Interplay of Pyrrolidine Units with Homo/Hetero Chirality and CF3-Aryl Substituents on Secondary Structures of ß-Proline Tripeptides in Solution.

All possible variants of ß-proline functionalized tripeptides consisting of homo/hetero chiral monomeric all-cis 5-arylpyrrolidine-2,4-dicarboxylate units were synthesized for the first time by a nonpeptidic coupling method based on 1,3-dipolar cycloaddition chemistry of azomethine ylides. Secondary structures of ß-proline tripeptides in solution were determined using the NMR spectroscopy data. o-(Trifluoromethyl)phenyl substituent contributes to stereoselectivity of 1,3-dipolar cycloaddition and structural features of ß-proline tripeptides. A ß-proline CF3-tripeptide with alternating absolute chirality between adjacent pyrrolidine units mimics natural PPII helix secondary structure.

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase.

The elongation of single-stranded DNA repeats at the 3'-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids-telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

A Binuclear Zinc Interaction Fold Discovered in the Homodimer of Alzheimer's Amyloid-ß Fragment with Taiwanese Mutation D7H.

Zinc-induced oligomerization of amyloid-ß peptide (Aß) produces potentially pathogenic agents of Alzheimer's disease. Mutations and modifications in the metal binding domain 1-16 of Aß peptide crucially affect its zinc-induced oligomerization by changing intermolecular zinc mediated interface. The 3D structure of this interface appearing in a range of Aß species is a prospective drug target for disease modifying therapy. Using NMR spectroscopy, EXAFS spectroscopy, mass spectrometry, and isothermal titration calorimetry the interaction of zinc ions with Aß fragments 1-7 and 1-10 carrying familial Taiwanese mutation D7H was studied. Zinc ions induce formation of a stable homodimer formed by the two peptide chains fastened by two zinc ions and stacking interactions of imidazole rings. A binuclear zinc interaction fold in the dimer structure was discovered. It can be used for designing zinc-regulated proteins and zinc-mediated self-assembling peptides.

MERA: a webserver for evaluating backbone torsion angle distributions in dynamic and disordered proteins from NMR data.

MERA (Maximum Entropy Ramachandran map Analysis from NMR data) is a new webserver that generates residue-by-residue Ramachandran map distributions for disordered proteins or disordered regions in proteins on the basis of experimental NMR parameters. As input data, the program currently utilizes up to 12 different parameters. These include three different types of short-range NOEs, three types of backbone chemical shifts ((15)N, (13)C(α), and (13)C'), six types of J couplings ((3)JHNHα, (3)JC'C', (3)JC'Hα, (1)JHαCα, (2)JCαN and (1)JCαN), as well as the (15)N-relaxation derived J(0) spectral density. The Ramachandran map distributions are reported in terms of populations of their 15° × 15° voxels, and an adjustable maximum entropy weight factor is available to ensure that the obtained distributions will not deviate more from a newly derived coil library distribution than required to account for the experimental data. MERA output includes the agreement between each input parameter and its distribution-derived value. As an application, we demonstrate performance of the program for several residues in the intrinsically disordered protein α-synuclein, as well as for several static and dynamic residues in the folded protein GB3.

Synthesis and Biological Evaluation of a Series of New Hybrid Amide Derivatives of Triazole and Thiazolidine-2,4-dione.

A series of hybrid compounds with triazole and thiazolidine nuclei connected by a linker has been synthesized and extensively studied. Various synthetic methods for the target compounds have been tested. A microbiological assessment of the obtained compounds was carried out on strains of pathogenic fungi C. albicans, C. non-albicans, multidrug-resistant C. auris, Rhizopus arrhizus, Aspergillus spp. and some dermatophytes and other yeasts. The lowest obtained MIC values for target compounds lie between 0.003 µg/mL and 0.5 µg/mL and therefore the compounds are not inferior or several times better than commercial azole drugs. The length of the acylpiperazine linker has a limited effect on antifungal activity. Some bioisosteric analogues were tested in microbiological analysis, but turned out to be weaker than the leader in activity. The highest activity was demonstrated by a compound with para-chlorobenzylidene substituent in the thiazolidine fragment. Molecular modelling was used to predict binding modes of synthesized molecules and rationalize experimentally observed SAR. The leader compound is twice more effective in inhibiting the formation of germ tubes by Candida albicans yeast cells compared to voriconazole. An increased level of Pdr5, an azoles drug efflux pump was observed, but the increase is lower than that caused by azoles. The results can be useful for further development of more powerful and safe antifungal agents.

AlphaFold accelerates artificial intelligence powered drug discovery: efficient discovery of a novel CDK20 small molecule inhibitor.

