Development of selective inhibitors of phosphatidylinositol 3-kinase C2α

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer

Identification of 2,4-Disubstituted Imidazopyridines as Hemozoin Formation Inhibitors with Fast-Killing Kinetics and In Vivo Efficacy in the Plasmodium falciparum NSG Mouse Model

A series of 2,4-disubstituted imidazopyridines, originating from a SoftFocus Kinase library, was identified from a high throughput phenotypic screen against the human malaria parasite Plasmodium falciparum. Hit compounds showed moderate asexual blood stage activity. During lead optimization, several issues were flagged such as cross-resistance against the multidrug-resistant K1 strain, in vitro cytotoxicity, and cardiotoxicity and were addressed through structure–activity and structure–property relationship studies. Pharmacokinetic properties were assessed in mice for compounds showing desirable in vitro activity, a selectivity window over cytotoxicity, and microsomal metabolic stability. Frontrunner compound 37 showed good exposure in mice combined with good in vitro activity against the malaria parasite, which translated into in vivo efficacy in the P. falciparum NOD-scid IL-2Rγnull (NSG) mouse model. Preliminary mechanistic studies suggest inhibition of hemozoin formation as a contributing mode of action

Development of selective inhibitors of phosphatidylinositol 3-kinase C2α

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.Supplementary Data 1: Data of the supra-hexagonal plot in Figure 2ASupplementary Data 2: Complete dataset of all MMV PRB compounds’ activity on Plasmodium life cycle stagesSupplementary Data 3: Full SMFA dataset to support Figure 5CSupplementary Data 4: Transcriptome analysis of MMV1580488 (ML324) treated parasites to support Figure 6C.The Medicines for Malaria Venture and South African Technology Innovation Agency (TIA). This project was in part supported by the South African Medical Research Council with funds received from the South African Department of Science and Innovation, in partnership with the Medicines for Malaria Venture; and the DST/NRF South African Research Chairs Initiative Grant; and CSIR Parliamentary Grant funding as well as the Bill and Melinda Gates Foundation and the Australian NHMRC (APP1072217).http://www.nature.com/ncommshj2021BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC

