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LAT-1, the large neutral amino acid transporter-1, plays a crucial role in cancer growth and proliferation, making it a promising biomarker for imaging and treating human malignancies such as brain cancers. Thus, growing attention is being paid to the development of LAT-targeted drug delivery systems with blood-brain penetration ability [1, 2]. For this purpose, gold nanoparticles (AuNPs) functionalized with L-DOPA have been developed [1]. The great potential of such systems to be used in brain cancer therapy prompts the need for evaluation of the processes occurring at the nano-bio interface [3]. In the presence of gold salts, L-DOPA can undergo various transformations such as oxidation, cyclization, and polymerization [4]. To avoid binding structurally altered molecules to the nanosurface and prevent any negative impact on human health, it is important to consider these transformation patterns. This study aimed to investigate the behavior of L-DOPA upon interaction with a gold nanosurface by using a combination of nuclear magnetic resonance (NMR) spectroscopy and computational methods. Experimental results showed that the final form of L-DOPA on the AuNPs surface is dependent on the molar ratio of reactants used during synthesis. When there was an excess of L-DOPA compared to Au, no alterations in the structure of L-DOPA were observed. However, an excess of Au was found to promote the oxidation of L-DOPA, which could result in various oxidation products being bound to the gold nanosurface. NMR spectra revealed that L-DOPA underwent intramolecular cyclization but could not differentiate oxidation products. On the other hand, computational simulations using density functional theory (DFT) and molecular dynamics (MD) techniques identified the most probable oxidation products for binding to the AuNP surface. The highest affinity to gold nanosurface was obtained for dopachrome compared to its tautomer and leukodopachrome. The obtained results represent valuable mechanistic data about the binding events at the surface of AuNPs to encourage their application as a drug-delivery systems in cancer therapy

The great potential of gold nanoparticles (AuNPs) functionalized with catechols, dopamine and L-DOPA, in biomedical applications prompts the need for evaluation of their interaction at the nano-bio interface.[1] In the presence of gold slats, catechols are known to transform by various pathways including oxidation, cyclization and polymerization.[2] Thus, different transformation patterns should be considered in order to avoid binding the structurally changed molecule to the nanosurface while preventing unpredictable consequences for the health status of an organism. In this study, the fate of functionalization agents upon interaction with gold nanosurface was investigated by combining nuclear magnetic resonance (NMR) spectroscopy and a computational approach. Experimental results revealed that the final form of catechol on the AuNPs nanosurface depends on the molar ratio of reactant used in synthesis. The oxidation of both L-DOPA and dopamine was promoted in the case of molar excess of Au. However, their final products differ as L-DOPA underwent intramolecular cyclization. Computational simulations using density functional theory (DFT) and molecular dynamics (MD) methods revealed the mechanism that stays behind this difference and clarified the most likely candidates, among different oxidation products, for binding to the AuNPs surface. The obtained results represent valuable mechanistic data about the binding events at the surface of AuNPs to encourage their application in biomedicine.

This chapter reviews applications of solid-state NMR to three peripheral membrane proteins: cardiolipin-bound cytochrome c, phosphatidylinositol-binding Pleckstrin homology domain and membrane-associated myelin basic protein. We describe solutions to common challenges, such as the dynamics of the membrane-bound protein and the preparation of suitable samples with biological relevance. Modern NMR hardware, molecular biology and sample preparation methods pave the way for effective studies of many interesting peripheral membrane proteins by solid-state NMR.

Nonreducible cystine isosteres represent important peptide design elements in that they can maintain a near-native tertiary conformation of the peptide while simultaneously extending the in vitro and in vivo half-life of the biomolecule. Examples of these cystine mimics include dicarba, diselenide, thioether, triazole, and lactam bridges. Each has unique physicochemical properties that impact upon the resulting peptide conformation. Each also requires specific conditions for its formation via chemical peptide synthesis protocols. While the preparation of peptides containing two lactam bonds within a peptide is technically possible and reported by others, to date there has been no report of the chemical synthesis of a heterodimeric peptide linked by two lactam bonds. To examine the feasibility of such an assembly, judicious use of a complementary combination of amine and acid protecting groups together with nonfragment-based, total stepwise solid phase peptide synthesis led to the successful preparation of an analogue of the model peptide, insulin-like peptide 3 (INSL3), in which both of the interchain disulfide bonds were replaced with a lactam bond. An analogue containing a single disulfide-substituted interchain lactam bond was also prepared. Both INSL3 analogues retained significant cognate RXFP2 receptor binding affinity.

