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1.
PLoS One ; 19(3): e0299541, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38551930

RESUMO

The activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes.


Assuntos
Corantes Fluorescentes , Fosfatidilinositóis , Fosfolipase C gama , Diester Fosfórico Hidrolases , Cumarínicos/farmacologia , Fosfolipases Tipo C
2.
Alzheimers Dement (N Y) ; 9(4): e12429, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38023622

RESUMO

INTRODUCTION: The risk of developing Alzheimer's disease is associated with genes involved in microglial function. Inositol polyphosphate-5-phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target. METHODS: We identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho-AKT levels provided further evidence of on-target pharmacology. A high-content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies. RESULTS: SHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration. DISCUSSION: 3-((2,4-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies. Highlights: Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain-contaning inositol phosphatase 1 (SHIP1) target engagement and on-target activity in cellular assays.A phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health.SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic.The chemical probe 3-((2,4-dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine is recommended to explore SHIP1 pharmacology.

3.
SLAS Discov ; 28(4): 170-179, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36933698

RESUMO

A rare coding variant in PLCγ2 (P522R) expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type. This mutation is reported to be protective against the cognitive decline associated with late-onset Alzheimer's disease (LOAD) and therefore, activation of wild-type PLCγ2 has been suggested as a potential therapeutic target for the prevention and treatment of LOAD. Additionally, PLCγ2 has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLCγ2 activity have been identified. Here, pharmacological inhibition may provide a therapeutic effect. In order to facilitate our investigation of the activity of PLCγ2, we developed an optimized fluorogenic substrate to monitor enzymatic activity in aqueous solution. This was accomplished by first exploring the spectral properties of various "turn-on" fluorophores. The most promising turn-on fluorophore was incorporated into a water-soluble PLCγ2 reporter substrate, which we named C8CF3-coumarin. The ability of PLCγ2 to enzymatically process C8CF3-coumarin was confirmed, and the kinetics of the reaction were determined. Reaction conditions were optimized to identify small molecule activators, and a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed with the goal of identifying small molecule activators of PLCγ2. The optimized screening conditions allowed identification of potential PLCγ2 activators and inhibitors, thus demonstrating the feasibility of this approach for high-throughput screening.


Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Corantes Fluorescentes , Fosfolipase C gama/genética , Ensaios de Triagem em Larga Escala , Cumarínicos
4.
J Glaucoma ; 26(2): e101-e102, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28002188

RESUMO

PURPOSE: To date, there have only been 5 reported cases of orbital cellulitis following implantation of an aqueous tube shunt for glaucoma. Previously reported cases have involved eyes with significant comorbidities and successful management has often required the removal of the device alongside systemic antibiotic therapy. PATIENTS AND METHODS: We present a 53-year-old man with severe orbital cellulitis, 3 months after routine implantation of a Baerveldt tube shunt for primary open angle glaucoma. The patient was managed medically, with topical and systemic antibiotic therapy. The patient went on to make a full recovery with the tube in situ. RESULTS AND CONCLUSIONS: We report that a more conservative approach (without tube removal) to be successful in a case where there is no evidence of tube exposure. It is important to appreciate that in some cases of orbital cellulitis without clear signs of intraocular involvement, a tube can be left in situ.


Assuntos
Implantes para Drenagem de Glaucoma , Glaucoma de Ângulo Aberto/cirurgia , Celulite Orbitária/etiologia , Complicações Pós-Operatórias , Combinação Amoxicilina e Clavulanato de Potássio/uso terapêutico , Antibacterianos/uso terapêutico , Quimioterapia Combinada , Floxacilina/uso terapêutico , Humanos , Pressão Intraocular/fisiologia , Levofloxacino/uso terapêutico , Masculino , Pessoa de Meia-Idade , Celulite Orbitária/diagnóstico , Celulite Orbitária/tratamento farmacológico , Estudos Retrospectivos , Tomografia Computadorizada por Raios X
5.
J Med Chem ; 59(8): 3840-53, 2016 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-27070999

RESUMO

Several indenoisoquinolines have shown promise as anticancer agents in clinical trials. Incorporation of a nitrogen atom into the indenoisoquinoline scaffold offers the possibility of favorably modulating ligand-binding site interactions, physicochemical properties, and biological activities. Four series of aza-A-ring indenoisoquinolines were synthesized in which the nitrogen atom was systematically rotated through positions 1, 2, 3, and 4. The resulting compounds were tested to establish the optimal nitrogen position for topoisomerase IB (Top1) enzyme poisoning activity and cytotoxicity to human cancer cells. The 4-aza compounds were the most likely to yield derivatives with high Top1 inhibitory activity. However, the relationship between structure and cytotoxicity was more complicated since the potency was influenced strongly by the side chains on the lactam nitrogen. The most cytotoxic azaindenoisoquinolines 45 and 46 had nitrogen in the 2- or 3-positions and a 3'-dimethylaminopropyl side chain, and they had MGM GI50 values that were slightly better than the corresponding indenoisoquinoline 64.


