency. Selected inhibitors were additionally evaluated in an orthogonal, malachite green assay versus the natural substrate ATP. The most potent CD39 inhibitors of the present series were ARL67156 and its derivatives 31 and 33 with values of around 1 µM. Selectivity studies showed that all three nucleotide analogs additionally blocked CD73 acting as dual-target inhibitors. Docking studies provided plausible binding modes to both targets. The present study provides a full characterization of the frequently applied CD39 inhibitor ARL67156, presents structure-activity relationships, and provides a basis for future optimization towards selective CD39 and dual CD39/CD73 inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphar.2020.01294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508162PMC
September 2020

Superior Pyrimidine Derivatives as Selective ABCG2 Inhibitors and Broad-Spectrum ABCB1, ABCC1, and ABCG2 Antagonists.

J Med Chem 2020 09 11;63(18):10412-10432. Epub 2020 Sep 11.

Pharmaceutical and Cellbiological Chemistry, Pharmaceutical Institute, Rheinische Friedrich-Wilhelms-University Bonn, An der Immenburg 4, 53121 Bonn, Germany.

In the search for highly effective modulators addressing ABCG2-mediated MDR, 23 pyrimidines were synthesized and biologically assessed. Seven derivatives with (a) nitrogen- and/or halogen-containing residue(s) had extraordinary potencies against ABCG2 (IC < 150 nM). The compounds competitively inhibited ABCG2-mediated Hoechst 33342 transport but were not substrates of ABCG2. The most potent MDR reverser, compound , concentration-dependently increased SN-38-mediated cancer cell death at 11 nM (EC), time-dependently doubled SN-38 toxicity in a period of 7 days at 10 nM, and half-maximally accelerated cell death combined with SN-38 at 17 nM. No induction of ABCG2 was observed. Furthermore, 11 pyrimidines were revealed as triple ABCB1/ABCC1/ABCG2 inhibitors. Five possessed IC values below 10 μM against each transporter, classifying them as some of the 50 most potent multitarget ABC transporter inhibitors. The most promising representative, compound , reversed ABCB1-, ABCC1-, and ABCG2-mediated MDR, making it one of the three most potent ABC transporter inhibitors and reversers of ABC transporters-mediated MDR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c00961DOI Listing
September 2020

Computational Investigations on the Binding Mode of Ligands for the Cannabinoid-Activated G Protein-Coupled Receptor GPR18.

Biomolecules 2020 04 29;10(5). Epub 2020 Apr 29.

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical & Medicinal Chemistry, University of Bonn, 53121 Bonn, Germany.

GPR18 is an orphan G protein-coupled receptor (GPCR) expressed in cells of the immune system. It is activated by the cannabinoid receptor (CB) agonist ∆-tetrahydrocannabinol (THC). Several further lipids have been proposed to act as GPR18 agonists, but these results still require unambiguous confirmation. In the present study, we constructed a homology model of the human GPR18 based on an ensemble of three GPCR crystal structures to investigate the binding modes of the agonist THC and the recently reported antagonists which feature an imidazothiazinone core to which a (substituted) phenyl ring is connected via a lipophilic linker. Docking and molecular dynamics simulation studies were performed. As a result, a hydrophobic binding pocket is predicted to accommodate the imidazothiazinone core, while the terminal phenyl ring projects towards an aromatic pocket. Hydrophobic interaction of Cys251 with substituents on the phenyl ring could explain the high potency of the most potent derivatives. Molecular dynamics simulation studies suggest that the binding of imidazothiazinone antagonists stabilizes transmembrane regions TM1, TM6 and TM7 of the receptor through a salt bridge between Asp118 and Lys133. The agonist THC is presumed to bind differently to GPR18 than to the distantly related CB receptors. This study provides insights into the binding mode of GPR18 agonists and antagonists which will facilitate future drug design for this promising potential drug target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom10050686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277601PMC
April 2020

Cell-permeable high-affinity tracers for G proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors.

