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1.
Nat Chem Biol ; 20(1): 93-102, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37679459

RESUMO

Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.


Assuntos
Ciclinas , Ubiquitina-Proteína Ligases , Ciclinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteólise , Relação Estrutura-Atividade
2.
Bioconjug Chem ; 29(1): 35-43, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29243926

RESUMO

Hyperbranched polyamidoamines (h-PAMAM) were prepared using a one-pot reaction to have similar molecular weight to third generation PAMAM (G3-PAMAM) dendrimers, and then functionalized with N-diazeniumdiolate nitric oxide (NO) donors. A wide range of NO storage capacities (∼1-2.50 µmol mg-1) and NO-release kinetics (t1/2 ∼30-80 min) were achieved by changing the extent of propylene oxide (PO) modification. The therapeutic potential of these materials was evaluated by studying their antibacterial activities and toxicity against common dental pathogens and human gingival fibroblast cells, respectively. Our results indicate that the combination of NO release and PO modification is necessary to yield h-PAMAM materials with efficient bactericidal action without eliciting unwarranted cytotoxicity. Of importance, NO-releasing PO-modified h-PAMAM polymers exhibited comparable biological properties (i.e., antibacterial action and cytotoxicity) to defect-free G3-PAMAM dendrimers, but at a substantially lower synthetic burden.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Dendrímeros/química , Dendrímeros/farmacologia , Doadores de Óxido Nítrico/química , Doadores de Óxido Nítrico/farmacologia , Compostos Azo/química , Compostos Azo/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Linhagem Celular , Humanos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade
3.
Biomacromolecules ; 19(4): 1189-1197, 2018 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-29551064

RESUMO

Low and high molecular weight alginate biopolymers were chemically modified to store and release potentially therapeutic levels of nitric oxide (NO). Carbodiimide chemistry was first used to modify carboxylic acid functional groups with a series of small molecule alkyl amines. The resulting secondary amines were subsequently converted to N-diazeniumdiolate NO donors via reaction with NO gas under basic conditions. NO donor-modified alginates stored between 0.4-0.6 µmol NO·mg-1. In aqueous solution, the NO-release kinetics were diverse (0.3-13 h half-lives), dependent on the precursor amine structure. The liberated NO showed bactericidal activity against Pseudomonas aeruginosa and Staphylococcus aureus with pathogen eradication efficiency dependent on both molecular weight and NO-release kinetics. The combination of lower molecular weight (∼5 kDa) alginates with moderate NO-release durations (half-life of ∼4 h) resulted in enhanced killing of both planktonic and biofilm-based bacteria. Toxicity against human respiratory epithelial (A549) cells proved negligible at NO-releasing alginate concentrations required to achieve a 5-log reduction in viability in the biofilm eradication assay.


Assuntos
Alginatos/farmacologia , Biopolímeros/química , Proliferação de Células/efeitos dos fármacos , Óxido Nítrico/farmacologia , Células A549 , Alginatos/química , Aminas/química , Antibacterianos/química , Antibacterianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos Azo/química , Biopolímeros/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Peso Molecular , Óxido Nítrico/química , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos
4.
Mol Pharm ; 12(10): 3569-74, 2015 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-26287799

RESUMO

The rapid decomposition of nitric oxide (NO) donors in aqueous environments remains a limitation for applications requiring extended NO release. Herein, we report the synthesis of dipalmitoylphosphatidylcholine-based liposomes capable of extended NO release using low molecular weight NO donors and a reverse-phase evaporation technique. The encapsulation of the NO donors within the liposomes enabled both prolonged NO release and enhanced storage compared to free NO donors alone. The NO-releasing liposomes also demonstrated enhanced efficacy against human pancreatic cancer cells. These NO-release vehicles represent attractive anticancer therapeutics due to their potential to store the majority of their NO payload until reaching cancerous tissue at which time the lower pH inherent to such environments will trigger an avalanche of NO.


Assuntos
Compostos Azo/administração & dosagem , Composição de Medicamentos/métodos , Lipossomos/uso terapêutico , Doadores de Óxido Nítrico/administração & dosagem , Compostos Azo/uso terapêutico , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Estabilidade de Medicamentos , Humanos , Lipossomos/administração & dosagem , Neoplasias Pancreáticas/tratamento farmacológico
5.
Mol Pharm ; 11(2): 645-50, 2014 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-24423090

RESUMO

Heparin is a widely used anticoagulant due to its ability to inhibit key components in the coagulation cascade such as Factor Xa and thrombin (Factor IIa). Its potential to preferentially bind to antithrombin (ATIII) results in a conformational change and activation that leads to the prevention of fibrin formation from fibrinogen and ultimately obstructs a hemostatic plug from forming. Nitric oxide (NO) exhibits potent antiplatelet activity attributed to its capacity to increase the amount of cyclic guanosine monophosphate (cGMP) within platelets, which decreases the Ca(2+) concentration required for platelet activation. Currently there is no single agent that combines the functions of both antiplatelet and anticoagulant (anti-Xa and anti-IIa) activities to effectively block both the extrinsic and the intrinsic coagulation pathways. The research reported herein demonstrates the ability to combine the physiological capabilities of both heparin and NO into one functional compound via use of a spermine derivative of heparin, thus enabling formation of a novel diazeniumdiolate (NONOate). The heparin-spermine NONOate has a half-life of 85 min at 25 °C (pH 7.4). The heparin backbone of the conjugate maintains its anticoagulant activity as demonstrated via an anti-Xa assay, providing an anticoagulant conversion of 3.6 µg/mL of the heparin-spermine-NONO conjugate being equivalent to 2.5 µg/mL (0.50 IU/mL) of underivatized heparin in terms of anti-Xa activity. Using standard platelet aggregometry, it is shown that the functionality of the NO release portion of the heparin conjugate prevents (nearly 100%) platelet aggregation in the presence of adenosine diphosphate (ADP, platelet agonist).