The application of artificial intelligence (AI) has been considered a revolutionary change in drug discovery and development. In 2020, the AlphaFold computer program predicted protein structures for the whole human genome, which has been considered a remarkable breakthrough in both AI applications and structural biology. Despite the varying confidence levels, these predicted structures could still significantly contribute to structure-based drug design of novel targets, especially the ones with no or limited structural information. In this work, we successfully applied AlphaFold to our end-to-end AI-powered drug discovery engines, including a biocomputational platform PandaOmics and a generative chemistry platform Chemistry42. A novel hit molecule against a novel target without an experimental structure was identified, starting from target selection towards hit identification, in a cost- and time-efficient manner. PandaOmics provided the protein of interest for the treatment of hepatocellular carcinoma (HCC) and Chemistry42 generated the molecules based on the structure predicted by AlphaFold, and the selected molecules were synthesized and tested in biological assays. Through this approach, we identified a small molecule hit compound for cyclin-dependent kinase 20 (CDK20) with a binding constant Kd value of 9.2 ± 0.5 µM (n = 3) within 30 days from target selection and after only synthesizing 7 compounds. Based on the available data, a second round of AI-powered compound generation was conducted and through this, a more potent hit molecule, ISM042-2-048, was discovered with an average Kd value of 566.7 ± 256.2 nM (n = 3). Compound ISM042-2-048 also showed good CDK20 inhibitory activity with an IC50 value of 33.4 ± 22.6 nM (n = 3). In addition, ISM042-2-048 demonstrated selective anti-proliferation activity in an HCC cell line with CDK20 overexpression, Huh7, with an IC50 of 208.7 ± 3.3 nM, compared to a counter screen cell line HEK293 (IC50 = 1706.7 ± 670.0 nM). This work is the first demonstration of applying AlphaFold to the hit identification process in drug discovery.

NMR solution structure of rat aß(1-16): toward understanding the mechanism of rats' resistance to Alzheimer's disease.

In an attempt to reveal the mechanism of rats' resistance to Alzheimer's disease, we determined the structure of the metal-binding domain 1-16 of rat ß-amyloid (rat Aß(1-16)) in solution in the absence and presence of zinc ions. A zinc-induced dimerization of the domain was detected. The zinc coordination site was found to involve residues His-6 and His-14 of both peptide chains. We used experimental restraints obtained from analyses of NMR and isothermal titration calorimetry data to perform structure calculations. The calculations employed an explicit water environment and a simulated annealing molecular-dynamics protocol followed by quantum-mechanical/molecular-mechanical optimization. We found that the C-tails of the two polypeptide chains of the rat Aß(1-16) dimer are oriented in opposite directions to each other, which hinders the assembly of rat Aß dimers into oligomeric aggregates. Thus, the differences in the structure of zinc-binding sites of human and rat Aß(1-16), their ability to form regular cross-monomer bonds, and the orientation of their hydrophobic C-tails could be responsible for the resistance of rats to Alzheimer's disease.

Insights into the structure and function of Est3 from the Hansenula polymorpha telomerase.

Telomerase is a ribonucleoprotein enzyme, which maintains genome integrity in eukaryotes and ensures continuous cellular proliferation. Telomerase holoenzyme from the thermotolerant yeast Hansenula polymorpha, in addition to the catalytic subunit (TERT) and telomerase RNA (TER), contains accessory proteins Est1 and Est3, which are essential for in vivo telomerase function. Here we report the high-resolution structure of Est3 from Hansenula polymorpha (HpEst3) in solution, as well as the characterization of its functional relationships with other components of telomerase. The overall structure of HpEst3 is similar to that of Est3 from Saccharomyces cerevisiae and human TPP1. We have shown that telomerase activity in H. polymorpha relies on both Est3 and Est1 proteins in a functionally symmetrical manner. The absence of either Est3 or Est1 prevents formation of a stable ribonucleoprotein complex, weakens binding of a second protein to TER, and decreases the amount of cellular TERT, presumably due to the destabilization of telomerase RNP. NMR probing has shown no direct in vitro interactions of free Est3 either with the N-terminal domain of TERT or with DNA or RNA fragments mimicking the probable telomerase environment. Our findings corroborate the idea that telomerase possesses the evolutionarily variable functionality within the conservative structural context.

Theoretical and NMR Conformational Studies of ß-Proline Oligopeptides With Alternating Chirality of Pyrrolidine Units.