Br J Pharmacol 2020 04 11;177(8):1898-1916. Epub 2020 Feb 11.

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany.

Background And Purpose: G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The G protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up-regulated in cancer and inflammatory diseases. G inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for G proteins are lacking.

Experimental Approach: We have now developed G -specific, cell-permeable H-labelled high-affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM). The tracers served to specifically label and quantify G proteins in their native conformation in cells and tissues with high accuracy.

Key Results: FR and YM displayed low nanomolar affinity for Gα , Gα and Gα expressed in CRISPR/Cas9 Gα -knockout cells, but not for Gα . The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a "dowel" effect of the pseudoirreversibly binding FR. A high-throughput binding assay led to the discovery of novel G inhibitors, which inhibited G signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation.

Conclusions And Implications: The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bph.14960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070167PMC
April 2020

Ligand binding and activation of UTP-activated G protein-coupled P2Y and P2Y receptors elucidated by mutagenesis, pharmacological and computational studies.

Biochim Biophys Acta Gen Subj 2020 03 5;1864(3):129501. Epub 2019 Dec 5.

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical & Medicinal Chemistry, University of Bonn, Germany. Electronic address:

The nucleotide receptors P2Y and P2Y are the most closely related G protein-coupled receptors (GPCRs) of the P2Y receptor (P2YR) family. Both subtypes couple to G proteins and are activated by the pyrimidine nucleotide UTP, but only P2YR is also activated by the purine nucleotide ATP. Agonists and antagonists of both receptor subtypes have potential as drugs e.g. for neurodegenerative and inflammatory diseases. So far, potent and selective, "drug-like" ligands for both receptors are scarce, but would be required for target validation and as lead structures for drug development. Structural information on the receptors is lacking since no X-ray structures or cryo-electron microscopy images are available. Thus, we performed receptor homology modeling and docking studies combined with mutagenesis experiments on both receptors to address the question how ligand binding selectivity for these closely related P2YR subtypes can be achieved. The orthosteric binding site of P2YR appeared to be more spacious than that of P2YR. Mutation of Y197 to alanine in P2YR resulted in a gain of ATP sensitivity. Anthraquinone-derived antagonists are likely to bind to the orthosteric or an allosteric site depending on their substitution pattern and the nature of the orthosteric binding site of the respective P2YR subtype. These insights into the architecture of P2Y- and P2YRs and their interactions with structurally diverse agonists and antagonist provide a solid basis for the future design of potent and selective ligands.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2019.129501DOI Listing
March 2020

Substituted 4-phenylthiazoles: Development of potent and selective A, A and dual A/A adenosine receptor antagonists.

Eur J Med Chem 2020 Jan 15;186:111879. Epub 2019 Nov 15.

Pharmaceutical Institute, Department of Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121, Bonn, Germany; Pharma Center Bonn, University of Bonn, Brühlerstr. 7, D-53121, Bonn, Germany. Electronic address:

Adenosine acts as a powerful signaling molecule via four distinct G protein-coupled receptors, designated A, A, A and A adenosine receptors (ARs). A and A ARs are G-coupled, while A and A ARs inhibit cAMP production via G proteins. Antagonists for A and A ARs may be useful for the treatment of (neuro)inflammatory diseases including acute kidney injury and kidney failure, pulmonary diseases, and Alzheimer's disease. In the present study, we optimized the versatile 2-amino-4-phenylthiazole scaffold by introducing substituents at N2 and C5 to obtain A and A AR antagonists including dual-target compounds. Selective A antagonists with (sub)nanomolar potency were produced, e.g. 11 and 13. These compounds showed species differences being significantly more potent at the rat as compared to the human A AR, and were characterized as inverse agonists. Several potent and selective A AR antagonists, e.g. 7, 8, 17 and 22 (K values of 5-9 nM at the human A AR) were prepared, which were much less potent at the rat orthologue. Moreover, dual A/A antagonists (10, 18) were developed showing K values between 8 and 42 nM. Docking and molecule dynamic simulation studies using the crystal structure of the A AR and a homology model of the A AR were performed to rationalize the observed structure-activity relationships.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2019.111879DOI Listing
January 2020

An Agonist Radioligand for the Proinflammatory Lipid-Activated G Protein-Coupled Receptor GPR84 Providing Structural Insights.

