Exploratory Photochemistry: Pushing the Boundaries of the p-Hydroxyphenacyl Photoremovable Protecting Group

Abstract Exploratory Photochemistry: Pushing the Boundaries of the p-Hydroxyphenacyl Photoremovable Protecting Group A. Exploratory and Mechanistic Photochemistry B. Novel Applications of the p-Hydroxyphenacyl Photoremovable Protecting Group By Chamani Perera, Ph.D. Department of Chemistry, November 2009 University of Kansas The p-hydroxyphenacyl (pHP) chromophore is an attractive addition to the family of photoremovable protecting groups in chemistry and biology. Substituent effects on the pHP moiety were explored using cyano substituent at otho and para positions, employing GABA and acetate leaving groups. The quantum yield for GABA release from 3-cyano pHP GABA in water was substantially higher (0.42) than that for the parent. Two novel applications of pHP photochemistry were demonstrated. A light sensitive release of a fluorophore from an almost non-fluorescent pHP fluorophore quencher ensembles resulted in regenerated fluorescence upon exposure to ultraviolet radiation. Carbazole as the quencher leaving group drove the photorelease toward an electron-transfer, radical mechanism. A second application demonstrated that fluorophilic pHP derivatives obtained by attaching a fluorous chain, could be employed to separate the products, often in 95-100% yield, after photorelease from the cage by using fluorous solid phase extraction and fluorous liquid-liquid extraction separation technology

Fluorescent Mimics of Cholesterol that Rapidly Bind Surfaces of Living Mammalian Cells

Mammalian cells acquire cholesterol, a critical membrane constituent, through multiple mechanisms. We synthesized mimics of cholesterol, fluorescent N-alkyl-3β-cholesterylamine-glutamic acids, that are rapidly incorporated into cellular plasma membranes compared with analogous cholesteryl amides, ethers, esters, carbamates, and a sitosterol analogue. This process was inhibited by ezetimibe, indicating a receptor-mediated uptake pathway

Antibody–Drug Conjugate that Exhibits Synergistic Cytotoxicity with an Endosome–Disruptive Peptide

Antibody–drug conjugates are an important class of cancer therapeutics. These agents generally bind a specific cell surface receptor, undergo receptor-mediated endocytosis, and enter the endosomal–lysosomal system, where the environment in these organelles facilitates the release of a membrane-permeable cytotoxin. By using a membrane-impermeable cytotoxin, we describe here a method that allows the cytotoxicity of an antibody conjugate to be triggered by co-administration with an endosome-disruptive peptide that exhibits low toxicity. This approach was validated by conjugation of an anionic derivative of the tubulin-binding cytotoxin colchinol methyl ether to lysine residues of the HER2-targeting antibody trastuzumab (Herceptin) via a disulfide. When this antibody binds HER2 on SKBR3 breast cancer cells and undergoes endocytosis, the membrane-impermeable cytotoxin is released, but it becomes trapped in endosomes, resulting in relatively low cytotoxicity (IC50 > 1 μM). However, co-administration with an essentially nontoxic (IC50 > 10 μM) cholesterol-linked endosome-disruptive peptide promotes the release of this small molecule into the cytoplasm, conferring subnanomolar cytotoxic potency (IC50 = 0.11 ± 0.07 nM). Studies of a structurally related fluorophore conjugate revealed that the endosome-disruptive peptide does not substantially enhance cleavage of the disulfide (t1/2 = 8 ± 2 h) within endosomes, suggesting that the mechanism of endosomal escape involves the efflux of some small molecules without facilitating substantial influx of reduced glutathione

