We designed a minimalistic zinc(II)-binding peptide featuring the Cys2His2 zinc-finger motif. To this aim, several tens of thousands of (His/Cys)-Xn-(His/Cys) protein fragments (n=2-20) were first extracted from the 3D protein structures deposited in Protein Data Bank (PDB). Based on geometrical constraints positioning two Cys (C) and two His (H) side chains at the vertices of a tetrahedron, approximately 22 000 sequences of the (H/C)-Xi-(H/C)-Xj-(H/C)-Xk-(H/C) type, satisfying Nmetal-binding H=Nmetal-binding C=2, were processed. Several other criteria, such as the secondary structure content and predicted fold stability, were then used to select the best candidates. To prove the viability of the computational design experimentally, three peptides were synthesized and subjected to isothermal calorimetry (ITC) measurements to determine the binding constants with Zn2+, including the entropy and enthalpy terms. For the strongest Zn2+ ions binding peptide, P1, the dissociation constant was shown to be in the nanomolar range (KD=~220 nM; corresponding to ΔGbind=-9.1 kcal mol-1). In addition, ITC showed that the [P1 : Zn2+] complex forms in 1 : 1 stoichiometry and two protons are released upon binding, which suggests that the zinc coordination involves both cysteines. NMR experiments also indicated that the structure of the [P1 : Zn2+] complex might be quite similar to the computationally predicted one. In summary, our proof-of-principle study highlights the usefulness of our computational protocol for designing novel metal-binding peptides.

• Klíčová slova
• Computer design, Isothermal calorimetry, Metal-binding peptide, NMR, QM modeling, Zinc(II),
• MeSH
• molekulární modely MeSH
• peptidy * chemie   metabolismus   chemická syntéza   MeSH
• sekvence aminokyselin MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• zinek * chemie   metabolismus   MeSH
• zinkové prsty MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• peptidy * MeSH
• zinek * MeSH

Lipid-mediated delivery of active pharmaceutical ingredients (API) opened new possibilities in advanced therapies. By encapsulating an API into a lipid nanocarrier (LNC), one can safely deliver APIs not soluble in water, those with otherwise strong adverse effects, or very fragile ones such as nucleic acids. However, for the rational design of LNCs, a detailed understanding of the composition-structure-function relationships is missing. This review presents currently available computational methods for LNC investigation, screening, and design. The state-of-the-art physics-based approaches are described, with the focus on molecular dynamics simulations in all-atom and coarse-grained resolution. Their strengths and weaknesses are discussed, highlighting the aspects necessary for obtaining reliable results in the simulations. Furthermore, a machine learning, i.e., data-based learning, approach to the design of lipid-mediated API delivery is introduced. The data produced by the experimental and theoretical approaches provide valuable insights. Processing these data can help optimize the design of LNCs for better performance. In the final section of this Review, state-of-the-art of computer simulations of LNCs are reviewed, specifically addressing the compatibility of experimental and computational insights.

• Klíčová slova
• ionizable lipid, lipid nanocarrier, lipid nanoparticle, liposome, molecular simulation, vesicle,
• MeSH
• léčivé přípravky chemie   MeSH
• lékové transportní systémy metody   MeSH
• lidé MeSH
• lipidy * chemie   MeSH
• nanočástice chemie   MeSH
• nosiče léků chemie   MeSH
• počítačová simulace MeSH
• simulace molekulární dynamiky * MeSH
• strojové učení MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• léčivé přípravky MeSH
• lipidy * MeSH
• nosiče léků MeSH

Recently published in vivo observations have highlighted the presence of cruciform structures within the genome, suggesting their potential significance in the rapid recognition of the target sequence for transcription factor binding. In this in vitro study, we investigate the organization and stability of the sense (coding) strand within the Serum Response Element of the c-Fos gene promoter (c-Fos SRE), specifically focusing on segments spanning 12 to 36 nucleotides, centered around the CArG-box. Through a thorough examination of UV absorption patterns with varying temperatures, we identified the emergence of a remarkably stable structure, which we conclusively characterized as a hairpin using complementary 1H NMR experiments. Our research decisively ruled out the formation of homoduplexes, as confirmed by supplementary fluorescence experiments. Utilizing molecular dynamics simulations with atomic distance constraints derived from NMR data, we explored the structural intricacies of the compact hairpin. Notably, the loop consisting of the six-membered A/T sequence demonstrated substantial stabilization through extensive stacking, non-canonical inter-base hydrogen bonding, and hydrophobic clustering of thymine methyl groups. These findings suggest the potential of the c-Fos SRE to adopt a cruciform structure (consisting of two opposing hairpins), potentially providing a topological recognition site for the SRF transcription factor under cellular conditions. Our results should inspire further biochemical and in vivo studies to explore the functional implications of these non-canonical DNA structures.