J Med Chem 2020 03 27;63(5):2391-2410. Epub 2019 Nov 27.

PharmaCenter Bonn, Pharmaceutical Institute, Department of Pharmaceutical and Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.

The orphan G protein-coupled receptor (GPCR) GPR84 is expressed on immune cells mediating proinflammatory and immunostimulatory effects. In this study, we prepared the fully efficacious, nonbiased GPR84 agonist 6-hexylamino-2,4(1,3)-pyrimidinedione () in tritium-labeled form ([H]PSB-1584) by hydrogenation of a hexenyl-substituted precursor with tritium gas. The radioligand was characterized by kinetic, saturation, and competition assays using membranes of Chinese hamster ovary cells recombinantly expressing the human GPR84. [H] reversibly labeled the receptor with high affinity ( 2.08 nM). Structurally diverse orthosteric and allosteric ligands, including newly designed and synthesized compounds, were studied in competition binding assays. A homology model of GPR84 was generated to perform docking studies rationalizing the experimental data. The radioligand was additionally used for labeling GPR84 in native cells and tissues. [H] constitutes the first GPR84 agonist radioligand representing a powerful tool for this poorly investigated GPCR, which has potential as a future drug target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.9b01339DOI Listing
March 2020

Agonists and Antagonists for Purinergic Receptors.

Methods Mol Biol 2020 ;2041:45-64

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, Bonn, Germany.

Membrane receptors that are activated by the purine nucleoside adenosine (adenosine receptors) or by purine or pyrimidine nucleotides (P2Y and P2X receptors) transduce extracellular signals to the cytosol. They play important roles in physiology and disease. The G protein-coupled adenosine receptors comprise four subtypes: A, A, A, and A. The G-protein-coupled P2Y receptors are subdivided into eight subtypes: P2Y, P2Y, P2Y, P2Y, P2Y, P2Y, P2Y, and P2Y, while the P2X receptors represent ATP-gated homomeric or heteromeric ion channels consisting of three subunits; the most important subunits are P2X1, P2X2, P2X3, P2X4, and P2X7. This chapter provides guidance for selecting suitable tool compounds for studying these large and important purine receptor families.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-9717-6_3DOI Listing
April 2020

Design and synthesis of sulfonamidophenylethylamides as novel cardiac myosin activator.

Bioorg Med Chem 2019 09 27;27(18):4110-4123. Epub 2019 Jul 27.

College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 34134, Republic of Korea. Electronic address:

The sulfonamidophenylethylamide analogues were explored for finding novel and potent cardiac myosin activators. Among them, N-(4-(N,N-dimethylsulfamoyl)phenethyl-N-methyl-5-phenylpentanamide (13, CMA at 10 µM = 48.5%; FS = 26.21%; EF = 15.28%) and its isomer, 4-(4-(N,N-dimethylsulfamoyl)phenyl-N-methyl-N-(3-phenylpropyl)butanamide (27, CMA at 10 µM = 55.0%; FS = 24.69%; EF = 14.08%) proved to be efficient cardiac myosin activators both in in vitro and in vivo studies. Compounds 13 (88.2 + 3.1% at 5 µM) and 27 (46.5 + 2.8% at 5 µM) showed positive inotropic effect in isolated rat ventricular myocytes. The potent compounds 13 and 27 were highly selective for cardiac myosin over skeletal and smooth muscle myosin, and therefore these potent and selective amide derivatives could be considered a new class of cardiac myosin activators for the treatment of systolic heart failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2019.07.041DOI Listing
September 2019

Overview of Recent Strategic Advances in Medicinal Chemistry.

