Maytansinol Functionalization: Towards Useful Probes for Studying Microtubule Dynamics.

Maytansinoids are a successful class of natural and semisynthetic tubulin binders, known for their potent cytotoxic activity. Their wider application as cytotoxins and chemical probes to study tubulin dynamics has been held back by the complexity of natural product chemistry. Here we report the synthesis of long-chain derivatives and maytansinoid conjugates. We confirmed that bulky substituents do not impact their high activity or the scaffold's binding mode. These encouraging results open new avenues for the design of new maytansine-based probes.

Maytansinol Functionalization: Towards Useful Probes for Studying Microtubule Dynamics.

Invited for the cover of this issue are the groups of Professors Passarella and Pieraccini at the University of Milan, in collaboration with some of the members of TubInTrain consortium. The image depicts work with the elements of nature, in particular the destabilising effect of maytansinol (the constellation) on microtubules (the trees). Read the full text of the article at 10.1002/chem.202203431.

Synthesis of SARS-CoV-2 Mpro inhibitors bearing a cinnamic ester warhead with in vitro activity against human coronaviruses.

COVID-19 now ranks among the most devastating global pandemics in history. The causative virus, SARS-CoV-2, is a new human coronavirus (hCoV) that spreads among humans and animals. Great efforts have been made to develop therapeutic agents to treat COVID-19, and among the available viral molecular targets, the cysteine protease SARS-CoV-2 Mpro is considered the most appealing one due to its essential role in viral replication. However, the inhibition of Mpro activity is an interesting challenge and several small molecules and peptidomimetics have been synthesized for this purpose. In this work, the Michael acceptor cinnamic ester was employed as an electrophilic warhead for the covalent inhibition of Mpro by endowing some peptidomimetic derivatives with such a functionality. Among the synthesized compounds, the indole-based inhibitors 17 and 18 efficiently impaired the in vitro replication of beta hCoV-OC-43 in the low micromolar range (EC50 = 9.14 µM and 10.1 µM, respectively). Moreover, the carbamate derivative 12 showed an antiviral activity of note (EC50 = 5.27 µM) against another hCoV, namely hCoV-229E, thus suggesting the potential applicability of such cinnamic pseudopeptides also against human alpha CoVs. Taken together, these results support the feasibility of considering the cinnamic framework for the development of new Mpro inhibitors endowed with antiviral activity against human coronaviruses.

Resonance Raman and Visible Micro-Spectroscopy for the In-Vivo and In-Vitro Characterization of Anthocyanin-Based Pigments in Blue and Violet Flowers: A Comparison with HPLC-ESI- MS Analysis of the Extracts.

Microanalysis techniques based on resonance Raman and reflection visible spectroscopy have been applied to the characterization of pigments responsible for the blue or violet coloration in flowers; in particular of Lobelia erinus, Campanula portenschlagiana, Cineraria, Viola tricolor, Anemone coronaria, Agapanthus, Platycodon, Salvia farinacea, Plumbago capensis, Ceratostigma plumbaginoides, Commelina communis and Salvia patens. The spectroscopic methods were applied both in vivo on the flower petals and in vitro on extracts obtained through a procedure based on SPE (solid-phase extraction) optimized for minimal quantities of vegetable raw material. Different patterns obtained for the Raman spectra have been correlated, also on the basis of density functional theory (DFT) calculations, with different schemes of substitution of the benzopyrilium nucleus of the anthocyanins and with various possible forms of copigmentation responsible for the stabilization of the blue color. The results obtained were verified by comparison with the analysis of the extracts by HPLC-ESI-MS (liquid chromatography-electrospray ionization-mass spectrometry).

Discovery of a Novel Trifluoromethyl Diazirine Inhibitor of SARS-CoV-2 Mpro.

SARS-CoV-2 Mpro is a chymotrypsin-like cysteine protease playing a relevant role during the replication and infectivity of SARS-CoV-2, the coronavirus responsible for COVID-19. The binding site of Mpro is characterized by the presence of a catalytic Cys145 which carries out the hydrolytic activity of the enzyme. As a consequence, several Mpro inhibitors have been proposed to date in order to fight the COVID-19 pandemic. In our work, we designed, synthesized and biologically evaluated MPD112, a novel inhibitor of SARS-CoV-2 Mpro bearing a trifluoromethyl diazirine moiety. MPD112 displayed in vitro inhibition activity against SARS-CoV-2 Mpro at a low micromolar level (IC50 = 4.1 µM) in a FRET-based assay. Moreover, an inhibition assay against PLpro revealed lack of inhibition, assuring the selectivity of the compound for the Mpro. Furthermore, the target compound MPD112 was docked within the binding site of the enzyme to predict the established intermolecular interactions in silico. MPD112 was subsequently tested on the HCT-8 cell line to evaluate its effect on human cells' viability, displaying good tolerability, demonstrating the promising biological compatibility and activity of a trifluoromethyl diazirine moiety in the design and development of SARS-CoV-2 Mpro binders.

Maytansinol Derivatives: Side Reactions as a Chance for New Tubulin Binders.

Maytansinol is a valuable precursor for the preparation of maytansine derivatives (known as maytansinoids). Inspired by the intriguing structure of the macrocycle and the success in targeted cancer therapy of the derivatives, we explored the maytansinol acylation reaction. As a result, we were able to obtain a series of derivatives with novel modifications of the maytansine scaffold. We characterized these molecules by docking studies, by a comprehensive biochemical evaluation, and by determination of their crystal structures in complex with tubulin. The results shed further light on the intriguing chemical behavior of maytansinoids and confirm the relevance of this peculiar scaffold in the scenario of tubulin binders.