• Publikační typ
• časopisecké články MeSH

This roadmap reviews the new, highly interdisciplinary research field studying the behavior of condensed matter systems exposed to radiation. The Review highlights several recent advances in the field and provides a roadmap for the development of the field over the next decade. Condensed matter systems exposed to radiation can be inorganic, organic, or biological, finite or infinite, composed of different molecular species or materials, exist in different phases, and operate under different thermodynamic conditions. Many of the key phenomena related to the behavior of irradiated systems are very similar and can be understood based on the same fundamental theoretical principles and computational approaches. The multiscale nature of such phenomena requires the quantitative description of the radiation-induced effects occurring at different spatial and temporal scales, ranging from the atomic to the macroscopic, and the interlinks between such descriptions. The multiscale nature of the effects and the similarity of their manifestation in systems of different origins necessarily bring together different disciplines, such as physics, chemistry, biology, materials science, nanoscience, and biomedical research, demonstrating the numerous interlinks and commonalities between them. This research field is highly relevant to many novel and emerging technologies and medical applications.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Along with the known kaempferol-3-O-α-l-rhamnopyranosyl-(1 → 2)-[6-O-(3-hydroxy-3-methylglutaryl)]-β-d-galactopyranoside (1), five new flavonoids, containing the rarely isolated aglycon tamarixetin, were isolated from a methanolic extract of the endemic Balkan species Astragalus thracicus Griseb. Three of the new compounds are substituted with 3-hydroxy-3-methylglutaryl residue (HMG), untypical for the genus Astragalus. The compounds were identified as tamarixetin-3-O-α-l-rhamnopyranosyl-(1 → 2)-[6-O-(3-hydroxy-3-methylglutaryl)]-β-d-galactopyranoside (2), tamarixetin-3-O-(2,6-di-O-α-l-rhamnopyranosyl)-β-d-galactopyranoside (3), tamarixetin 3-O-β-d-apiofuranosyl-(1 → 2)-β-d-galactopyranoside (4), tamarixetin-3-O-β-d-apiofuranosyl-(1 → 2)-[6-O-(3-hydroxy-3-methylglutaryl)]-β-d-galactopyranoside (5), and tamarixetin-3-O-β-d-apiofuranosyl-(1 → 2)-[α-l-rhamnopyranosyl-(1 → 6)]-β-d-galactopyranoside (6). Selected compounds from A. thracicus were tested to evaluate their anticollagenase activity. The greatest effect was observed for quercetin-3-O-β-d-apiofuranosyl-(1 → 2)-β-d-galactopyranoside, possibly due to the presence of an ortho-dihydroxy arrangement of flavonoid ring B. The effect on collagenase and elastase was further evaluated also by in silico study, and the test compounds showed some level of in silico interaction.

• Publikační typ
• časopisecké články MeSH

Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1 O2 ). However, its 1 O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1 O2 production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective 1 O2 production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low 1 O2 production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.

• Klíčová slova
• LOV2 domain, flavin cofactor, genetically encoded photosensitizers, miniSOG, singlet oxygen,
• MeSH
• aminokyseliny MeSH
• flavinmononukleotid chemie   MeSH
• flavoproteiny * chemie   metabolismus   MeSH
• fotosenzibilizující látky * MeSH
• proteinové domény MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• flavinmononukleotid MeSH
• flavoproteiny * MeSH
• fotosenzibilizující látky * MeSH

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 μM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 μM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

• MeSH
• antiparazitární látky terapeutické užití   MeSH
• Chagasova nemoc * farmakoterapie   MeSH
• leucylaminopeptidasa chemie   farmakologie   terapeutické užití   MeSH
• lidé MeSH
• objevování léků MeSH
• trypanocidální látky * terapeutické užití   MeSH
• Trypanosoma cruzi * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiparazitární látky MeSH
• leucylaminopeptidasa MeSH
• trypanocidální látky * MeSH