J Med Chem 2019 11 28;62(21):9375-9414. Epub 2019 May 28.

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences , Shandong University , 44 West Culture Road , 250012 Ji'nan , Shandong , P. R. China.

Introducing novel strategies, concepts, and technologies that speed up drug discovery and the drug development cycle is of great importance both in the highly competitive pharmaceutical industry as well as in academia. This Perspective aims to present a "big-picture" overview of recent strategic innovations in medicinal chemistry and drug discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.9b00359DOI Listing
November 2019

Identification of Thienopyrimidine Scaffold as an Inhibitor of the ABC Transport Protein ABCC1 (MRP1) and Related Transporters Using a Combined Virtual Screening Approach.

J Med Chem 2019 05 30;62(9):4383-4400. Epub 2019 Apr 30.

Pharmaceutical Chemistry II, Pharmaceutical Institute , Rheinische Friedrich-Wilhelms-University of Bonn , An der Immenburg 4 , 53121 Bonn , Germany.

A virtual screening protocol with combination of similarity search and pharmacophore modeling was applied to virtually screen a large compound library to gain new scaffolds regarding ABCC1 inhibition. Biological investigation of promising candidates revealed four compounds as ABCC1 inhibitors, three of them with scaffolds not associated with ABCC1 inhibition until now. The best hit molecule-a thienopyrimidine-was a moderately potent, competitive inhibitor of the ABCC1-mediated transport of calcein AM which also sensitized ABCC1-overexpressing cells toward daunorubicin. Further evaluation showed that it was a moderately potent, competitive inhibitor of the ABCB1-mediated transport of calcein AM, and noncompetitive inhibitor of the ABCG2-mediated pheophorbide A transport. In addition, the thienopyrimidine could also sensitize ABCB1- as well as ABCG2-overexpressing cells toward daunorubicin and SN-38, respectively, in concentration ranges that qualified it as one of the ten best triple ABCC1/ABCB1/ABCG2 inhibitors in the literature. Besides, three more new multitarget inhibitors were identified by this virtual screening approach.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b01821DOI Listing
May 2019

Structure-Activity Relationship of Purine and Pyrimidine Nucleotides as Ecto-5'-Nucleotidase (CD73) Inhibitors.

J Med Chem 2019 04 21;62(7):3677-3695. Epub 2019 Mar 21.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases , National Institutes of Health , Bethesda , Maryland 20892 , United States.

Cluster of differentiation 73 (CD73) converts adenosine 5'-monophosphate to immunosuppressive adenosine, and its inhibition was proposed as a new strategy for cancer treatment. We synthesized 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of purine and pyrimidine nucleosides, which represent nucleoside diphosphate analogues, and compared their CD73 inhibitory potencies. In the adenine series, most ribose modifications and 1-deaza and 3-deaza were detrimental, but 7-deaza was tolerated. Uracil substitution with N-methyl, but not larger groups, or 2-thio, was tolerated. 1,2-Diphosphono-ethyl modifications were not tolerated. N-(Aryl)alkyloxy-cytosine derivatives, especially with bulky benzyloxy substituents, showed increased potency. Among the most potent inhibitors were the 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of 5-fluorouridine (4l), N-benzoyl-cytidine (7f), N-[ O-(4-benzyloxy)]-cytidine (9h), and N-[ O-(4-naphth-2-ylmethyloxy)]-cytidine (9e) ( K values 5-10 nM at human CD73). Selected compounds tested at the two uridine diphosphate-activated P2Y receptor subtypes showed high CD73 selectivity, especially those with large nucleobase substituents. These nucleotide analogues are among the most potent CD73 inhibitors reported and may be considered for development as parenteral drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.9b00164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526958PMC
April 2019

A Adenosine Receptor Antagonists with Picomolar Potency.

J Med Chem 2019 04 15;62(8):4032-4055. Epub 2019 Apr 15.