High-Throughput Screening for Bacterial Glycosyltransferase Inhibitors

This work is licensed under a Creative Commons Attribution 4.0 International License.The enteropathogenic and enterohemorrhagic Escherichia coli NleB proteins as well as the Salmonella enterica SseK proteins are type III secretion system effectors that function as glycosyltransferase enzymes to post-translationally modify host substrates on arginine residues. This modification is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles, and is distinct from the activity of the mammalian O-linked N-acetylglucosaminyltransferase. We conducted high-throughput screening assays to identify small molecules that inhibit NleB/SseK activity. Two compounds, 100066N and 102644N, both significantly inhibited NleB1, SseK1, and SseK2 activities. Addition of these compounds to cultured mammalian cells was sufficient to inhibit NleB1 glycosylation of the tumor necrosis factor receptor type 1-associated DEATH domain protein. These compounds were also capable of inhibiting Salmonella enterica strain ATCC 14028 replication in mouse macrophage-like cells. Neither inhibitor was significantly toxic to mammalian cells, nor showed in vitro cross-reactivity with the mammalian O-linked N-acetylglucosaminyltransferase. These compounds or derivatives generated from medicinal chemistry refinements may have utility as a potential alternative therapeutic strategy to antibiotics or as reagents to further the study of bacterial glycosyltransferases.National Institute of Allergy and Infectious Diseases grant number AI127973COBRE P20GM113117COBRE P20GM103638CMLD Legacy (GM111385) gran

Gas-phase Fragmentation of Deprotonated p-Hydroxyphenacyl Derivatives

Electrospray ionization of methanolic solutions of p-hydroxyphenacyl derivatives HO-C6H4-C(O)-CH2-X (X = leaving group) provides abundant signals for the deprotonated species which are assigned to the corresponding phenolate anions −O-C6H4-C(O)-CH2-X. Upon collisional activation in the gas phase, these anions inter alia undergo loss of a neutral “C8H6O2” species concomitant with formation of the corresponding anions X−. The energies required for the loss of neutral roughly correlate with the gas phase acidities of the conjugate acids (HX). Extensive theoretical studies performed for X = CF3COO in order to reveal the energetically most favorable pathway for the formation of neutral “C8H6O2” suggest three different routes of similar energy demands, involving a spirocyclopropanone, epoxide formation, and a diradical, respectively

Sterol profiling of Leishmania parasites using a new HPLC-tandem mass spectrometry-based method and antifungal azoles as chemical probes reveals a key intermediate sterol that supports a branched ergosterol biosynthetic pathway

Human leishmaniasis is an infectious disease caused by Leishmania protozoan parasites. Current chemotherapeutic options against the deadly disease have significant limitations. The ergosterol biosynthetic pathway has been identified as a drug target in Leishmania. However, remarkable differences in the efficacy of antifungal azoles that inhibit ergosterol biosynthesis have been reported for the treatment of leishmaniasis. To better understand the sterol biosynthetic pathway in Leishmania and elucidate the mechanism underlying the differential efficacy of antifungal azoles, we developed a new LC-MS/MS method to study sterol profiles in promastigotes of three Leishmania species, including two L. donovani, one L. major and one L. tarentolae strains. A combination of distinct precursor ion masses and LC retention times allowed for specific detection of sixteen intermediate sterols between lanosterol and ergosterol using the newly developed LC-MS/MS method. Although both posaconazole and fluconazole are known inhibitors of fungal lanosterol 14α-demethylase (CYP51), only posaconazole led to a substantial accumulation of lanosterol in azole-treated L. donovani promastigotes. Furthermore, a key intermediate sterol accumulated by 40- and 7-fold when these parasites were treated with posaconazole and fluconazole, respectively, which was determined as 4α,14α-dimethylzymosterol by high resolution mass spectrometry and NMR spectroscopy. The identification of 4α,14α-dimethylzymosterol supports a branched ergosterol biosynthetic pathway in Leishmania, where lanosterol C4- and C14-demethylation reactions occur in parallel rather than sequentially. Our results suggest that selective inhibition of leishmanial CYP51 is insufficient to effectively prevent parasite growth and dual inhibitors of both CYP51 and the unknown sterol C4-demethylase may be required for optimal antiparasitic effect

