Protein Kinase C and Anaplastic Lymphoma Kinase Targeted Compounds

The protein kinases (PKs) belong to the largest single family of enzymes, phosphotransferases, which catalyze the phosphorylation of other enzymes and proteins and function primarily in signal transduction. Consequently, PKs regulate cell mechanisms such as growth, differentiation, and proliferation. Dysfunction of these cellular mechanisms may lead to cancer, a major predicament in health care. Even though there is a range of clinically available cancer-fighting drugs, increasing number of cancer cases and setbacks such as drug resistance, constantly keep cancer research active. At the commencement of this study an isophthalic acid derivative had been suggested to bind to the regulatory domain of protein kinase C (PKC). In order to investigate the biological effects and structure-activity relationships (SARs) of this new chemical entity, a library of compounds was synthesized. The best compounds induced apoptosis in human leukemia HL-60 cells and were not cytotoxic in Swiss 3T3 fibroblasts. In addition, the best apoptosis inducers were neither cytotoxic nor mutagenic. Furthermore, results from binding affinity assays of PKC isoforms revealed the pharmacophores of these isophthalic acid derivatives. The best inhibition constants of the tested compounds were measured to 210 nM for PKCα and to 530 nM for PKCδ. Among natural compounds targeting the regulatory domain of PKC, the target of bistramide A has been a matter of debate. It was initially found to activate PKCδ; however, actin was recently reported as the main target. In order to clarify and to further study the biological effects of bistramide A, the total syntheses of the natural compound and two isomers were performed. Biological assays of the compounds revealed accumulation of 4n polyploid cells as the primary mode of action and the compounds showed similar overall antiproliferative activities. However, each compound showed a distinct distribution of antimitotic effect presumably via actin binding, proapoptotic effect presumably via PKCδ, and pro-differentiation effect as evidenced by CD11b expression. Furthermore, it was shown that the antimitotic and proapoptotic effects of bistramide A were not secondary effects of actin binding but independent effects. The third aim in this study was to synthesize a library of a new class of urea-based type II inhibitors targeted at the kinase domain of anaplastic lymphoma kinase (ALK). The best compounds in this library showed IC50 values as low as 390 nM for ALK while the initial low cellular activities were successfully increased even by more than 70 times for NPM-ALK- positive BaF3 cells. More importantly, selective antiproliferative activity on ALK-positive cell lines was achieved; while the best compound affected the BaF3 and SU-DHL-1 cells with IC50 values of 0.5 and 0.8 μM, respectively, they were less toxic to the NPM-ALK-negative human leukemic cells U937 (IC50 = 3.2 μM) and BaF3 parental cells (IC50 = 5.4 μM). Furthermore, SAR studies of the synthesized compounds revealed functional groups and positions of the scaffold, which enhanced the enzymatic and cellular activities.Proteinkinaser är enzymer som katalyserar fosforyleringen av proteiner och tillhör den största enskilda familjen av enzymer, fosfotransferaserna. Proteinkinaserna har sin huvudsakliga uppgift i signaltransduktionen, d.v.s. de för vidare extracellulära signaler till intracellulära molekyler och ger på detta vis upphov till en cellulär respons. Till dessa hör t.ex. celltillväxten, celldifferentieringen och celldelningen. Dysfunktion av dessa cellulära mekanismer