Pharmaceutical Chemistry I, PharmaCenter Bonn, Pharmaceutical Institute , Rheinische Friedrich-Wilhelms-Universitat Bonn , An der Immenburg 4 , D-53121 Bonn , Germany.

The A adenosine receptor (AAR) was proposed as a novel target for the (immuno)therapy of cancer since AAR blockade results in antiproliferative, antiangiogenic, antimetastatic, and immunostimulatory effects. In this study, we explored the structure-activity relationships of xanthin-8-yl-benzenesulfonamides mainly by introducing a variety of linkers and substituents attached to the sulfonamide residue. A new, convergent strategy was established, which facilitated the synthesis of the target compounds. Many of the new compounds exhibited subnanomolar affinity for the AAR combined with high selectivity. Functional groups were introduced, which will allow the attachment of dyes and other reporter groups. 8-(4-((4-(4-Bromophenyl)piperazin-1-yl)sulfonyl)phenyl)-1-propylxanthine (34, PSB-1901) was the most potent A-antagonist ( K 0.0835 nM, K 0.0598 nM, human AAR) with >10 000-fold selectivity versus all other AR subtypes. It was similarly potent and selective at the mouse AAR, making it a promising tool for preclinical studies. Computational studies predicted halogen bonding to contribute to the outstanding potency of 34.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.9b00071DOI Listing
April 2019

The Journey of HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) from Lab to Clinic.

J Med Chem 2019 05 27;62(10):4851-4883. Epub 2018 Dec 27.

Department of Pharmacology and Experimental Neuroscience , University of Nebraska Medical Center , Omaha 68198-5880 , United States.

Human immunodeficiency virus (HIV) infection is now pandemic. Targeting HIV-1 reverse transcriptase (HIV-1 RT) has been considered as one of the most successful targets for the development of anti-HIV treatment. Among the HIV-1 RT inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have gained a definitive place due to their unique antiviral potency, high specificity, and low toxicity in antiretroviral combination therapies used to treat HIV. Until now, >50 structurally diverse classes of compounds have been reported as NNRTIs. Among them, six NNRTIs were approved for HIV-1 treatment, namely, nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), etravirine (ETR), rilpivirine (RPV), and doravirine (DOR). In this perspective, we focus on the six NNRTIs and lessons learned from their journey through development to clinical studies. It demonstrates the obligatory need of understanding the physicochemical and biological principles (lead optimization), resistance mutations, synthesis, and clinical requirements for drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b00843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049092PMC
May 2019

Development of a selective and highly sensitive fluorescence assay for nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39).

Analyst 2018 Nov;143(22):5417-5430

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.

Ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) is a major ectonucleotidase that hydrolyzes proinflammatory ATP via ADP to AMP, which is subsequently converted by ecto-5'-nucleotidase (CD73) to immunosuppressive adenosine. Activation of CD39 has potential for treating inflammatory diseases, while inhibition was suggested as a novel strategy for the immunotherapy of cancer. In the present study, we developed a selective and highly sensitive capillary electrophoresis (CE) assay using a novel fluorescent CD39 substrate, a fluorescein-labelled ATP (PSB-170621A) that is converted to its AMP derivative. To accelerate the assays, a two-directional (forward and reverse) CE system was implemented using 96-well plates, which is suitable for the screening of compound libraries (Z'-factor: 0.7). The detection limits for the forward and reverse operation were 11.7 and 2.00 pM, respectively, indicating a large enhancement in sensitivity as compared to previous methods (e.g. malachite-green assay: 1 000 000-fold, CE-UV assay: 500 000-fold, fluorescence polarization immunoassay: 12 500-fold). Enzyme kinetic studies at human CD39 revealed a Km value of 19.6 μM, and a kcat value of 119 × 10-3 s-1 for PSB-170621A, which shows similar substrate properties as ATP (11.4 μM and 82.5 × 10-3 s-1). The compound displayed similar properties at rat and mouse CD39. Subsequent docking studies into a homology model of human CD39 revealed a hydrophobic pocket that accommodates the fluorescein tag. PSB-170621A was found to be preferably hydrolyzed by CD39 as compared to other ectonucleotidases. The new assay was validated by performing inhibition assays with several standard CD39 inhibitors yielding results that were consonant with data using the natural substrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c8an01108gDOI Listing
November 2018

Radiosynthesis and in vivo evaluation of a fluorine-18 labeled pyrazine based radioligand for PET imaging of the adenosine A receptor.