High-Throughput Screening for Bacterial Glycosyltransferase Inhibitors

The enteropathogenic and enterohemorrhagic Escherichia coli NleB proteins as well as the Salmonella enterica SseK proteins are type III secretion system effectors that function as glycosyltransferase enzymes to post-translationally modify host substrates on arginine residues. This modification is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles, and is distinct from the activity of the mammalian O-linked N-acetylglucosaminyltransferase. We conducted high-throughput screening assays to identify small molecules that inhibit NleB/SseK activity. Two compounds, 100066N and 102644N, both significantly inhibited NleB1, SseK1, and SseK2 activities. Addition of these compounds to cultured mammalian cells was sufficient to inhibit NleB1 glycosylation of the tumor necrosis factor receptor type 1-associated DEATH domain protein. These compounds were also capable of inhibiting Salmonella enterica strain ATCC 14028 replication in mouse macrophage-like cells. Neither inhibitor was significantly toxic to mammalian cells, nor showed in vitro cross-reactivity with the mammalian O-linked N-acetylglucosaminyltransferase. These compounds or derivatives generated from medicinal chemistry refinements may have utility as a potential alternative therapeutic strategy to antibiotics or as reagents to further the study of bacterial glycosyltransferases

Successful Treatment of Clonal Eosinophilia with FIP1L1-PDGFRA Rearrangement with Low Dose IMATINIB – A Case Report

Clonal eosinophilia with FIP1L1-PDGFRA rearrangement is a rare disorder which is almost exclusively seen in males.  Targeted therapy with Imatinib has led to successful achievement of molecular remission of this disorder but no evidence exists for continuous or maintenance therapy or treatment free remission yet. This is a case of clonal eosinophilia with FIP1L1-PDGFRA rearrangement in a young man, who presented with very high eosinophil and neutrophil counts, moderate thrombocytopenia, hepatosplenomegaly and skin and lung involvement. Imatinib at 100mg daily was started after initial treatment with cytoreductive therapy and steroids. He responded well to low dose Imatinib with complete absence of symptoms and normalization of counts within 3 months and disappearance of molecular evidence of the disease following 2 years of therapy. Imatinib was then tailed off and he remains asymptomatic on a weekly dose of 100mg Imatinib. Workup towards a diagnosis of eosinophilia has been made easy with the new classification and inclusion of molecular evaluation. The high eosinophil count, presence of organomegaly and thrombocytopenia favoured a clonal aetiology .Low dose Imatinib gives excellent clinical, haematological and molecular remission and can be safely reduced thereafter to weekly maintenance

Fungally Derived Isoquinoline Demonstrates Inducer-Specific Tau Aggregation Inhibition

The microtubule-associated protein tau promotes the stabilization of the axonal cytoskeleton in neurons. In several neurodegenerative diseases, such as Alzheimer’s disease, tau has been found to dissociate from microtubules, leading to the formation of pathological aggregates that display an amyloid fibril-like structure. Recent structural studies have shown that the tau filaments isolated from different neurodegenerative disorders have structurally distinct fibril cores that are specific to the disease. These “strains” of tau fibrils appear to propagate between neurons in a prion-like fashion that maintains their initial template structure. In addition, the strains isolated from diseased tissue appear to have structures that are different from those made by the most commonly used in vitro modeling inducer molecule, heparin. The structural differences among strains in different diseases and in vitro-induced tau fibrils may contribute to recent failures in clinical trials of compounds designed to target tau pathology. This study identifies an isoquinoline compound (ANTC-15) isolated from the fungus Aspergillus nidulans that can both inhibit filaments induced by arachidonic acid (ARA) and disassemble preformed ARA fibrils. When compared to a tau aggregation inhibitor currently in clinical trials (LMTX, LMTM, or TRx0237), ANTC-15 and LMTX were found to have opposing inducer-specific activities against ARA and heparin in vitro-induced tau filaments. These findings may help explain the disappointing results in translating potent preclinical inhibitor candidates to successful clinical treatments