kan till exempel leda till cancer som utgör ett stort dilemma inom vården. Även om det finns ett utbud av kliniskt tillgängliga cancerläkemedel, håller ökande antal av cancerfall och motgångar, såsom resistens mot läkemedel, ständigt cancerforskningen aktiv. Denna doktorsavhandling behandlar proteinkinaserna proteinkinas C (PKC) och anaplastisk lymfomkinas (ALK) och mera ingående utvecklingen av nya föreningar designade att binda deras respektive regulatoriska och katalytiska domäner. PKC är en intracellulär kinas som fosforylerar serin och treonin i proteiner. ALK är däremot en receptortyrosinkinas som har en förmodad extracellulär ligandbindande domän och en intracellulär katalytisk domän och som fosforylerar tyrosin i proteiner. Båda kinaserna är involverade i cancer. Den första delen av undersökningen behandlar isoftalsyraderivat som designades att binda till den regulatoriska domänen av PKC. Ett bibliotek av föreningar syntetiserades för att undersöka de biologiska effekterna och struktur-aktivitetssambandet hos dessa föreningar. De bästa föreningarna orsakade apoptos (programmerad celldöd) i humana leukemiceller (HL-60) men inte i schweiziska 3T3-fibroblaster. Dessutom var de bästa apoptosframkallande derivaten varken cytotoxiska eller mutagena. Resultaten från bindingsaffinitetsstudier med dessa isoftalsyraderivat på PKC isoformer åskådliggjorde de viktiga funktionella grupperna på föreningarna. De bästa inhibitorkonstanterna uppmättes till 210 nM för PKCα och 530 nM för PKCδ. Bistramid A är en bioaktiv cyklisk polyeter som kan isoleras från sjöpungar (Lissoclinum bistratum) och det har diskuterats om den binder den regulatoriska domänen hos PKC. Ursprungligen fann man att bistramid A aktiverar PKCδ medan de senaste rapporterna däremot har visat att aktin skulle vara det huvudsakliga proteinmålet. Totalsyntesen av denna naturprodukt samt två av dess strukturisomerer utfördes för att kunna klargöra och för att ytterligare kunna studera de biologiska effekterna av bistramid A. Syntesen av den centrala delen av bistramid A, som utgör en del av denna undersökning, utfördes modifierat enligt litteraturen. Resultat från biologiska aktivitetsstudier av föreningarna visade ansamling av 4n polyploida celler som den primära verkningsmekanismen och föreningarna visade liknande allmänna antiproliferativa aktiviteter. Föreningarna visade dock olika fördelningar av antimitotisk effekt förmodligen via bindningen till aktin, proapoptotisk effekt förmodligen via PKCδ, och pro-differentierings effekt som framgår av expressionen av CD11b. Resultaten visade dessutom att bistramid A:s mitoshämmande och proapoptotiska effekter inte var sekundära effekter av bindningen till aktin men oberoende effekter. Det tredje målsättningen i denna studie var att syntetisera ett bibliotek av en ny klass av urea-baserade typ II-inhibitorer designade att binda till kinasdomänen på ALK. De bästa föreningarna i detta bibliotek gav 50 % inhibition vid koncentrationer (IC50) så låga som 390 nM för ALK medan de preliminärt låga cellulära aktiviteterna framgångsrikt ökades mer än 70 gånger för NPM-ALK-positiva BaF3-celler. Ett ännu viktigare resultat var att selektiv antiproliferativ aktivitet för ALK-positiva cellinjer kunde uppnås. De bästa föreningarna inhiberade ALK-positiva BaF3- och SU-DHL-1-celler med IC50-värden på 0,5 och 0,8 μM, respektive, medan de var mindre toxiska för NPM-ALK-negativa humana leukemiceller (IC50-värden för U937 och BaF3 var 3,2 och 5,4 μM, respektive). Dessutom visade resultaten från studierna struktur-aktivitetssambandena hos föreningarna som modulerade de enzymatiska och cellulära aktiviteterna