Bioorg Med Chem 2018 09 29;26(16):4650-4663. Epub 2018 Jul 29.

Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Department of Neuroradiopharmaceuticals, Leipzig, Germany. Electronic address:

On the basis of a pyrazine core structure, three new adenosine A receptor ligands (7a-c) were synthesized containing a 2-fluoropyridine moiety suitable for F-labeling. Compound 7a was docked into a homology model of the A receptor based on X-ray structures of the related A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A receptor, 7a was radiolabeled as the most suitable candidate (K(A) = 4.24 nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [F]7a with a standardized uptake value (SUV) of ≈1 at 5 min post injection followed by a fast wash out. Metabolism studies of [F]7a in mice revealed the formation of a blood-brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2018.07.045DOI Listing
September 2018

2,4,6-Substituted Quinazolines with Extraordinary Inhibitory Potency toward ABCG2.

J Med Chem 2018 09 20;61(17):7952-7976. Epub 2018 Aug 20.

Pharmaceutical Institute University of Bonn An der Immenburg 4 53121 Bonn , Germany.

Several members of the ABC transporter superfamily play a decisive role in the development of multidrug resistance (MDR) in cancer. One of these MDR associated efflux transporters is ABCG2. One way to overcome this MDR is the coadministration of potent inhibitors of ABCG2. In this study, we identified novel inhibitors containing a 2,4,6-substituted quinazoline scaffold. Introduction of a 6-nitro function led to extraordinarily potent compounds that were highly selective for ABCG2 and also able to reverse the MDR toward the chemotherapeutic drugs SN-38 and mitoxantrone. The binding of substrate Hoechst 33342 and the two potent inhibitors 31 and 41 which differ in their mechanism of inhibition was rationalized using the recently published cryo-EM structures of ABCG2. For a better understanding of the interaction between the inhibitors and ABCG2, additional investigations regarding the ATPase activity, the interaction with Hoechst 33342, and with the conformational sensitive 5D3 antibody were carried out.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b01011DOI Listing
September 2018

3-(2-Carboxyethyl)indole-2-carboxylic Acid Derivatives: Structural Requirements and Properties of Potent Agonists of the Orphan G Protein-Coupled Receptor GPR17.

J Med Chem 2018 09 5;61(18):8136-8154. Epub 2018 Sep 5.

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical Chemistry I , University of Bonn , An der Immenburg 4 , 53121 Bonn , Germany.

The orphan receptor GPR17 may be a novel drug target for inflammatory diseases. 3-(2-Carboxyethyl)-4,6-dichloro-1 H-indole-2-carboxylic acid (MDL29,951, 1) was previously identified as a moderately potent GPR17 agonist. In the present study, we investigated the structure-activity relationships (SARs) of 1. Substitution of the indole 1-, 5-, or 7-position was detrimental. Only small substituents were tolerated in the 4-position while the 6-position accommodated large lipophilic residues. Among the most potent compounds were 3-(2-carboxyethyl)-1 H-indole-2-carboxylic acid derivatives containing the following substituents: 6-phenoxy (26, PSB-1737, EC 270 nM), 4-fluoro-6-bromo (33, PSB-18422, EC 27.9 nM), 4-fluoro-6-iodo (35, PSB-18484, EC 32.1 nM), and 4-chloro-6-hexyloxy (43, PSB-1767, EC 67.0 nM). (3-(2-Carboxyethyl)-6-hexyloxy-1 H-indole-2-carboxylic acid (39, PSB-17183, EC 115 nM) behaved as a partial agonist. Selected potent compounds tested at human P2Y receptor subtypes showed high selectivity for GPR17. Docking into a homology model of the human GPR17 and molecular dynamic simulation studies rationalized the observed SARs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.7b01768DOI Listing
September 2018

6-(Ar)Alkylamino-Substituted Uracil Derivatives: Lipid Mimetics with Potent Activity at the Orphan G Protein-Coupled Receptor 84 (GPR84).