Assuntos
Heparina/química , Óxido Nítrico/química , Anticoagulantes/farmacologia , Heparina/farmacologia , Estrutura Molecular , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Espermina/química , Espermina/farmacologia
6.
ACS Biomater Sci Eng ; 6(1): 433-441, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-32671191

RESUMO

Nitric oxide (NO) is a broad-spectrum antibacterial agent, making it an attractive alternative to traditional antibiotics for treating infections. To date, a direct comparison of the antibacterial activity of gaseous NO (gNO) versus water-soluble NO-releasing biopolymers has not been reported. In this study, the bactericidal action of NO-releasing chitosan oligosaccharides was compared to gNO treatment against cystic fibrosis-relevant Gram-positive and Gram-negative bacteria. A NO exposure chamber was constructed to enable the dosing of bacteria with gNO at concentrations up to 800 ppm under both aerobic and anaerobic conditions. Bacteria viability, solution properties (i.e., pH, NO concentration), and toxicity to mammalian cells were monitored to ensure a thorough understanding of bactericidal action and reproducibility for each delivery method. The NO-releasing chitosan oligosaccharides required significantly lower NO doses relative to gNO therapy to elicit antibacterial action against Pseudomonas aeruginosa and Staphylococcus aureus under both aerobic and anaerobic conditions. Reduced NO doses required for bacteria eradication using water-soluble NO-releasing chitosan were attributed to the release of NO in solution, removing the need to transfer from gas to liquid phase and the associated long diffusion distances of gNO treatment.


Assuntos
Antibacterianos , Óxido Nítrico , Animais , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Reprodutibilidade dos Testes
7.
RSC Adv ; 7(84): 53236-53246, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-30740219

RESUMO

In this study, fast and slow nitric oxide (NO)-releasing liposomes (half-lives of 2.5 and >72 h, respectively) were prepared by encapsulation of N-propyl-1,3-propanediamine/NO (PAPA/NO) and diethylenetriamine/NO (DETA/NO), respectively, via reverse phase evaporation. The anticancer activity of the otherwise equivalent fast and slow NO-releasing systems was evaluated against several distinct pancreatic, colorectal, and breast cancer cell lines. The anticancer assays (via cytotoxicity) over 72 h revealed that the slower NO-releasing liposomes consistently required lower NO payloads (LD50 <3 µg/mL) relative to the fast NO-release system (LD50 >6 µg/mL) to elicit cytotoxicity. The mechanism of intracellular NO build-up in cancer cells was studied using confocal fluorescence microscopy and flow cytometry, the results of which indicated that a more gradual NO accumulation was characteristic of the slow NO-release system. Protein expression via Western blot analysis revealed that slower NO release resulted in more necrotic/apoptotic cells, while faster release reduced the number of mitotic cells to a greater extent. Overall, these studies demonstrate the potential of NO-releasing liposomes for anticancer therapy and highlight the significance of release kinetics (and NO payloads) required to induce cell death.

8.
ACS Biomater Sci Eng ; 3(9): 2136-2143, 2017 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32309633

RESUMO

We report the ability to readily tune NO release from N-diazeniumdiolate-encapsulated liposomal structures by altering the NO donor molecule structure and/or phospholipid composition (independently or in combination). While encapsulating more stable NO donors expectedly enhanced the NO release (up to 48 h) from the liposomes, the phospholipid headgroup surface area proved equally useful in controlling NO-release kinetics by influencing the water uptake and concomitant N-diazeniumdiolate NO donor breakdown (to NO). The potential therapeutic utility of the NO-releasing liposomes was further assessed in biological/proteinaceous fluids. The NO-release kinetics were similar in buffer and serum.

9.
Polym Chem ; 8(16): 2552-2558, 2017 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-29057011

RESUMO

Chitosan was selectively monophosphorylated via reaction with phosphorus oxychloride (POCl3) to enhance water solubility while avoiding polyphosphate formation. The use of POCl3 resulted in negligible product degradation (i.e., breakdown of O-glycosidic bonds) even after a 3 d reaction period (<5% weight loss). X-ray photoelectron spectroscopy (XPS) characterization of the POCl3-phosphorylated chitosan (P-chitosan) revealed a phosphorus to nitrogen (P/N) atomic ratio of 0.30. Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy verified the monophosphorylation of chitosan's primary and secondary alcohols, and primary amines. The calcium chelation efficiency for the phosphorylated product approached 0.05 mg Ca2+ per mg of P-chitosan as measured by inductively coupled plasma-optical emission spectrometry (ICP-OES), indicating improved chelation over native chitosan. This selective monophosphorylation approach proved useful for modifying other biopolymers, including cellulose and alginate.

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