Benzoylphosphonates as photocrosslinkable phosphotyrosine mimetics

Fehlregulationen des Phosphorylierungsgrads von Proteinen führen zu zahlreichen Krankheiten, wie Krebs und Diabetes. Als Grundlage für die Entwicklung neuer Medikamente ist eine genaue Kenntnis der molekularen Wechselwirkungen unabdingbar. Photoaffinitätsmarkierungen stellen in diesem Zusammenhang ein äußerst wichtiges Werkzeug dar. Der erste Teil der Arbeit bestand in der Entwicklung eines photovernetzbaren Phosphotyrosinmimetikums basierend auf Benzoylphosphonat. Die Photoreaktivität wurde zunächst anhand der Photodimerisierung von Benzoylphosphonat 19 untersucht. Dabei konnten erstmals die Photodimerisierungsprodukte isoliert und strukturell charakterisiert werden. Benzoylphosphonat 19 wies sehr gute Affinitäten zur PTP-Domäne von MptpA und zur SH2-Domäne von STAT5b mit Ki-Werten von 186 µM bzw. 326 µM auf. Die Bestrahlung bei 365 nm führte zu einer ca. zehnfachen (MptpA) bzw. zweifachen (STAT5b) Erhöhung der Aktivität. Der Einbau des Benzoylphosphonats in eine Peptidsequenz führte zu hochaffinen PAL-Sonden für STAT5b mit Bindungskonstanten zwischen 0.90 µM und 11 µM, wenngleich diese nicht ganz so aktiv waren wie die Phosphotyrosinpeptide. Die Bestrahlung der PAL-Sonde Ac-GkYLSLPPW-NH2 48 in Gegenwart von STAT5b resultierte in einer Verdoppelung der Aktivität. Die Bestrahlung der Phosphotyrosinpeptide hatte hingegen keinen Effekt. Untersuchungen der PAL-Sonde FAM-GkYLSLPPW-NH2 45 zeigten eine zeit- und konzentrationsabhängige Sättigung der Photovernetzung mit STAT5b. Die Spezifität der Photovernetzung wurde u.a. durch Unterdrückung der Vernetzung durch einen Verdränger nachgewiesen. In ersten Experimenten mit einer Biotin-markierten Sonde konnte STAT5b photovernetzt und über Avidin- Harzkugeln isoliert werden, was nach tryptischem Verdau durch Massenspektrometrie bestätigt werden konnte. Der Einsatz von Benzoylphosphonaten als photovernetzbare Phosphotyrosinmimetika in PAL-Sonden bietet viele Anwendungsmöglichkeiten in der Proteomik, z.B. bei der Untersuchung von Protein-Ligand-Wechselwirkungen. Der zweite Teil der Arbeit bestand in der Untersuchung des Inhibitionsmechanismus von Pyrimidotriazindionen gegenüber Proteintyrosinphosphatasen (PTPs). Diese Substanzklasse wird in Gegenwart von Reduktanden wie DTT oder NADH zu Dihydropyrimidotriazindionen reduziert. Die anschließende Reoxidation führt unter aeroben Bedingungen zur Bildung von Wasserstoffperoxid, welches über einen Europium-Tetracyclin-Assay quantifiziert wurde. Inhibitionsstudien von Pyrimidotriazinen gegenüber der Proteintyrosinphosphatase MptpA zeigten IC50-Werte im hohen nanomolaren Bereich. Steigende Konzentrationen von DTT hatten hingegen eine Reduktion der Inhibitionswirkung zur Folge, was auf eine Reaktivierung des durch H2O2 oxidierten Cysteinrests schließen lässt. Ein Vergleich der durch Pyrimidotriazine erzeugten Mengen H2O2 und der zur PTP- Inhibition benötigten Menge ließen eine alleinige Inhibition durch H2O2 vermuten. Dies wurde durch den Verlust der Inhibitionswirkung in Gegenwart von Katalase bestätigt. Darüber hinaus wurde die Produktion von mikromolaren Mengen Wasserstoffperoxid durch nanomolare Pyrimidotriazinkonzentrationen auch in Jurkat-Zellen ohne Reduktionsmittelzusatz durch FACS nachgewiesen. Im Gegensatz zu direkter H2O2-Zugabe zeigte sich bei den Pyrimidotriazinen jedoch keine Sättigung des intrazellulären Wasserstoffperoxids. Es ist denkbar, dass Pyrimidotriazine bei der Erforschung redoxregulierter Systeme zur selektiven Produktion von Wasserstoffperoxid in Zellen eingesetzt werden könnten.Failure in the regulation of phosphorylation in proteins leads to a number of diseases like cancer and diabetes. As a basis for drug development, an exact knowledge of the molecular interactions is required. In this area photoaffinity labeling is a very powerful tool for the elucidation of protein function. The first part of the thesis was concerned with the development of a photocrosslinkable phosphotyrosine mimetic based on benzoylphosphonate. The photoreactivity of benzoylphosphonate 19 was investigated through photodimerization. To my knowledge, photodimerization products could be isolated and structurally characterized for the first time. Benzoylphosphonate 19 showed a very good affinity towards the protein tyrosine phosphatase (PTP) domain of MptpA and the SH2 domain of STAT5b with Ki values of 186 µM and 326 µM respectively. Irradiation of the samples at 365 nm led to a ten-fold (MptpA) and a two-fold (STAT5b) increase of affinity. The incorporation of the benzoylphosphonate into peptide sequences led to high affinity PAL probes for STAT5b with binding constants between 0.90 µM and 11 µM, although they were not as active as the native phosphotyrosine peptides. Irradiation of the probe Ac-GkYLSLPPW- NH2 48 resulted in a two-fold increase of activity. Irradiation of the phosphotyrosine peptides had no effect. Investigations of PAL probe FAM-GkYLSLPPW-NH2 45 showed a time and concentration dependent saturation of photocrosslinking. The crosslinking specificity was proven amongst others by crosslinking inhibition upon addition of a ligand displacing the PAL probe. In trial experiments with a biotinylated probe, STAT5b could be photocrosslinked and isolated with avidin beads. STAT5b was identified by mass spectrometry after tryptic digestion of the corresponding gel band. PAL probes based on benzoylphosphonates as photocrosslinkable phosphotyrosine mimetics provide a powerful tool in proteomics, e.g. in exploring protein ligand interactions. In the second part of the thesis, the mechanism of inhibition for pyrimidotriazines against PTPs was examined. This class of substances is reduced by reducing agents such as DTT or NADH resulting in the formation of dihydropyrimidotriazines. The following reoxidation under aerobic conditions is accompanied by hydrogen peroxide production which was quantified using a europium tetracycline assay. Inhibition studies of pyrimidotriazines against the Mycobacterium tuberculosis protein tyrosine phosphatase A (MptpA) revealed IC50 values in the high nanomolar range. Increasing concentrations of DTT resulted in decreasing inhibitory activity indicating a partial reactivation of the oxidized active cystein residue. A comparison of the amount of hydrogen peroxide produced by pyrimidotriazines with that needed for PTP inhibition led to the conclusion that inhibition is mediated solely by H2O2. This was confirmed by experiments in the presence of catalase which resulted in a complete loss of inhibition. Furthermore production of micromolar amounts of H2O2 by nanomolar pyrimidotriazine concentrations was also shown to work in Jurkat cells using FACS. In contrast to direct addition of H2O2, pyrimidotriazines showed no saturation of intracellular H2O2. It is conceivable that pyrimidotriazines could be used as tools in redox regulation research to selectively produce hydrogen peroxide in cells