ACS Omega 2018 Mar 20;3(3):3365-3383. Epub 2018 Mar 20.

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.

GPR84, a G protein-coupled receptor that is activated by medium-chain (hydroxy)fatty acids, appears to play an important role in inflammation, immunity, and cancer. Recently, 6-octylaminouracil () has been reported to act as an agonist at GPR84. Here, we describe the synthesis of 69 derivatives and analogs of , 66 of which represent new compounds. They were evaluated in (a) cyclic adenosine monophosphate accumulation and (b) β-arrestin assays in human GPR84-expressing cells. Potent nonbiased as well as G protein-biased agonists were developed, e.g., 6-hexylamino-2,4(1,3)-pyrimidinedione (, PSB-1584, EC 5.0 nM (a), 3.2 nM (b), bias factor: 0) and 6-((-chloro- and -bromo-phenylethyl)amino)-2,4(1,3)-pyrimidinedione (, PSB-16434, EC 7.1 nM (a), 520 nM (b), bias factor: 1.9 = 79-fold G pathway-selective; , PSB-17365, EC 2.5 nM (a), 100 nM (b), bias factor 1.3 = 20-fold selective), which were selective versus other free fatty acid-activated receptors. Compounds and were found to be metabolically stable upon incubation with human liver microsomes. A pharmacophore model was created on the basis of structurally diverse lipidlike GPR84 agonists.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsomega.7b02092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044507PMC
March 2018

Probing Substituents in the 1- and 3-Position: Tetrahydropyrazino-Annelated Water-Soluble Xanthine Derivatives as Multi-Target Drugs With Potent Adenosine Receptor Antagonistic Activity.

Front Chem 2018 26;6:206. Epub 2018 Jun 26.

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany.

Tetrahydropyrazino-annelated theophylline (1,3-dimethylxanthine) derivatives have previously been shown to display increased water-solubility as compared to the parent xanthines due to their basic character. In the present study, we modified this promising scaffold by replacing the 1,3-dimethyl residues by a variety of alkyl groups including combinations of different substituents in both positions. Substituted benzyl or phenethyl residues were attached to the N8 of the resulting 1,3-dialkyl-tetrahydropyrazino[2,1- ]purinediones with the aim to obtain multi-target drugs that block human A and A adenosine receptors (ARs) and monoaminoxidase B (MAO-B). 1,3-Diethyl-substituted derivatives showed high affinity for A ARs, e.g., (PSB-18339, 8--bromobenzyl-substituted) displayed a K value of 13.6 nM combined with high selectivity. 1-Ethyl-3-propargyl-substituted derivatives exhibited increased A AR affinity. The 8-phenethyl derivative was selective for the A AR (K 149 nM), while the corresponding 8-benzyl-substituted compound (PSB-1869) blocked A and A ARs with equal potency (K A, 180 nM; A, 282 nM). The 1-ethyl-3-methyl-substituted derivative (PSB-18405) bearing a -dichlorobenzyl residue at N8 blocked all three targets, A ARs (K 396 nM), A ARs (K 1,620 nM), and MAO-B (IC 106 nM) with high selectivity vs. the other subtypes (A and A ARs, MAO-A), and can thus be considered as a multi-target drug. Our findings were rationalized by molecular docking studies based on previously published X-ray structures of the protein targets. The new drugs have potential for the treatment of neurodegenerative diseases, in particular Parkinson's disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fchem.2018.00206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028563PMC
June 2018
-->