The TAP1-TAP2 complex transports antigenic peptide substrates into the endoplasmic reticulum (ER). In ER, the peptides are further processed and loaded on the major histocompatibility class (MHC) I molecules by the peptide loading complex (PLC). The TAP transporters are linked with the PLC; a target for cancers and viral immune evasion. But the mechanisms whereby the cancer-derived mutations in TAP1-TAP2 or viral factors targeting the PLC, interfere peptide transport are only emerging. This study describes that transit of peptides through TAP can take place via two different channels (4 or 8 helices) depending on peptide length and sequence. Molecular dynamics and binding affinity predictions of peptide-transporters demonstrated that smaller peptides (8-10 mers; e.g. AAGIGILTV, SIINFEKL) can transport quickly through the transport tunnel compared to longer peptides (15-mer; e.g. ENPVVHFFKNIVTPR). In line with a regulated and selective peptide transport by TAPs, the immunopeptidome upon IFN-γ treatment in melanoma cells induced the shorter length (9-mer) peptide presentation over MHC-I that exhibit a relatively weak binding affinity with TAP. A conserved distance between N and C terminus residues of the studied peptides in the transport tunnel were reported. Furthermore, by adversely interacting with the TAP transport passage or affecting TAPNBD domains tilt movement, the viral proteins and cancer-derived mutations in TAP1-TAP2 may induce allosteric effects in TAP that block conformation of the tunnel (closed towards ER lumen). Interestingly, some cancer-associated mutations (e.g. TAP1R372Q and TAP2R373H) can specifically interfere with selective transport channels (i.e. for longer-peptides). These results provide a model for how viruses and cancer-associated mutations targeting TAP interfaces can affect MHC-I antigen presentation, and how the IFN-γ pathway alters MHC-I antigen presentation via the kinetics of peptide transport.

• Klíčová slova
• Cancer mutations, IFN-γ, Immunopeptidome, In silico peptide screening, Kinetics, MHC-I, Molecular dynamics, Peptide, TAP1, TAP2, Transporters, Viral factors,
• Publikační typ
• časopisecké články MeSH

The authors report on the synthesis and biological evaluation of new compounds whose structure combines tacrine and indole moieties. Tacrine-indole heterodimers were designed to inhibit cholinesterases and β-amyloid formation, and to cross the blood-brain barrier. The most potent new acetylcholinesterase inhibitors were compounds 3c and 4d (IC50 = 25 and 39 nM, respectively). Compound 3c displayed considerably higher selectivity for acetylcholinesterase relative to human plasma butyrylcholinesterase in comparison to compound 4d (selectivity index: IC50 [butyrylcholinesterase]/IC50 [acetylcholinesterase] = 3 and 0.6, respectively). Furthermore, compound 3c inhibited β-amyloid-dependent amyloid nucleation in the yeast-based prion nucleation assay and displayed no dsDNA destabilizing interactions with DNA. Compounds 3c and 4d displayed a high probability of crossing the blood-brain barrier. The results support the potential of 3c for future development as a dual-acting therapeutic agent in the prevention and/or treatment of Alzheimer's disease.

• Klíčová slova
• 7-methoxytacrine, Alzheimer's disease, cholinesterases, in vitro, indole, tacrine,
• MeSH
• acetylcholinesterasa metabolismus   MeSH
• Alzheimerova nemoc farmakoterapie   MeSH
• amyloidní beta-protein metabolismus   MeSH
• cholinesterasové inhibitory chemie   farmakologie   MeSH
• cílená molekulární terapie MeSH
• dimerizace MeSH
• DNA chemie   MeSH
• hematoencefalická bariéra MeSH
• indoly chemie   farmakologie   MeSH
• inhibiční koncentrace 50 MeSH
• lidé MeSH
• ligandy MeSH
• neuroprotektivní látky chemie   farmakologie   MeSH
• preklinické hodnocení léčiv MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• takrin chemie   farmakologie   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• acetylcholinesterasa MeSH
• amyloidní beta-protein MeSH
• cholinesterasové inhibitory MeSH
• DNA MeSH
• indoly MeSH
• ligandy MeSH
• neuroprotektivní látky MeSH
• takrin MeSH

BACKGROUND: Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. RESULTS: We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. CONCLUSIONS: The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

• Klíčová slova
• Deorphanization, Functional evolution, HEK293 cells, Odorant receptor, Pest insect, Pheromone receptor, Xenopus oocyte,
• MeSH
• hmyzí proteiny chemie   genetika   MeSH
• ligandy MeSH
• nosatcovití chemie   genetika   MeSH
• receptory pachové chemie   genetika   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hmyzí proteiny MeSH
• ligandy MeSH
• receptory pachové MeSH