No abstract

The delivery of genetic material into plants has been historically challenging due to the cell wall barrier, which blocks the passage of many biomolecules. Carbon nanotube-based delivery has emerged as a promising solution to this problem and has been shown to effectively deliver DNA and RNA into intact plants. Mitochondria are important targets due to their influence on agronomic traits, but delivery into this organelle has been limited to low efficiencies, restricting their potential in genetic engineering. This work describes the use of a carbon nanotube-polymer hybrid modified with functional peptides to deliver DNA into intact plant mitochondria with almost 30 times higher efficiency than existing methods. Genetic integration of a folate pathway gene in the mitochondria displays enhanced plant growth rates, suggesting its applications in metabolic engineering and the establishment of stable transformation in mitochondrial genomes. Furthermore, the flexibility of the polymer layer will also allow for the conjugation of other peptides and cargo targeting other organelles for broad applications in plant bioengineering.

Human enzyme aldo-keto reductase family member 1B10 (AKR1B10) has evolved as a tumor marker and promising antineoplastic target. It shares high structural similarity with the diabetes target enzyme aldose reductase (AR). Starting from the potent AR inhibitor IDD388, we have synthesized a series of derivatives bearing the same halophenoxyacetic acid moiety with an increasing number of bromine (Br) atoms on its aryl moiety. Next, by means of IC measurements, X-ray crystallography, WaterMap analysis, and advanced binding free energy calculations with a quantum-mechanical (QM) approach, we have studied their structure-activity relationship (SAR) against both enzymes. The introduction of Br substituents decreases AR inhibition potency but improves it in the case of AKR1B10. Indeed, the Br atoms in ortho position may impede these drugs to fit into the AR prototypical specificity pocket. For AKR1B10, the smaller aryl moieties of MK181 and IDD388 can bind into the external loop A subpocket. Instead, the bulkier MK184, MK319, and MK204 open an inner specificity pocket in AKR1B10 characterized by a π-π stacking interaction of their aryl moieties and Trp112 side chain in the native conformation (not possible in AR). Among the three compounds, only MK204 can make a strong halogen bond with the protein (-4.4 kcal/mol, using QM calculations), while presenting the lowest desolvation cost among all the series, translated into the most selective and inhibitory potency AKR1B10 (IC = 80 nM). Overall, SAR of these IDD388 polyhalogenated derivatives have unveiled several distinctive AKR1B10 features (shape, flexibility, hydration) that can be exploited to design novel types of AKR1B10 selective drugs.

Cell-penetrating peptides (CPPs) have been widely utilized as efficient molecular tools for the delivery of bioactive cargoes such as peptides, proteins, and genetic material. However, to improve their versatility as tools in biological environments, the resistance of CPPs to enzymatic degradation and their structural stability must be improved to achieve long-term efficacy. Here we designed and synthesized novel artificial CPPs, poly(LysAibXaa), containing periodic α-aminoisobutyric acid (Aib) and L-lysine by chemoenzymatic polymerization. Poly(LysAibAla) tended to form 3 10 -and α-helical structures under the amphiphilic cell-membrane-mimicking environment. Poly(LysAibXaa) exhibited long-term internalization and thus high accumulation in live cells, which is attributed to the improvement in the resistance to proteolytic digestion as a result of the incorporation of Aib residues into the peptide backbone. We presented a simple molecular design and synthesis of efficient CPPs applicable to both human and plant cells with long-term stability and negligible cytotoxicity.

Human aldo-keto reductase 1B15 (AKR1B15) is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower k cat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers all-trans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15.