C1 domain-targeted isophthalates as protein kinase C modulators : structure-based design, structure-activity relationships and biological activities

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C1 Domain-Targeted Isophthalate Derivatives Induce Cell Elongation and Cell Cycle Arrest in HeLa Cells

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A miniaturised 3D printed polypropylene reactor for online reaction analysis by mass spectrometry

Correction: Reaction Chemistry & Engineering, vol. 2:5, p. 811 DOI: 81110.1039/C7RE90018JA miniaturised polypropylene reactor was fabricated by 3D printing using fused deposition modeling. A stainless steel nanoelectrospray ionisation capillary and a magnetic stir bar were integrated into the reactor during the printing process. The integrated nanoelectrospray ionisation capillary allows direct sampling of a reaction solution without external pumping. It also allows ionisation of the analytes. Therefore, very rapid online mass spectrometric chemical reaction monitoring is possible. Operation of the miniaturised reactor is shown by the online nanoelectrospray mass spectrometry characterisation of a Diels–Alder reaction and the subsequent retro Diels–Alder reaction.Peer reviewe

Current Status and Future Prospects of C1 Domain Ligands as Drug Candidates

The second messenger diacylglycerol (DAG) plays a central role in the signal transduction of G-protein coupled receptors and receptor tyrosine kinases by binding to C1 domain of effector proteins. C1 domain was first identified in protein kinase C (PKC) which comprises a family of ten isoforms that play roles in diverse cellular processes such as proliferation, apoptosis and differentiation. Aberrant signaling through PKC isoforms and other C1 domain-containing proteins has been implicated in several pathological disorders. Drug discovery concerning C1 domains has exploited both natural products and rationally designed compounds. Currently, molecules from several classes of C1 domain-binding compounds are in clinical trials; however, still more have the potential to enter the drug development pipeline. This review gives a summary of the recent developments in C1 domain-binding compounds.Peer reviewe

Simple 3D printed stainless steel microreactors for online mass spectrometric analysis

A simple flow chemistry microreactor with an electrospray ionization tip for real time mass spectrometric reaction monitoring is introduced. The microreactor was fabricated by a laser-based additive manufacturing technique from acid-resistant stainless steel 316L. The functionality of the microreactor was investigated by using an inverse electron demand Diels-Alder and subsequent retro Diels-Alder reaction for testing. Challenges and problems encountered are discussed and improvements proposed. Adsorption of reagents to the rough stainless steel channel walls, short length of the reaction channel, and making a proper ESI tip present challenges, but the microreactor is potentially useful as a disposable device.Peer reviewe

Design, synthesis, and biological activity of isophthalic acid derivatives targeted to the C1 domain of protein kinase C

Protein kinase C (PKC) is a widely studied molecular target for the treatment of cancer and other diseases. We have approached the issue of modifying PKC function by targeting the C1 domain in the regulatory region of the enzyme. Using the X-ray crystal structure of the PKC delta C1b domain, we have discovered conveniently synthesizable derivatives of dialkyl 5-(hydroxymethyl)isophthalate that can act as potential C1 domain ligands. Structure-activity studies confirmed that the important functional groups predicted by modeling were indispensable for binding to the C1 domain and that the modifications of these groups diminished binding. The most promising compounds were able to displace radiolabeled phorbol ester ([H-3]PDBu) from PKC alpha and delta at K-i values in the range of 200-900 nM. Furthermore, the active isophthalate derivatives could modify PKC activation in living cells either by inducing PKC-dependent ERK phosphorylation or by inhibiting phorbol-induced ERK phosphorylation. In conclusion, we report here, for the first time. that derivatives of isophthalic acid represent an attractive novel group of C1 domain ligands that can be used as research tools or further modified for potential drug development.Protein kinase C (PKC) is a widely studied molecular target for the treatment of cancer and other diseases. We have approached the issue of modifying PKC function by targeting the C1 domain in the regulatory region of the enzyme. Using the X-ray crystal structure of the PKC delta C1b domain, we have discovered conveniently synthesizable derivatives of dialkyl 5-(hydroxymethyl)isophthalate that can act as potential C1 domain ligands. Structure-activity studies confirmed that the important functional groups predicted by modeling were indispensable for binding to the C1 domain and that the modifications of these groups diminished binding. The most promising compounds were able to displace radiolabeled phorbol ester ([H-3]PDBu) from PKC alpha and delta at K-i values in the range of 200-900 nM. Furthermore, the active isophthalate derivatives could modify PKC activation in living cells either by inducing PKC-dependent ERK phosphorylation or by inhibiting phorbol-induced ERK phosphorylation. In conclusion, we report here, for the first time. that derivatives of isophthalic acid represent an attractive novel group of C1 domain ligands that can be used as research tools or further modified for potential drug development.Protein kinase C (PKC) is a widely studied molecular target for the treatment of cancer and other diseases. We have approached the issue of modifying PKC function by targeting the C1 domain in the regulatory region of the enzyme. Using the X-ray crystal structure of the PKC delta C1b domain, we have discovered conveniently synthesizable derivatives of dialkyl 5-(hydroxymethyl)isophthalate that can act as potential C1 domain ligands. Structure-activity studies confirmed that the important functional groups predicted by modeling were indispensable for binding to the C1 domain and that the modifications of these groups diminished binding. The most promising compounds were able to displace radiolabeled phorbol ester ([H-3]PDBu) from PKC alpha and delta at K-i values in the range of 200-900 nM. Furthermore, the active isophthalate derivatives could modify PKC activation in living cells either by inducing PKC-dependent ERK phosphorylation or by inhibiting phorbol-induced ERK phosphorylation. In conclusion, we report here, for the first time. that derivatives of isophthalic acid represent an attractive novel group of C1 domain ligands that can be used as research tools or further modified for potential drug development.Peer reviewe