Benzoylphosphonate-based photoactive phosphopeptide mimetics for modulation of protein tyrosine phosphatases and highly specific labeling of SH2 domains

A light switch for phosphotyrosine- recognizing proteins: Irradiation of the bioisosteric benzoylphosphonate suffices to "turn off" the activity of target proteins and to label them covalently. Photoactive bioisosters may find applications in functional cell biology, bioanalytics, and proteome research

5-Aryl-2-(naphtha-1-yl)sulfonamido-thiazol-4(5H)-ones as clathrin inhibitors

The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (2), rhodanine-based lead that led to the Pitstop® 2 family of clathrin inhibitors is described herein. Head group substitution and bioisosteric replacement of the rhodanine core with a 2-aminothiazol-4(5H)-one scaffold eliminated off target dynamin activity. A series of N-substituents gave first phenylglycine (20, IC50 ∼ 20 μM) then phenyl (25, IC50 ∼ 7.1 μM) and 1-napthyl sulfonamide (26, Pitstop® 2 compound, IC50 ∼ 1.9 μM) analogues with good activity, validating this approach. A final library exploring the head group resulted in three analogues displaying either slight improvements or comparable activity (33, 38, and 29 with IC50 ∼ 1.4, 1.6 and 1.8 μM respectively) and nine others with IC50 < 10 μM. These results were rationalized using in silico docking studies. Docking studies predicted enhanced Pitstop® 2 family binding, not a loss of binding, within the Pistop® groove of the reported clathrin mutant invalidating recent assumptions of poor selectivity for this family of clathrin inhibitors

Development of selective inhibitors of phosphatidylinositol 3 kinase C2 alpha

Phosphatidylinositol 3 kinase type 2 amp; 945; PI3KC2 amp; 945; and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three kinase Class twO INhibitors PITCOINs , potent and highly selective small molecule inhibitors of PI3KC2 amp; 945; catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2 amp; 945; due to their unique mode of interaction with the ATP binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2 amp; 945; mediated synthesis of phosphatidylinositol 3 phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet dependent thrombus formation. PITCOINs are potent and selective cell permeable inhibitors of PI3KC2 amp; 945; function with potential biomedical applications ranging from thrombosis to diabetes and cance