Predrag Cudic

Effects of cyclic lipodepsipeptide structural modulation on stability, antibacterial activity, and human cell toxicity.

Bacterial infections are becoming increasingly difficult to treat due to the development and spread of antibiotic resistance. Therefore, identifying novel antibacterial targets and new antibacterial agents capable of treating infections by drug‐resistant bacteria is of vital importance. The structurally simple yet potent fusaricidin or LI‐F class of natural products represents a particularly attractive source of candidates for the development of new antibacterial agents. We synthesized 18 fusaricidin/LI‐F analogues and investigated the effects of structure modification on their conformation, serum stability, antibacterial activity, and toxicity toward human cells. Our findings show that substitution of an ester bond in depsipeptides with an amide bond may afford equally potent analogues with improved stability and greatly decreased cytotoxicity. The lower overall hydrophobicity/amphiphilicity of amide analogues in comparison with their parent depsipeptides, as indicated by HPLC retention times, may explain the dissociation of antibacterial activity and human cell cytotoxicity. These results indicate that amide analogues may have significant advantages over fusaricidin/LI‐F natural products and their depsipeptide analogues as lead structures for the development of new antibacterial agents.

In vitro and in vivo activities of novel cyclic lipopeptides against staphylococcal biofilms.

A worldwide public health problem has resulted from the alarming spread of multi-drug resistant bacteria combined with the frequent occurrence of biofilm-type infections, creating a growing need for new therapies. In this study, we have demonstrated that novel cyclic lipopeptides, such as 1, cyclo-[D-Ala-(12-guanidinododecanoyl)Thr-D-Val-Val-DaThr-D-Asn], and 2, cyclo-[D-Ala-(12-guanidinododecanoyl)Dap-D-Val-Val-D-aThr-D-Asn], derived from the fusaricidin/ LI-F natural products efficiently inhibit the growth of Staphylococcus aureus biofilm in vitro at their minimum inhibitory concentrations (MICs). Complete S. aureus biofilm eradication was observed at 3 x MIC for 1 and 4 x MIC for 2. Promising in vivo activity was demonstrated by the ability of depsipeptide 1 to reduce the proliferation of methicillinresistant S. aureus US300 in a porcine wound model. Due to their unique structure and potent antibacterial and antibiofilm activities, cyclic lipopeptides that belong to the fusaricidin/LI-F family of antibiotics represent particularly attractive lead structures for the development of new antibacterial agents capable of treating complicated biofilm-associated infections.

Development of second generation peptides modulating cellular adiponectin receptor responses.

The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM—low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10–1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.

Effect of ester to amide or N-methylamide substitution on bacterial membrane depolarization and antibacterial activity of novel cyclic lipopeptides.

Cyclic lipopeptides derived from the fusaricidin/LI‐F family of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents. In comparison with natural products, these derivatives may offer better stability under physiologically relevant conditions and lower nonspecific toxicity, while preserving their antibacterial activity. In this study we assessed the ability of cyclic lipodepsipeptide 1 and its analogues—amide 2, N‐methylamide 3, and linear peptide 4—to interact with the cytoplasmic membranes of selected Gram‐positive bacteria. We also investigated their bacteriostatic/bactericidal modes of action and in vivo potency by using a Galleria mellonella model of MRSA infection. Cyclic lipopeptides 1 and 2 depolarize the cytoplasmic membranes of Gram‐positive bacteria in a concentration‐dependent manner. The degree of membrane depolarization was influenced by the structural and physical properties of 1 and 2, with the more flexible and hydrophobic peptide 1 being most efficient. However, membrane depolarization does not correlate with bacterial cell lethality, suggesting that membrane‐targeting activity is not the main mode of action for this class of antibacterial peptides. Conversely, substitution of the depsipeptide bond in 1 with an N‐methylamide bond in 3, or its hydrolysis to peptide 4, lead to a complete loss of antibacterial activity and indicate that the conformation of cyclic lipopeptides plays a role in their antibacterial activities. Cyclic lipopeptides 1 and 2 are also capable of improving the survival of G. mellonella larvae infected with MRSA at varying efficiencies, reflecting their in vitro activities. Gaining more insight into the structure–activity relationship and mode of action of these cyclic lipopeptides may enable the development of new antibiotics of this class with improved antibacterial activity.

Solid‐phase synthesis of fusaricidin/li‐f class of cyclic lipopeptides: Guanidinylation of resin‐bound peptidyl amines

Fusaricidins/LI‐Fs and related cyclic lipopeptides represent an interesting new class of antibacterial peptides with the potential to meet the challenge of antibiotic resistance in bacteria. Our previous study (Bionda et al. ChemMedChem 2012, 7, 871–882) revealed the significance of the guanidinium group located at the termini of the lipidic tails of these cyclic lipopeptides for their antibacterial activities. Therefore, devising a synthetic strategy that will allow incorporation of guanidinium functionality into their structure is of particular practical importance. Since appropriately protected guanidino fatty acid building blocks are not commercially available, our strategy toward guanidinylated fusaricidin/LI‐F analogs include solid‐phase synthesis of a cyclic lipopeptide precursor possessing a lipidic tail with a terminal amino group followed by its conversion into corresponding guanidine. To find the optimal method for this conversion, we have examined commonly used guanidinylation reagents under the conditions compatible with standard solid‐phase peptide synthesis. Described experimental results demonstrated superiority of N,N′‐di‐Boc‐N″‐triflylguanidine in solid‐phase preparation of fusaricidin/LI‐F class of cyclic lipopeptides. The triflylguanidine reagent gave a single monoguanidinylated product in excellent yield independently of the type of solid‐support.