Mechanism of the Oxidation of Heptafulvenes to Tropones Studied by Online Mass Spectrometry and Density Functional Theory Calculations

We have identified the most likely reaction mechanism for oxidizing heptafulvenes to the corresponding tropones by experimental and theoretical investigations. The experimental studies were done by coupling a three-dimensional printed miniaturized reactor with an integrated electrospray ionization needle to a mass spectrometer. Using the experimentally observed ions as a basis, nine alternative reaction pathways were investigated with density functional theory calculations. The lowest energy reaction pathway starts with the formation of an epoxide that is opened upon the addition of a second equivalent of the oxidizing species meta-chloroperoxybenzoic acid. The adduct formed then undergoes a Criegee-like rearrangement to yield a positively charged hemiketal, which on deprotonation dissociates into acetone resembles a Hock-like rearrangement. and tropone. Overall, the reaction mechanism resembles a Hock-like rearrangement.Peer reviewe

Adenosine analogs bearing phosphate isosteres as human MDO1 ligands

The human O-acetyl-ADP-ribose deacetylase MDO1 is a mono-ADP-ribosylhydrolase involved in the reversal of post-translational modifications. Until now MDO1 has been poorly characterized, partly since no ligand is known besides adenosine nucleotides. Here, we synthesized thirteen compounds retaining the adenosine moiety and bearing bioisosteric replacements of the phosphate at the ribose 50-oxygen. 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 such as phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biochemical evaluation showed that two compounds, one from both series, inhibited ADP-ribosyl hydrolysis mediated by MDO1 in high concentrations. (C) 2018 Elsevier Ltd. All rights reserved.Peer reviewe

Chemical analysis using 3D printed glass microfluidics

Additive manufacturing (3D printing) is a disruptive technology that is changing production systems globally. In addition, microfluidic devices are increasingly being used for chemical analysis and continuous production of chemicals. Printing of materials such as polymers and metals is already a reality, but additive manufacturing of glass for microfluidic systems has received minor attention. We characterize microfluidic devices (channel cross-section dimensions down to a scale of 100 mm) that have been produced by additive manufacturing of molten soda-lime glass in tens of minutes and report their mass spectrometric and Raman spectroscopic analysis examples. The functionality of a microfluidic glass microreactor is shown with online mass spectrometric analysis of linezolid synthesis. Additionally, the performance of a direct infusion device is demonstrated by mass spectrometric analysis of drugs. Finally, the excellent optical quality of the glass structures is demonstrated with in-line Raman spectroscopic measurements. Our results promise a bright future for additively manufactured glass microdevices in diverse fields of science.Peer reviewe