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1.
J Control Release ; 361: 694-716, 2023 09.
Article in English | MEDLINE | ID: mdl-37567507

ABSTRACT

Extracellular vesicles (EVs) are nanosized intercellular messengers that bear enormous application potential as biological drug delivery vehicles. Much progress has been made for loading or decorating EVs with proteins, peptides or RNAs using genetically engineered donor cells, but post-isolation loading with synthetic drugs and using EVs from natural sources remains challenging. In particular, quantitative and unambiguous data assessing whether and how small molecules associate with EVs versus other components in the samples are still lacking. Here we describe the systematic and quantitative characterisation of passive EV loading with small molecules based on hydrophobic interactions - either through direct adsorption of hydrophobic compounds, or by membrane anchoring of hydrophilic ligands via cholesterol tags. As revealed by single vesicle imaging, both ligand types bind to CD63 positive EVs (exosomes), however also non-specifically to other vesicles, particles, and serum proteins. The hydrophobic compounds Curcumin and Terbinafine aggregate on EVs with no apparent saturation up to 106-107 molecules per vesicle as quantified by liquid chromatography - high resolution mass spectrometry (LC-HRMS). For both compounds, high density EV loading resulted in the formation of a population of large, electron-dense vesicles as detected by quantitative cryo-transmission electron microscopy (TEM), a reduced EV cell uptake and a toxic gain of function for Curcumin-EVs. In contrast, cholesterol tagging of a hydrophilic mdm2-targeted cyclic peptide saturated at densities of ca 104-105 molecules per vesicle, with lipidomics showing addition to, rather than replacement of endogenous cholesterol. Cholesterol anchored ligands did not change the EVs' size or morphology, and such EVs retained their cell uptake activity without inducing cell toxicity. However, the cholesterol-anchored ligands were rapidly shed from the vesicles in presence of serum. Based on these data, we conclude that (1) both methods allow loading of EVs with small molecules but are prone to unspecific compound binding or redistribution to other components if present in the sample, (2) cholesterol anchoring needs substantial optimization of formulation stability for in vivo applications, whereas (3) careful titration of loading densities is warranted when relying on hydrophobic interactions of EVs with hydrophobic compounds to mitigate changes in physicochemical properties, loss of EV function and potential cell toxicity.


Subject(s)
Curcumin , Extracellular Vesicles , Ligands , Extracellular Vesicles/metabolism , Hydrophobic and Hydrophilic Interactions , Cholesterol/metabolism
2.
J Med Chem ; 65(12): 8345-8379, 2022 06 23.
Article in English | MEDLINE | ID: mdl-35500094

ABSTRACT

Balanced pan-class I phosphoinositide 3-kinase inhibition as an approach to cancer treatment offers the prospect of treating a broad range of tumor types and/or a way to achieve greater efficacy with a single inhibitor. Taking buparlisib as the starting point, the balanced pan-class I PI3K inhibitor 40 (NVP-CLR457) was identified with what was considered to be a best-in-class profile. Key to the optimization to achieve this profile was eliminating a microtubule stabilizing off-target activity, balancing the pan-class I PI3K inhibition profile, minimizing CNS penetration, and developing an amorphous solid dispersion formulation. A rationale for the poor tolerability profile of 40 in a clinical study is discussed.


Subject(s)
Antineoplastic Agents , Phosphatidylinositol 3-Kinases , Aminopyridines/pharmacology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Organic Chemicals , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use
3.
RSC Chem Biol ; 2(6): 1661-1668, 2021 Dec 02.
Article in English | MEDLINE | ID: mdl-34977581

ABSTRACT

Mouse double minute 2 homolog (MDM2, Hdm2) is an important negative regulator of the tumor suppressor p53. Using a mRNA based display technique to screen a library of >1012 in vitro-translated cyclic peptides, we have identified a macrocyclic ligand that shows picomolar potency on MDM2. X-Ray crystallography reveals a novel binding mode utilizing a unique pharmacophore to occupy the Phe/Trp/Leu pockets on MDM2. Conjugation of a cyclic cell-penetrating peptide (cCPP) to the initially non cell-permeable ligand enables cellular uptake and a pharmacodynamic response in SJSA-1 cells. The demonstrated enhanced intracellular availability of cyclic peptides that are identified by a display technology exemplifies a process for the application of intracellular tools for drug discovery projects.

5.
J Med Chem ; 63(21): 12542-12573, 2020 11 12.
Article in English | MEDLINE | ID: mdl-32930584

ABSTRACT

FGF19 signaling through the FGFR4/ß-klotho receptor complex has been shown to be a key driver of growth and survival in a subset of hepatocellular carcinomas, making selective FGFR4 inhibition an attractive treatment opportunity. A kinome-wide sequence alignment highlighted a poorly conserved cysteine residue within the FGFR4 ATP-binding site at position 552, two positions beyond the gate-keeper residue. Several strategies for targeting this cysteine to identify FGFR4 selective inhibitor starting points are summarized which made use of both rational and unbiased screening approaches. The optimization of a 2-formylquinoline amide hit series is described in which the aldehyde makes a hemithioacetal reversible-covalent interaction with cysteine 552. Key challenges addressed during the optimization are improving the FGFR4 potency, metabolic stability, and solubility leading ultimately to the highly selective first-in-class clinical candidate roblitinib.


Subject(s)
Piperazines/chemistry , Protein Kinase Inhibitors/chemistry , Pyridines/chemistry , Receptor, Fibroblast Growth Factor, Type 4/antagonists & inhibitors , Amino Acid Sequence , Animals , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Cysteine/chemistry , Dogs , Drug Design , Half-Life , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Liver Neoplasms/drug therapy , Mice , Microsomes, Liver/metabolism , Molecular Dynamics Simulation , Piperazines/metabolism , Piperazines/pharmacology , Piperazines/therapeutic use , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyridines/metabolism , Pyridines/pharmacology , Pyridines/therapeutic use , Rats , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Structure-Activity Relationship , Xenograft Model Antitumor Assays
6.
ACS Med Chem Lett ; 9(3): 215-220, 2018 Mar 08.
Article in English | MEDLINE | ID: mdl-29541363

ABSTRACT

As part of a project to identify FGFR4 selective inhibitors, scaffold morphing of a 2-formylquinoline amide hit identified series of 2-formylpyridine ureas (2-FPUs) with improved potency and physicochemical properties. In particular, tetrahydronaphthyridine urea analogues with cellular activities below 30 nM have been identified. Consistent with the hypothesized reversible-covalent mechanism of inhibition, the 2-FPUs exhibited slow binding kinetics, and the aldehyde, as the putative electrophile, could be demonstrated to be a key structural element for activity.

7.
Rech Soins Infirm ; (110): 113-21, 2012 Sep.
Article in French | MEDLINE | ID: mdl-23091921

ABSTRACT

The research presented below examines the focus charting model in French nursing practice. Between the objectives targeted by this model and actual practice, where to place the reflective process of the nurse? To answer this question, the methodology used is the comprehensive approach. It is characterized by the production of semi-structured interviews of nurses using the model studied, but also by the in situ observation of practices. The results show that nurses engaged a reflexive process in the use of the focus charting model. This reflexive process is "in" the action rather than "on" or "for" the action. Nurse's position vis a vis at their disposal is investigated.


Subject(s)
Nursing Records , Observation , Humans
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