Identification of activators of methionine sulfoxide reductases A and B.

The methionine sulfoxide reductase (Msr) family of enzymes has been shown to protect cells against oxidative damage. The two major Msr enzymes, MsrA and MsrB, can repair oxidative damage to proteins due to reactive oxygen species, by reducing the methionine sulfoxide in proteins back to methionine. A role of MsrA in animal aging was first demonstrated in Drosophila melanogaster where transgenic flies over-expressing recombinant bovine MsrA had a markedly extended life span. Subsequently, MsrA was also shown to be involved in the life span extension in Caenorhabditis elegans. These results supported other studies that indicated up-regulation, or activation, of the normal cellular protective mechanisms that cells use to defend against oxidative damage could be an approach to treat age related diseases and slow the aging process. In this study we have identified, for the first time, compounds structurally related to the natural products fusaricidins that markedly activate recombinant bovine and human MsrA and human MsrB.

Dissociation of antimicrobial and hemolytic activities of gramicidin S through N-methylation modification.

β‐Sheet antimicrobial peptides (AMPs) are well recognized as promising candidates for the treatment of multidrug‐resistant bacterial infections. To dissociate antimicrobial activity and hemolytic effect of β‐sheet AMPs, we hypothesize that N‐methylation of the intramolecular hydrogen bond(s)‐forming amides could improve their specificities for microbial cells over human erythrocytes. We utilized a model β‐sheet antimicrobial peptide, gramicidin S (GS), to study the N‐methylation effects on the antimicrobial and hemolytic activities. We synthesized twelve N‐methylated GS analogues by replacement of residues at the β‐strand and β‐turn regions with N‐methyl amino acids, and tested their antimicrobial and hemolytic activities. Our experiments showed that the HC50 values increased fivefold compared with that of GS, when the internal hydrogen‐bonded leucine residue was methylated. Neither hemolytic effect nor antimicrobial activity changed when proline alone was replaced with N‐methylalanine in the β‐turn region. However, analogues containing N‐methylleucine at β‐strand and N‐methylalanine at β‐turn regions exhibited a fourfold increase in selectivity index compared to GS. We also examined the conformation of these N‐methylated GS analogues using 1H NMR and circular dichroism (CD) spectroscopy in aqueous solution, and visualized the backbone structures and residue orientations using molecular dynamics simulations. The results show that N‐methylation of the internal hydrogen bond‐forming amide affected the conformation, backbone shape, and side chain orientation of GS.

Direct access to side chain N,N′-diaminoalkylated derivatives of basic amino acids suitable for solid-phase peptide synthesis

A simple and efficient one-pot procedure that enables rapid access to orthogonally protected N,N′-diaminoalkylated basic amino acid building blocks fully compatible with standard Boc and Fmoc solid-phase peptide synthesis is reported. Described synthetic approach includes double reductive alkylation of Nα-protected diamino acids with N-protected amino aldehydes in the presence of sodium cyanoborohydride. This approach allows preparation of symmetrical, as well as unsymmetrical, basic amino acid derivatives with branched side-chains that can be further modified, enhancing their synthetic utility. The suitability of the synthesized branched basic amino acid building blocks for use in standard solid-phase peptide synthesis has been demonstrated by synthesis of an indolicidin analogue in which the lysine residue was substituted with the synthetic derivative Nα-(9H-fluorenyl-9-methoxycarbonyl)-Nβ,Nβ′-bis[2-(tert-butoxycarbonylamino)ethyl]-l-2,3-diaminopropionic acid. This substitution resulted in an analogue with more ordered secondary structure in 2,2,2-trifluoroethanol and enhanced antibacterial activity without altering hemolytic activity.Graphical abstract

Synthesis of anthrose lipidic derivative as mimic of B. anthracis BclA glycoprotein for use in ELISA-like binding assays

ABSTRACT The surfaces of Bacillus anthracis endospores expose anthrose-containing oligosaccharides, which have been considered for use as a target for specific detection of the spores. In this direction, we have developed an efficient and straightforward synthetic strategy toward anthrose lipidic derivate tetradecyl 4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-β-d-glucopyranoside 16 as a model target for B. anthracis spores. The ability of the prepared anthrose and glucose (for control purposes) lipidic derivatives to display on a multiwell plate was demonstrated by a colorimetric phenol-sulfuric acid assay and their potential utility in multiwell binding assays was assessed using fluorescein-labeled concanavalin A (ConA-FITC) and Aleuria aurantia (AAL-FITC). GRAPHICAL ABSTRACT