Synthetic ß-peptides are potential functional mimetics of native α-proteins. A recently developed, novel, synthetic approach provides an effective route to the broad group of ß-proline oligomers with alternating patterns of stereogenic centers. Conformation of the pyrrolidine ring, Z/E isomerism of ß-peptide bonds, and hindered rotation of the neighboring monomers determine the spatial structure of this group of ß-proline oligopeptides. Preferences in their structural organization and corresponding thermodynamic properties are determined by NMR spectroscopy, restrained molecular dynamics and quantum mechanics. The studied ß-proline oligopeptides exist in dimethyl sulfoxide solution in a limited number of conformers, with compatible energy of formation and different spatial organization. In the ß-proline tetrapeptide with alternating chirality of composing pyrrolidine units, one of three peptide bonds may exist in an E configuration. For the alternating ß-proline pentapeptide, the presence of an E configuration for at least of one ß-peptide bond is mandatory. In this case, three peptide bonds synchronously change their configurations. Larger polypeptides may only exist in the presence of several E configurations of ß-peptide bonds forming a wave-like extended structure.

N-domain of angiotensin-converting enzyme hydrolyzes human and rat amyloid-ß(1-16) peptides as arginine specific endopeptidase potentially enhancing risk of Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder. Amyloid-ß (Aß) aggregation is likely to be the major cause of AD. In contrast to humans and other mammals, that share the same Aß sequence, rats and mice are invulnerable to AD-like neurodegenerative pathologies, and Aß of these rodents (ratAß) has three amino acid substitutions in the metal-binding domain 1-16 (MBD). Angiotensin-converting enzyme (ACE) cleaves Aß-derived peptide substrates, however, there are contradictions concerning the localization of the cleavage sites within Aß and the roles of each of the two ACE catalytically active domains in the hydrolysis. In the current study by using mass spectrometry and molecular modelling we have tested a set of peptides corresponding to MBDs of Aß and ratAß to get insights on the interactions between ACE and these Aß species. It has been shown that the N-domain of ACE (N-ACE) acts as an arginine specific endopeptidase on the Aß and ratAß MBDs with C-amidated termini, thus assuming that full-length Aß and ratAß can be hydrolyzed by N-ACE in the same endopeptidase mode. Taken together with the recent data on the molecular mechanism of zinc-dependent oligomerization of Aß, our results suggest a modulating role of N-ACE in AD pathogenesis.

Heparin Modulates the Kinetics of Zinc-Induced Aggregation of Amyloid-ß Peptides.

Zinc-induced aggregation of amyloid-ß peptides (Aß) is considered to contribute to the pathogenesis of Alzheimer's disease. While glycosaminoglycans (GAGs) that are commonly present in interneuronal space are known to enhance Aß self-aggregation in vitro, the impact of GAGs on the formation of zinc-induced amorphous Aß aggregates has not yet been thoroughly studied. Here, employing dynamic light scattering, bis-ANS fluorimetry, and sedimentation assays, we demonstrate that heparin serving as a representative GAG modulates the kinetics of zinc-induced Aß42 aggregation in vitro by slowing the rate of aggregate formation and aggregate size growth. By using synthetic Aß16 peptides to model the Aß metal-binding domain (MBD), heparin was found to effectively interact with MBDs in complex with zinc ions. We suggest that heparin adsorbs to the surface of growing zinc-induced Aß42 aggregates via electrostatic interactions, thus creating a steric hindrance that inhibits further inclusion of monomeric and/or oligomeric zinc-Aß42 complexes. Furthermore, the adsorbed heparin can interfere with the zinc-bridging mechanism of Aß42 aggregation, requiring the formation of two zinc-mediated interaction interfaces in the MBD. As revealed by computer simulations of the zinc-Aß16 homodimer complexed with a heparin chain, heparin can interact with the MBD via polar contacts with residues Arg-5 and Tyr-10, resulting in a conformational rearrangement that hampers the formation of the second zinc-mediated interaction in the MBD interface. The findings of this study suggest that GAGs, which are common in the in vivo macromolecular environment, may have a substantial impact on the time course of zinc-induced Aß aggregation.

Zinc-induced heterodimer formation between metal-binding domains of intact and naturally modified amyloid-beta species: implication to amyloid seeding in Alzheimer's disease?

Zinc ions and modified amyloid-beta peptides (Aß) play a critical role in the pathological aggregation of endogenous Aß in Alzheimer's disease (AD). Zinc-induced Aß oligomerization is mediated by the metal-binding domain (MBD) which includes N-terminal residues 1-16 (Aß1-16). Earlier, it has been shown that Aß1-16 as well as some of its naturally occurring variants undergoes zinc-induced homodimerization via the interface in which zinc ion is coordinated by Glu11 and His14 of the interacting subunits. In this study using surface plasmon resonance technique, we have found that in the presence of zinc ions Aß1-16 forms heterodimers with MBDs of two Aß species linked to AD: Aß containing isoAsp7 (isoAß) and Aß containing phosphorylated Ser8 (pS8-Aß). The heterodimers appear to possess the same interface as the homodimers. Simulation of 200 ns molecular dynamic trajectories in two constructed models of dimers ([Aß1-16/Zn/Aß1-16] and [isoAß1-16/Zn/Aß1-16]), has shown that conformational flexibility of the N-terminal fragments of the dimer subunits is controlled by the structure of corresponding sites 6-8. The data suggest that isoAß and pS8-Aß can be involved in the AD pathogenesis by means of their zinc-dependent interactions with endogenous Aß resulting in the formation of heterodimeric seeds for amyloid aggregation.

