mu-Opioid receptor szelektív peptidek kísérleti és elméleti konfomációanalízise = Experimental and theoretical conformation analysis of mu-opioid receptor selective peptide ligands

A támogatási periódus alatt természetes és szintetikus m-opioid receptor ligandumok szerkezet-hatás összefüggéseit vizsgáltuk és egy kombinált kísérleti-elméleti szerkezetvizsgálati protokoll kidolgozásán munkálkodtunk. Tíz ismert affinitású m-opioid receptor ligandum molekuladinamikai szimulációkon alapuló szerkezetanalízise alapján egy négy szerkezeti paraméterrel leírható bioaktív szerkezeti modellt állítottunk fel. Az IR és VCD spektroszkópiai méréseken, valamint kvantumkémiai spektrum-modellezésen alapuló kombinált szerkezetvizsgálati protokoll kidolgozásakor előzetes elgondolásainkat követve nem kaptunk várakozásainkat kielégítő eredményeket. Ez szükségszerűvé tette hipotézisünk újraértékelését és alternatív megoldások, módszerek bevezetését. Ezen a téren a kutatás még folyamatban van. Az előbbiekben említett alapkutatási ágon elért eredményeink viszont lehetővé tették, hogy egy új kutatási vonalat nyitva potenciális m-opioid ligandumokat tervezzünk. A tisztán elméleti úton nyert adatokra alapozott molekulatervezés és tesztelés a vártnál is jobb eredményekkel zárult. Az újonnan tervezett, előállított ligandumok tesztjei megerősítették korábban emlitett, bioaktív szerekezeti feltevéseinket. Eredményeinket négy folyóiratcikk, egy konferenciacikk, egy összefoglaló cikk és egy közlésre benyújtott kézirat rögzíti. | During the funding period we studied the structure-activity relationships of naturally occurring and synthetic m-opioid receptor ligands. Additionally we worked on the development of structural analysis protocol, which combines experimental and theoretical methods. On the basis of structural analyses of ten m-opioid receptor ligands of known affinity we proposed a bioactive structure model, which is defined by four specific structural parameter. Results obtained during the development of structural analysis protocol, which combines IR and VCD spectroscopy and quantum chemical spectrum modeling did not fulfill our expectations. Therefore, re-evaluation of our hypothesis and introduction of alternative methods and solutions were needed. Work is still in progress on this branch of our studies. However, our proposed structure-activity model allowed us to design new potential m-opioid ligands, hereby opening a new direction in our research. The new ligands, designed on the basis of theoretical results, were proved to be bioactive therefore reinforcing our previously proposed model. Our results were published in the form of four journal articles, one conference proceeding, one review article and one manuscript, which is now submitted for publication

All-Atom Molecular Dynamics Simulations Indicated the Involvement of a Conserved Polar Signaling Channel in the Activation Mechanism of the Type I Cannabinoid Receptor

The type I cannabinoid G protein-coupled receptor (CB1, GPCR) is an intensely investigated pharmacological target, owing to its involvement in numerous physiological functions as well as pathological processes such as cancers, neurodegenerative diseases, metabolic disorders and neuropathic pain. In order to develop modern medications that exert their effects through binding to the CB1 receptor, it is essential to understand the structural mechanism of activation of this protein. The pool of atomic resolution experimental structures of GPCRs has been expanding rapidly in the past decade, providing invaluable information about the function of these receptors. According to the current state of the art, the activity of GPCRs involves structurally distinct, dynamically interconverting functional states and the activation is controlled by a cascade of interconnecting conformational switches in the transmembrane domain. A current challenge is to uncover how different functional states are activated and what specific ligand properties are responsible for the selectivity towards those different functional states. Our recent studies of the mu-opioid and beta(2)-adrenergic receptors (MOP and beta(2)AR, respectively) revealed that the orthosteric binding pockets and the intracellular surfaces of these receptors are connected through a channel of highly conserved polar amino acids whose dynamic motions are in high correlation in the agonist- and G protein-bound active states. This and independent literature data led us to hypothesize that, in addition to consecutive conformational transitions, a shift of macroscopic polarization takes place in the transmembrane domain, which is furnished by the rearrangement of polar species through their concerted movements. Here, we examined the CB1 receptor signaling complexes utilizing microsecond scale, all-atom molecular dynamics (MD) simulations in order to see if our previous assumptions could be applied to the CB1 receptor too. Apart from the identification of the previously proposed general features of the activation mechanism, several specific properties of the CB1 have been indicated that could possibly be associated with the signaling profile of this receptor

