Francesc Rabanal

Gram-negative outer and inner membrane models: insertion of cyclic cationic lipopeptides.

Most Gram-negative bacteria are susceptible to polymyxin B (PxB), and development of resistance to this cationic lipopeptide is very rare. PxB mechanism of action involves interaction with both the outer membrane (OM) and the inner membrane (IM) of bacteria. For the design of new antibiotics based on the structure of PxB and with improved therapeutic indexes, it is essential to establish the key features of PxB that are important for activity. We have used an approach based on mimicking the outer layers of the OM and the IM of Gram-negative bacteria using monolayers of lipopolysaccharide (LPS) or anionic 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), respectively, and using a combination of penetration assay, analysis of pressure/area curves, and Brewster angle microscopy to monitor surface morphology changes. Synthetic analogue sp-B maintains the basic structural characteristics of the natural compound and interacts with the OM and the IM in a similar way. Analogue sp-C, with a mutation of the sequence [d-Phe6-Leu7] into [d-Phe6-Dab7], shows that this hydrophobic domain is involved in LPS binding. The significant role of the positive charges is demonstrated with sp-Dap analogue, where l-alpha,gamma-diaminobutyric acid residues Dab1 and Dab8 are replaced by l-alpha,gamma-diaminopropionic acid (Dap), resulting in lower degrees of insertion in both LPS and PG monolayers. The importance of the N-terminal acyl chain is demonstrated with polymyxin B nonapeptide (PxB-np). PxB-np shows lower affinity for LPS compared to PxB, sp-B, or sp-C, but it does not insert into PG monolayers, although it binds superficially to the anionic film. Since PxB microbial killing appears to be mediated by osmotic instability due to OM-IM phospholipid exchange, the ability of the different peptides to induce membrane-membrane lipid exchange has been studied by use of phospholipid unilamellar vesicles. Results indicate that cationic amphipathicity determines peptide activity.

Use of 2,2′-dithiobis(5-nitropyridine) for the heterodimerization of cysteine containing peptides. Introduction of the 5-nitro-2-pyridinesulfenyl group

Abstract 2,2′-Dithiobis(5-nitropyridine) has proven to be a suitable reagent for the activation of the thiol function of cysteine in peptides and subsequent asymmetric disulfide formation. The utility of this reagent is tested in the preparation of de novo designed cytochrome model heterodimeric 62-mer peptides.

SOLID-PHASE SYNTHESIS OF HEAD-TO-TAIL CYCLIC PEPTIDES VIA LYSINE SIDE-CHAIN ANCHORING

Abstract The N,N′ -disuccinimidyl carbonate (DSC) has been successfully used for the efficient conversion of conventional hydroxymethyl resins into active carbonate resins, which are suitable for the incorporation of protected amino acids via an amino function, allowing the preparation of “head-to-tail” cyclic lysine containing peptides.

Active carbonate resins for solid-phase synthesis through the anchoring of a hydroxyl function. Synthesis of cyclic and alcohol peptides

Abstract N , N ′-Disuccinimidyl carbonate (DSC) has been successfully used for the efficient conversion of 4-hydroxymethylpolystyrene and 4-hydroxymethyl-3-nitrobenzamido (Nbb) resins into active carbonate resins, which are suitable for the incorporation of molecules via a hydroxyl function. This methodology has been applied to the preparation of the growth hormone inhibitor, Sandostatin.

Active carbonate resins: Application to the solid-phase synthesis of alcohol, carbamate and cyclic peptides☆

Abstract N,N′-disuccinimidyl carbonate (DSC) has been successfully used to generate carbonates and carbamates on conventional hydroxymethyl and aminomethyl based resins. This methodology extends the applicability of such linkers, which were initially designed for the anchoring of carboxylic acids. Thus, amino and hydroxy groups have been attached onto classical resins to give straightforward access to the solid-phase synthesis of alcohols, carbamates, and cyclic peptides with an evident pharmaceutical interest.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

Engineering photosynthesis: synthetic redox proteins

Abstract The understanding of the principal parameters naturally selected to engineer biological electron transfer systems has been combined with techniques of de novo design and chemical synthesis of peptides to initiate a program of synthesis of simple proteins equipped with multiple redox cofactors. Our primary goal is to assemble functional redox protein maquettes of the large, complicated natural proteins such as the photosynthetic reaction center. Attention is focused on strategies for engineering and construction of well defined, stable four-helix bundles containing suitable redox cofactors optimized for efficient electron transfer.

Synthesis and applications of a new base-labile fluorene derived linker for solid-phase peptide synthesis

Abstract The handle N-[(9-hydroxymethyl)-2-fluorenyl]succinamic acid (HMFS) is reported for the preparation of protected peptide segments in combination with a Boc/Bzl protection scheme. Treatment of peptide-resins with morpholine in DMF renders protected peptides in high yields and purities.

Synthesis of novel proteins

De novo and rational protein design are progressing towards the chemical synthesis of proteins with pre-selected structure and function. The data illustrate diverse experimental and computational approaches which test our comprehension of protein structure, hydrophobic core packing and global stability, especially of coiled-coil proteins. The incorporation of biological cofactors, including hemes, as well as active sites, such as that of iron superoxide dismutase, into designed proteins provides an exciting next step towards the synthesis of proteins with enzymatic function.

An overview of antimicrobial peptides and the latest advances in their development

ABSTRACT Introduction: The recent dramatic increase in the incidence of antimicrobial resistance has been recognized by organizations such as the United Nations and World Health Organization as well as the governments of the USA and several European countries. A relatively new weapon in the fight against severe infections caused by multi-drug resistant bacteria is antimicrobial peptides (AMPs). These include colistin, currently regarded as the last line of antimicrobial therapy against multi-drug resistant microorganisms. Areas covered: Here, the authors provide an overview of the current research on AMPs. The focus is AMPs currently being developed for the treatment of recalcitrant bacterial infections, the synergies of AMPs and antibiotics, and the activity of AMPs against biofilm. This review also includes a brief introduction into the use of AMPs in infections caused by Mycobacterium, fungi, and parasites. Expert opinion: In research into new antimicrobials, AMPs are gaining increasing attention. While many are natural and are produced by a wide variety of organisms, others are being newly designed and chemically synthesized in the laboratory to achieve novel antimicrobial agents. The same strategy to fight infections in nature is thus being effectively exploited to safeguard human and animal health.