Octapeptins are cyclic lipopeptides that do not exhibit cross-resistance with polymyxins and have a broader spectrum of activity that includes fungi and Gram-positive bacteria. In the present study we investigated the interaction of octapeptin A3 with asymmetric outer membrane models of the Gram-negative pathogen Pseudomonas aeruginosa, using neutron reflectometry, together with fluorimetric and calorimetry methods. For the first time our neutron reflectometry results reveal that the interaction of octapeptin A3 with the Gram-negative outer membrane involves an initial transient polar interaction with the phospholipid and lipid A head groups, followed by the penetration of the entire octapeptin molecule into the fatty acyl core of the outer membrane. This mechanism contrasts that of polymyxin B which specifically targets lipid A, whereas octapeptins appear to target both lipid A and phospholipids. Furthermore, the mechanism of octapeptins does not appear to be highly dependent on an initial complimentary electrostatic interaction with lipid A, which accounts for their ability to bind to the lipid A of polymyxin-resistant Gram-negative bacteria which is modified with cationic moieties that act to electrostatically repel the cationic polymyxin molecule. The presented findings shed new light on the mechanism whereby octapeptins penetrate the outer membrane of polymyxin-resistant Gram-negative pathogens and highlight their potential as candidates for development as new antibiotics against problematic multi-drug resistant pathogens.

The development of new approaches in the treatment of Parkinson's disease presents a challenge due to the presence of the blood-brain barrier, which hinders effective drug delivery to the brain. Due to their unique physicochemical properties, nanoparticles have the potential to improve conventional therapeutic approaches and enable targeted drug delivery across the blood-brain barrier. The main goal of this doctoral thesis was to synthesize and characterize differently functionalized gold and selenium nanoparticles and to examine their interaction with catechols (levodopa and dopamine) in order to design more effective and safer therapeutic systems for Parkinson's disease. The process of drug binding to the nano-surface was investigated using various spectroscopic, laser and microscopic methods, while the bloodbrain barrier permeability of prepared nano-systems was tested under in vitro settings. The obtained results have provided a better understanding of the processes that occur during the binding of catechol drugs to gold and selenium nanoparticles. This will potentially enable the development of a nano-formulation with targeted drug delivery to the brain with the aim of improving the therapy of Parkinson's disease. Razvoj novih pristupa u liječenju Parkinsonove bolesti predstavlja izazov zbog prisutnosti krvno-moždane barijere koja otežava učinkovitu dostavu lijekova u mozak. Zahvaljujući jedinstvenim fizikalno-kemijskim svojstvima, nanočestice imaju potencijal poboljšati konvencionalne terapijske pristupe i omogućiti ciljanu isporuku lijekova preko krvnomoždane barijere. Glavni cilj ovog doktorskog rada bio je sintetizirati i karakterizirati različito funkcionalizirane nanočestice zlata i selena te ispitati njihovo međudjelovanje s kateholima (levodopa i dopamin) u svrhu dizajniranja učinkovitijih i sigurnijih terapijskih sustava za liječenje Parkinsonove bolesti. Proces vezanja lijekova na nano-površinu ispitivao se pomoću različitih spektroskopskih, laserskih i mikroskopskih metoda, a zatim su se provela istraživanja propusnosti krvno-moždane barijere za novopripravljene nanosustave u in vitro uvjetima. Dobiveni rezultati omogućili su bolje razumijevanje procesa koji se događaju prilikom vezanja lijekova kateholne strukture i novih nanočestica zlata i selena. Na taj način potiče se razvoj nano-formulacija s ciljanom isporukom lijekova u mozak s ciljem poboljšanja terapije za Parkinsonovu bolest.

We shall describe two approaches for rapidly finding novel antibiotic peptide classes from animals also in view of the loss of such natural antibiotics due to accelerated species extinction. Both approaches are also designed to breach homology bottleneck when looking for promising lead peptide candidates for the development of peptide antibiotics. The first approach deals with identifying elusive selectivity properties of antimicrobial peptides as quantified by measured therapeutic index (TI). Introduced sequence moments has helped us to construct algorithms not only for predicting a TI from the primary structure in the case of natural amphibian peptides, but also for designing peptide antibiotics with optimized TI, non-homologous to any known natural or synthetic antimicrobial peptides. For one class of such antibiotics, the adepantins [1], we shall compare the theoretical predictions with experimental activity and selectivity results. The second approach allowed us to identify numerous novel putative antimicrobial peptides in the EST database, by applying our observation that, as a rule, specific subclasses of conserved signal sequences exist that are exclusively associated with antimicrobial peptides. As it turns out, at the present time this observation has been most useful for identifying novel amphibian and teleost antimicrobial peptides. We shall discuss these results, their implications, and experimental verifications. [1] D. Juretić, D. Vukičević, N. Ilić, N. Antcheva, A.Tossi, (2009) J Chem Inf Model 49, 2873.