Universal Properties and Specificities of the beta(2)-Adrenergic Receptor-G(s) Protein Complex Activation Mechanism Revealed by All-Atom Molecular Dynamics Simulations

G protein-coupled receptors (GPCRs) are transmembrane proteins of high pharmacological relevance. It has been proposed that their activity is linked to structurally distinct, dynamically interconverting functional states and the process of activation relies on an interconnecting network of conformational switches in the transmembrane domain. However, it is yet to be uncovered how ligands with different extents of functional effect exert their actions. According to our recent hypothesis, based on indirect observations and the literature data, the transmission of the external stimulus to the intracellular surface is accompanied by the shift of macroscopic polarization in the transmembrane domain, furnished by concerted movements of highly conserved polar motifs and the rearrangement of polar species. In this follow-up study, we have examined the beta(2)-adrenergic receptor (beta(2)AR) to see if our hypothesis drawn from an extensive study of the mu-opioid receptor (MOP) is fundamental and directly transferable to other class A GPCRs. We have found that there are some general similarities between the two receptors, in agreement with previous studies, and there are some receptor-specific differences that could be associated with different signaling pathways

Mapping and Identification of Antifungal Peptides in the Putative Antifungal Protein AfpB from the Filamentous Fungus Penicillium digitatum

Antifungal proteins (AFPs) from Ascomycetes are small cysteine-rich proteins that are abundantly secreted and show antifungal activity against non-producer fungi. A gene coding for a class B AFP (AfpB) was previously identified in the genome of the plant pathogen Penicillium digitatum. However, previous attempts to detect the AfpB protein were not successful despite the high expression of the corresponding afpB gene. In this work, the structure of the putative AfpB was modeled. Based on this model, four synthetic cysteine-containing peptides, PAF109, PAF112, PAF118, and PAF119, were designed and their antimicrobial activity was tested and characterized. PAF109 that corresponds to the gamma-core motif present in defensin-like antimicrobial proteins did not show antimicrobial activity. On the contrary, PAF112 and PAF118, which are cationic peptides derived from two surface-exposed loops in AfpB, showed moderate antifungal activity against P. digitatum and other filamentous fungi. It was also confirmed that cyclization through a disulfide bridge prevented peptide degradation. PAF116, which is a peptide analogous to PAF112 but derived from the Penicillium chrysogenum antifungal protein PAF, showed activity against P. digitatum similar to PAF112, but was less active than the native PAF protein. The two AfpB-derived antifungal peptides PAF112 and PAF118 showed positive synergistic interaction when combined against P. digitatum. Furthermore, the synthetic hexapeptide PAF26 previously described in our laboratory also exhibited synergistic interaction with the peptides PAF112, PAF118, and PAF116, as well as with the PAF protein. This study is an important contribution to the mapping of antifungal motifs within the AfpB and other AFPs, and opens up new strategies for the rational design and application of antifungal peptides and proteins

Mitragynine/Corynantheidine Pseudoindoxyls As Opioid Analgesics with Mu Agonism and Delta Antagonism, Which Do Not Recruit beta-Arrestin-2.

Natural products found in Mitragyna speciosa, commonly known as kratom, represent diverse scaffolds (indole, indolenine, and spiro pseudoindoxyl) with opioid activity, providing opportunities to better understand opioid pharmacology. Herein, we report the pharmacology and SAR studies both in vitro and in vivo of mitragynine pseudoindoxyl (3), an oxidative rearrangement product of the corynanthe alkaloid mitragynine. 3 and its corresponding corynantheidine analogs show promise as potent analgesics with a mechanism of action that includes mu opioid receptor agonism/delta opioid receptor antagonism. In vitro, 3 and its analogs were potent agonists in [35S]GTPgammaS assays at the mu opioid receptor but failed to recruit beta-arrestin-2, which is associated with opioid side effects. Additionally, 3 developed analgesic tolerance more slowly than morphine, showed limited physical dependence, respiratory depression, constipation, and displayed no reward or aversion in CPP/CPA assays, suggesting that analogs might represent a promising new generation of novel pain relievers