Matrix metalloproteinase-9 (MMP-9) and its inhibitors in cancer: A minireview

Fluorine is a valuable probe for investigating the interactions of biological molecules because of its favorable NMR characteristics, its small size, and its near total absence from biology. Advances in biosynthetic methods allow fluorine to be introduced into peptides and proteins with high precision, and the increasing sensitivity of NMR spectrometers has facilitated the use of 19F NMR to obtain molecular-level insights into a wide range of often-complex biological interactions. Here, we summarize the advantages of solution-state 19F NMR for studying the interactions of peptides and proteins with other biological molecules, review methods for the production of fluorine-labeled materials, and describe some representative recent examples in which 19F NMR has been used to study conformational changes in peptides and proteins and their interactions with other biological molecules.

Using 19F NMR to Probe Biological Interactions of Proteins and Peptides

ConspectusDeciphering the fluorine code is how we describe not only the focus of this Account, but also the systematic approach to studying the impact of fluorine’s incorporation on the properties of peptides and proteins used by our groups and others. The introduction of fluorine has been shown to impart favorable, but seldom predictable, properties to peptides and proteins, but up until about two decades ago the outcomes of fluorine modification of peptides and proteins were largely left to chance. Driven by the motivation to extend the application of the unique properties of the element fluorine from medicinal and agro chemistry to peptide and protein engineering we have established extensive research programs that enable the systematic investigation of effects that accompany the introduction of fluorine into this class of biopolymers. The introduction of fluorine into amino acids offers a universe of options for modifications with regard to number and position of fluorine substituents in the amino aci...

Deciphering the Fluorine Code—The Many Hats Fluorine Wears in a Protein Environment

Fluoropolymers have unique physicochemical properties such as hydrophobicity and lipophobicity, good chemical stability and bio-inertness, low surface energy and phase segregation. Owing to these properties, fluoropolymers have been widely used to prepare high performance materials. Especially, the use of fluoropolymers in biomedical applications has grown rapidly during the past decade. This critical review focuses on the recent advances of fluoropolymers in gene delivery, cytosolic protein delivery, drug delivery, magnetic resonance imaging, photodynamic therapy, anti-fouling and anti-bacterial applications, and tissue engineering. The mechanisms and features of fluoropolymers in these specific applications are discussed. Besides, we have reviewed the methods to synthesize water-soluble fluoropolymers for the applications and explained their supramolecular assembly behaviors in solutions. Finally, the opportunities and challenges of fluoropolymers in biomedical applications are discussed.

Fluoropolymers in biomedical applications: state-of-the-art and future perspectives.

Cytosolic delivery of peptides remains a challenging task owing to their susceptibility to enzymatic degradation and the existence of multiple intracellular barriers. Here, we report a new strategy to address these issues by decoration of a fluorous tag on the terminal of cargo peptides. The fluorous-tagged peptides were assembled into nanostructures, efficiently internalized by cells via several endocytic pathways and released into the cytosol after endosomal escape. They were relatively stable against enzymatic degradation and showed much higher efficiency than nonfluorinated analogs and cell penetrant peptide–conjugated ones. The proposed strategy also efficiently delivered a proapoptotic peptide into specific sites in the cells and restored the function of cargo peptide after cytosolic delivery. The fluorous-tagged proapoptotic peptide efficiently inhibited tumor growth in vivo. This study provides an efficient fluorination strategy to promote the cytosolic delivery of peptides.

Fluoroalkylation promotes cytosolic peptide delivery.

Fluorinated amino acids can have dramatic effects on protein stability and protein–protein interactions due to the unique stereoelectronic properties of fluorine. Previous approaches to assessing their properties have mainly focused on helical systems, even though fluoro-amino acids are known to exhibit lower intrinsic helix propensities than their hydrocarbon analogues. Fluorination of specific β-sheet positions within globular proteins has been shown to have a stabilizing effect, suggesting that fluorinated amino acids may generally be well suitable for modulating non-helical structures. Still, fluorinated amino acids have rarely been studied in amyloid forming peptides, which take on a characteristically high cross-β-sheet content. Here, we examine the substitution of natural amino acids within an amyloid forming model peptide by amino acids that contain different stoichiometries of fluorine in their side chains. This approach enables a systematic evaluation of the impact of fluorine on amyloid formation. We have investigated the impact of size, hydrophobicity and secondary structure propensities of the fluorinated amino acids on the amyloid formation process. The structure of the model peptide is based on an engineered coiled coil folding motif that was designed to provide an α-helical starting structure that can fold into β-sheet rich amyloids under controlled conditions. Substitution with fluorinated amino acids was accomplished for two neighboring valine residues that play a key role in the structural transition. The resulting peptides show an unexpected folding behavior as a consequence of the interplay of stereoelectronic effects, helix propensity, hydrophobicity and position of the particular substitution within the amyloid forming system.

Fluorinated amino acids in amyloid formation: a symphony of size, hydrophobicity and α-helix propensity

New matrix metalloproteinase inhibitors based on γ-fluorinated α-aminocarboxylic and α-aminohydroxamic acids.

To study MMP activity in vivo in disease, several radiolabeled MMP inhibitors functioning as radiotracers have been evaluated by means of SPECT and PET. Unfortunately, most of them suffer from metabolic instability, mainly hepatobiliary clearance and insufficient target binding. The introduction of a fluorine atom into MMPIs could contribute to target binding, enhance the metabolic stability and might shift the clearance towards more renal elimination. Recently developed α-sulfonylaminohydroxamic acid based γ-fluorinated inhibitors of MMP-2 and -9 provide promising fluorine interactions with the enzyme active site and high MMP inhibition potencies. The aim of this study is the (radio)synthesis of a γ-fluorinated MMP-2 and -9 inhibitor to evaluate its potential as a radiotracer to image MMP activity in vivo. Two new fluorine-containing, enantiomerically pure inhibitors for MMP-2 and -9 were synthesized in a six step sequence. Both enantiomers exhibited equal inhibition potencies in the low nanomolar and subnanomolar range. LogD value indicated better water solubility compared to the CGS 25966 based analog. The most potent inhibitor was successfully radiofluorinated. In vivo biodistribution in wild type mice revealed predominantly hepatobiliary clearance. Two major radioactive metabolites were found in different organs. Defluorination of the radiotracer was not observed. (Radio)synthesis of a CGS based γ-fluorinated MMP inhibitor was successfully accomplished. The (S)-enantiomer, which normally shows no biological activity, also exhibited high MMP inhibition potencies, which may be attributed to additional interactions of fluorine with enzyme’s active site. Despite higher hydrophilicity no significant differences in the clearance characteristics compared to non-fluorinated MMPIs was observed. Metabolically stabilizing effect of the fluorine was not monitored in vivo in wild type mice.

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Synthesis, radiosynthesis, in vitro and first in vivo evaluation of a new matrix metalloproteinase inhibitor based on γ-fluorinated α-sulfonylaminohydroxamic acid

Malte Behrends

Papers

Fluorinated amino acids in amyloid formation: a symphony of size, hydrophobicity and α-helix propensity

New matrix metalloproteinase inhibitors based on γ-fluorinated α-aminocarboxylic and α-aminohydroxamic acids.

Synthesis, radiosynthesis, in vitro and first in vivo evaluation of a new matrix metalloproteinase inhibitor based on γ-fluorinated α-sulfonylaminohydroxamic acid