Antimicrobial peptides (AMPs) are molecules involved in the defense mechanisms of a wide range of organisms. It has been shown for few families of host defense peptides that different C-terminal AMPs are derived from a single family of precursor polypeptides with unique features and remarkably well conserved sequence both within and between species. We showed that, for a large set of host defense peptide sequences taken from the antimicrobial database AMPB2, the signal peptide and acidic propeptide sequences of host defense peptides are highly conserved and specific for host defense peptides. We pointed out that processing of immature host defense peptides deserves further study since it may lead to discovery of highly conserved subset of signal recognition particles and peptidases specific for such peptides. Furthermore, we used this property of signal peptides to find new mature peptides (AMPs) by searching EST using signal peptide sequence as a query for the TBLASTP. In addition, we used cluster analysis for identification of signal peptide families.

book of abstracts

Antimicrobial peptides (AMPs) - natural peptide antibiotics produced by most organisms as part of the innate immune system - are an example of how nature can provide a solution to the increasingly serious problem of bacteria gaining resistance to normally used antibiotics. However, this type of potential therapeutic agent is often disadvantaged by an excessive cytotoxicity to host cells. In a recently published paper [1], using the Designer algorithm, we have used numerous AMP attributes to design and then test an example of an AMP selective for E. coli, that has an exceptionally low hemolytic activity, and shows little similarity to any known antimicrobial peptide. The peptide was named adepantin-1, an abbreviation derived from the Automatically Designed Peptide Antibiotic Number 1. Its precise mechanism of action has still to be determined, and we are also interested to know if several analogues, offered by the same algorithm, would be equally active and selective. Two additional adepantins from the same group, adepantin-2 and adepantin-3, have thus been synthesized. Furthermore, to test aspects of the possible mechanism of action, we have labeled them with the fluorescent probe BODIPY, and prepared covalent dimers. We shall report on the progress of our study of their conformation in membrane-mimetic solvent and mechanism of action in bacterial and host cells. [1] D.Juretić, D. Vukicević, N. Ilić, N. Antcheva, A.Tossi, Computational design of highly selective antimicrobial peptides, J Chem Inf Model. 2009 Dec ; 49(12):2873-2882.

Human dipeptidyl-peptidases (h.DPP III) hydrolyzes dipeptides from the N-terminal of its substrates. It has implied role in intracellular protein catabolism, as well in pain-modulatory system and endogenous defense against oxidative stress.1, 2, 3 We performed series of molecular dynamics (MD) simulations for the complexes between the enzyme, wild type (WT) and its H568N and W300L mutants, and both synthetic and natural ligands, Arg-Arg-2-naphthylamide (RRNA), Tyr-Phe-hydroxamate and endomorphine-1, respectively. We accomplished 30 ns of MD simulations for the complexes between WT and the H568N mutated DPP III with synthetic ligands. For their complexes with endomorphine-1 we performed 4 ns and for the free W300L mutant of DPP III and its complex with RRNA 3 ns of MD simulations. Systems simulated 30 ns show octahedral coordination of the central zinc ion, while for others the zinc coordination sphere is still no stable mainly due to fluctuations of the active site Glu508 and His450. MD simulations revealed the enzyme subsites S1, S2, S1' and S2' as well as the substrate N-terminus recognition site pointing out that both, synthetic and natural ligands bind into the DPPIII active site in a similar manner. Ligand binding induced reorganization of the binding site as well as its partial closure, involving a small flexible loop at the narrow end of the cleft. The binding free energies were evaluated using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA). Further MD simulations are required to elucidate: a) effects of the tryptophan to leucine mutation on the complex stability and b) to thoroughly investigate the endomorphine-1 binding into the active site. Also, using the QM/MM methodology we plan to shed some light on the hydrolysis that takes place in the DPP III active site.