Synthesis and Biological Studies on (KLAKLAK)2-NH2 Analog Containing Unnatural Amino Acid β-Ala and Conjugates with Second Pharmacophore

Abstract

(1) Background: Peptides are good candidates for anticancer drugs due to their natural existence in the body and lack of secondary effects. (KLAKLAK)2 is an antimicrobial peptide that also shows good anticancer properties. (2) Methods: The Solid Phase Peptide Synthesis (Fmoc-strategy) was used for the synthesis of target molecules, analogs of (KLAKLAK)2-NH2. The purity of all compounds was monitored by HPLC, and their structures were proven using mass spectrometry. Cytotoxicity and antiproliferative effects were studied using 3T3 NRU and MTT tests, respectively. For determination of antimicrobial activity, the disc-diffusion method was used. Hydrolytic stability at three pH values, which mimic the physiological pH in the body, was investigated by means of the HPLC technique. (3) Results: A good selective index against MCF-7 tumor cell lines, combined with good cytotoxicity and antiproliferative properties, was revealed for conjugates NphtG-(KLAKLAK)2-NH2 and Caf-(KLAKLAK)2-NH2. The same compounds showed very good antifungal properties and complete hydrolytic stability for 72 h. The compound Caf-(KLβ-AKLβ-AK)2-NH2 containing β-Ala in its structures exhibited good antimicrobial activity against Escherichia coli K12 407 and Bacillus subtilis 3562, in combination with very good antiproliferative and cytotoxic properties, as well as hydrolytic stability. (4) Conclusions: The obtained results reveal that all synthesized conjugates could be useful for medical practice as anticancer or antimicrobial agents.

Interaction of KLAKLAK-NH2 and Analogs with Biomimetic Membrane Models

“…The introduction of the β-amino analog of Ala into the primary structure of KLAKLAK-NH 2 peptides producing more rigid and conformationally confined amphiphilic structures (P2, P3 and P3-long peptides) results here in the most important alterations of the physicochemical properties of the lipid membrane. These alterations are related to the antiproliferative and antimicrobial activity of the studied peptides reported so far [ 18 , 19 ]. The short P3-analog has been shown to exhibit a high antiproliferative effect against mammary gland type A adenocarcinoma cells (MCF-7) [ 18 ], while the doubled P3-long peptide combines low cytotoxicity and high antibacterial activity [ 19 ].…”

“…The purity of obtained peptides and their structure were analyzed using HPLC-MS. The chromatographic purity was 100% for peptides P1, P2 and P3-long and 99.14% for the peptide P3 [ 18 , 19 ].…”

“…However, our previous studies on (KLAKLAK) 2 -NH 2 analogs revealed that a single KLAKLAK-NH 2 sequence has moderate activity against the Gram-negative test microorganism E. coli K12 407 at a 20 µM concentration, but its analogs containing unnatural amino acid β-Ala and nor-Leu in the primary structure do not show any activity [ 18 ]. In addition, if unnatural amino acids β-Ala and nor-Leu are added to the primary structure of double-sequenced analogues (KLβAKLβAK) 2 -NH 2 and (Knor-LAKnor-LAK) 2 -NH 2 , antibacterial activity is enlarged against the Gram-positive microorganisms of the strain Bacillus subtilis 3562 [ 19 , 20 ]. Often, peptide molecules are used as vehicles in order to transport active molecules in the cell due to their ability to penetrate the membrane [ 21 , 22 ].…”

“…In addition, our studies showed that KLAKLAK-NH 2 analogs could be successfully used as such as vehicles to transport a second pharmacophore, a very well-known naphtalimide with proven anticancer properties [ 23 ]. Moreover, both parts of the molecule showed a synergic antiproliferative effect [ 18 , 19 ].…”

Non‐canonical amino acid bioincorporation into antimicrobial peptides and its challenges

“…Another method gaining traction is the incorporation of non-canonical amino acids (ncAAs) into AMP sequences. Incorporation of ncAAs has resulted in decreased cytotoxicity, enhanced potency, improved bioavailability, and resistance to proteolysis among other benefits [20][21][22][23] and has to date been mostly facilitated by chemical synthesis of AMPs incorporating ncAAs.…”