Assuntos
Isoquinolinas/química , Isoquinolinas/farmacologia , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Relação Estrutura-Atividade
6.
Bioorg Med Chem ; 24(7): 1469-79, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26906474

RESUMO

Fluorine and chlorine are metabolically stable, but generally less active replacements for a nitro group at the 3-position of indenoisoquinoline topoisomerase IB (Top1) poisons. A number of strategies were employed in the present investigation to enhance the Top1 inhibitory potencies and cancer cell growth inhibitory activities of halogenated indenoisoquinolines. In several cases, the new compounds' activities were found to rival or surpass those of similarly substituted 3-nitroindenoisoquinolines, and several unusually potent analogs were discovered through testing in human cancer cell cultures. A hydroxyethylaminopropyl side chain on the lactam nitrogen of two halogenated indenoisoquinoline Top1 inhibitors was found to also impart inhibitory activity against tyrosyl DNA phosphodiesterases 1 and 2 (TDP1 and TDP2), which are enzymes that participate in the repair of DNA damage induced by Top1 poisons.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Indenos/farmacologia , Isoquinolinas/farmacologia , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/farmacologia , Relação Dose-Resposta a Droga , Humanos , Indenos/síntese química , Indenos/química , Isoquinolinas/síntese química , Isoquinolinas/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/química
7.
J Med Chem ; 58(9): 3997-4015, 2015 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-25909279

RESUMO

3-Nitroindenoisoquinoline human topoisomerase IB (Top1) poisons have potent antiproliferative effects on cancer cells. The undesirable nitro toxicophore could hypothetically be replaced by other functional groups that would retain the desired biological activities and minimize potential safety risks. Eleven series of indenoisoquinolines bearing 3-nitro bioisosteres were synthesized. The molecules were evaluated in the Top1-mediated DNA cleavage assay and in the National Cancer Institute's 60 cell line cytotoxicity assay. The data reveal that fluorine and chlorine may substitute for the 3-nitro group with minimal loss of Top1 poisoning activity. The new information gained from these efforts can be used to design novel indenoisoquinolines with improved safety.


Assuntos
Antineoplásicos/química , Indenos/química , Isoquinolinas/química , Inibidores da Topoisomerase I/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligação de Hidrogênio , Indenos/síntese química , Indenos/farmacologia , Isoquinolinas/síntese química , Isoquinolinas/farmacologia , Conformação Molecular , Ligação Proteica , Teoria Quântica , Estereoisomerismo , Relação Estrutura-Atividade , Termodinâmica , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/farmacologia
8.
J Med Chem ; 57(4): 1495-512, 2014 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-24517248

RESUMO

Carbohydrate moieties were strategically transported from the indolocarbazole topoisomerase I (Top1) inhibitor class to the indenoisoquinoline system in search of structurally novel and potent Top1 inhibitors. The syntheses and biological evaluation of 20 new indenoisoquinolines glycosylated with linear and cyclic sugar moieties are reported. Aromatic ring substitution with 2,3-dimethoxy-8,9-methylenedioxy or 3-nitro groups exerted strong effects on antiproliferative and Top1 inhibitory activities. While the length of the carbohydrate side chain clearly correlated with antiproliferative activity, the relationship between stereochemistry and biological activity was less clearly defined. Twelve of the new indenoisoquinolines exhibit Top1 inhibitory activity equal to or better than that of camptothecin. An advanced synthetic intermediate from this study was also used to efficiently prepare indotecan (LMP400) and indimitecan (LMP776), two anticancer agents currently under investigation in a Phase I clinical trial at the National Institutes of Health.


Assuntos
Antineoplásicos/síntese química , Benzodioxóis/síntese química , Isoquinolinas/síntese química , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/farmacologia , Carboidratos/química , Cromatografia Líquida de Alta Pressão , Ciclização , Avaliação Pré-Clínica de Medicamentos , Isoquinolinas/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Espectrometria de Massas por Ionização por Electrospray
9.
J Med Chem ; 56(6): 2581-605, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23472886

RESUMO

Nuclear receptors, such as the retinoid X receptor (RXR), are proteins that regulate a myriad of cellular processes. Molecules that function as RXR agonists are of special interest for the prevention and control of carcinogenesis. The majority of these ligands possess an acidic moiety that is believed to be key for RXR activation. This communication presents the design, synthesis, and biological evaluation of both acidic and nonacidic indenoisoquinolines as new RXR ligands. In addition, a comprehensive structure-activity relationship study is presented that identifies the important features of the indenoisoquinoline rexinoids. The ease of modification of the indenoisoquinoline core and the lack of the necessity of a carboxyl group for activity make them an attractive and unusual family of RXR agonists. This work establishes a structural foundation for the design of new and novel rexinoid cancer chemopreventive agents.


Assuntos
Anticarcinógenos/síntese química , Anticarcinógenos/farmacologia , Desenho de Fármacos , Isoquinolinas/síntese química , Isoquinolinas/farmacologia , Anticarcinógenos/química , Anticarcinógenos/metabolismo , Técnicas de Química Sintética , Humanos , Isoquinolinas/química , Isoquinolinas/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Receptores de Calcitriol/metabolismo , Receptor X Retinoide alfa/agonistas , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/metabolismo , Relação Estrutura-Atividade
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