Interplay of histidine residues of the Alzheimer's disease Aß peptide governs its Zn-induced oligomerization.

Conformational changes of Aß peptide result in its transformation from native monomeric state to the toxic soluble dimers, oligomers and insoluble aggregates that are hallmarks of Alzheimer's disease (AD). Interactions of zinc ions with Aß are mediated by the N-terminal Aß(1-16) domain and appear to play a key role in AD progression. There is a range of results indicating that these interactions trigger the Aß plaque formation. We have determined structure and functional characteristics of the metal binding domains derived from several Aß variants and found that their zinc-induced oligomerization is governed by conformational changes in the minimal zinc binding site 6HDSGYEVHH14. The residue H6 and segment 11EVHH14, which are part of this site are crucial for formation of the two zinc-mediated interaction interfaces in Aß. These structural determinants can be considered as promising targets for rational design of the AD-modifying drugs aimed at blocking pathological Aß aggregation.

Control of Azomethine Cycloaddition Stereochemistry by CF3 Group: Structural Diversity of Fluorinated ß-Proline Dimers.

ß-Proline-functionalized dimers consisting of homochiral monomeric units were synthesized by a non-peptidic coupling method for the first time. The applied synthetic methodology is based on 1,3-dipolar cycloaddition chemistry of azomethine ylides and provides absolute control over the ß-proline backbone stereogenic centers. An o-(trifluoromethyl)phenyl substituent contributes to appropriate stabilization of the definite acrylamide chiral cis conformation and to achieve the dipole reactivity that is not observed for aryl groups lacking strong electronegative character.

A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein, an intrinsically disordered protein.

α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential H(N) − H(α) and H(N) − H(N) NOEs, values for (3) JHNHα, (1) JHαCα, (2) JCαN, and (1) JCαN, as well as chemical shifts of (15)N, (13)C(α), and (13)C' nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20-30%) of the PPII region and a somewhat higher population of the potentially aggregation-prone ß region (20-40%) than seen in the database. A generally lower population of the αR region (10-20%) is found. Analysis of (1)H − (1)H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein.

Contact-based ligand-clustering approach for the identification of active compounds in virtual screening.

Evaluation of docking results is one of the most important problems for virtual screening and in silico drug design. Modern approaches for the identification of active compounds in a large data set of docked molecules use energy scoring functions. One of the general and most significant limitations of these methods relates to inaccurate binding energy estimation, which results in false scoring of docked compounds. Automatic analysis of poses using self-organizing maps (AuPosSOM) represents an alternative approach for the evaluation of docking results based on the clustering of compounds by the similarity of their contacts with the receptor. A scoring function was developed for the identification of the active compounds in the AuPosSOM clustered dataset. In addition, the AuPosSOM efficiency for the clustering of compounds and the identification of key contacts considered as important for its activity, were also improved. Benchmark tests for several targets revealed that together with the developed scoring function, AuPosSOM represents a good alternative to the energy-based scoring functions for the evaluation of docking results.

NMR solution structure and function of the C-terminal domain of eukaryotic class 1 polypeptide chain release factor.

Termination of translation in eukaryotes is triggered by two polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3. eRF1 is a three-domain protein that interacts with eukaryotic class 2 polypeptide chain release factor 3 via its C-terminal domain (C-domain). The high-resolution NMR structure of the human C-domain (residues 277-437) has been determined in solution. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure. The structure of the minidomain (residues 329-372), which was ill-defined in the crystal structure, has been determined in solution. The protein backbone dynamics, studied using (15)N-relaxation experiments, showed that the C-terminal tail 414-437 and the minidomain are the most flexible parts of the human C-domain. The minidomain exists in solution in two conformational states, slowly interconverting on the NMR timescale. Superposition of this NMR solution structure of the human C-domain onto the available crystal structure of full-length human eRF1 shows that the minidomain is close to the stop codon-recognizing N-terminal domain. Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor. The results provide new insights into the possible role of the C-domain in the process of translation termination.

NMR assignments of the C-terminal domain of human polypeptide release factor eRF1.

We report NMR assignments of the protein backbone of the C-terminal domain (163 a.a.) of human class 1 translation termination factor eRF1. It was found that several protein loop residues exist in two slowly interconverting conformational states.