Molecular Similarity Perception Based on Machine-Learning Models.

Molecular similarity is an impressively broad topic with many implications in several areas of chemistry. Its roots lie in the paradigm that 'similar molecules have similar properties'. For this reason, methods for determining molecular similarity find wide application in pharmaceutical companies, e.g., in the context of structure-activity relationships. The similarity evaluation is also used in the field of chemical legislation, specifically in the procedure to judge if a new molecule can obtain the status of orphan drug with the consequent financial benefits. For this procedure, the European Medicines Agency uses experts' judgments. It is clear that the perception of the similarity depends on the observer, so the development of models to reproduce the human perception is useful. In this paper, we built models using both 2D fingerprints and 3D descriptors, i.e., molecular shape and pharmacophore descriptors. The proposed models were also evaluated by constructing a dataset of pairs of molecules which was submitted to a group of experts for the similarity judgment. The proposed machine-learning models can be useful to reduce or assist human efforts in future evaluations. For this reason, the new molecules dataset and an online tool for molecular similarity estimation have been made freely available.

Exploring Orthogonality between Halogen and Hydrogen Bonding Involving Benzene.

The concept of orthogonality between halogen and hydrogen bonding, brought out by Ho and coworkers some years ago, has become a widely accepted idea within the chemists' community. While the original work was based on a common carbonyl oxygen as acceptor for both interactions, we explore here, by means of M06-2X, M11, ωB97X, and ωB97XD/aug-cc-PVTZ DFT calculations, the interdependence of halogen and hydrogen bonding with a shared π-electron system of benzene. The donor groups (specifically NCBr and H2O) were placed on either or the same side of the ring, according to a double T-shaped or a perpendicular geometry, respectively. The results demonstrate that the two interactions with benzene are not strictly independent on each other, therefore outlining that the orthogonality between halogen and hydrogen bonding, intended as energetical independence between the two interactions, should be carefully evaluated according to the specific acceptor group.

On-resin multicomponent 1,3-dipolar cycloaddition of cyclopentanone-proline enamines and sulfonylazides as an efficient tool for the synthesis of amidino depsipeptide mimics.

Depsipeptides are biologically active peptide derivatives that possess a high therapeutic interest. The development of depsipeptide mimics characterized by a chemical diversity could lead to compounds with enhanced features and activity. In this work, an on-resin multicomponent procedure for the synthesis of amidino depsipeptide mimics is described. This approach exploits a metal-free 1,3-dipolar cycloaddition of cyclopentanone-proline enamines and sulfonylazides. In this reaction, the obtained primary cycloadduct undergoes a ring opening and molecular rearrangement giving access to a linear sulfonyl amidine functionalized with both a peptide chain and a diazoalkane. The so-obtained diazo function "one pot" reacts with the carboxylic group of N-Fmoc-protected amino acids leading to amidino depsipeptide mimics possessing a C4 aliphatic chain. An important advantage of this procedure is the possibility to easily obtain amidino-functionalized derivatives that are proteolytically stable peptide bond bioisosteres. Moreover, the conformational freedom given by the alkyl chain could promote the obtainment of cyclic depsipeptide with a stabilized secondary structure as demonstrated with both in silico calculations and experimental conformational studies. Finally, labeled depsipeptide mimics can be also synthesized using a fluorescent sulfonylazide in the multicomponent reaction.

X-ray Constrained Spin-Coupled Wavefunction: a New Tool to Extract Chemical Information from X-ray Diffraction Data.

The X-ray constrained wavefunction (XCW) approach is a reliable and widely used method of quantum crystallography that allows the determination of wavefunctions compatible with X-ray diffraction data. So far, all the existing XCW techniques have been developed in the framework of molecular orbital theory and, consequently, provide only pictures of the "experimental" electronic structures that are far from the traditional chemical perception. Here a new strategy is proposed that, by combining the XCW philosophy with the spin-coupled method of valence bond theory, enables direct extraction of traditional chemical information (e.g., weights of resonance structures) from X-ray diffraction measurements. Preliminary results have shown that the new technique is really able to efficiently capture the effects of the crystal environment on the electronic structure, and can be considered as a new useful tool to perform chemically sound analyses of the X-ray diffraction data.

Assessment of DFT Functionals for QTAIM Topological Analysis of Halogen Bonds with Benzene.

Halogen bonding, a noncovalent interaction between a halogen atom and a nucleophilic site, is receiving a growing attention in the chemical community stimulating a large number of theoretical investigations. The density functional theory (DFT) approach revealed to be one of the most suitable methods owing to its accuracy and low computational cost. We report here a detailed analysis of the performance of an extensive set of DFT functionals in reproducing accurate binding energies and topological properties for the halogen-bonding interaction of either NCX or PhX molecules (X = F, Cl, Br, I) with the aromatic system of benzene in the T-shaped configuration. It was found that the better performance for both sets of properties is provided by a small subset of functionals able to take into account, implicitly or explicitly (by inclusion of an additive pairwise potential), the dispersion contribution, that is, ωB97X, M06-2X, M11, mPW2PLYP-D, and B2PLYP-D3.

Halogen bonds with benzene: an assessment of DFT functionals.

The performance of an extensive set of density functional theory functionals has been tested against CCSD(T) and MP2 results, extrapolated to the complete basis set (CBS) limit, for the interaction of either DCl or DBr (D = H, HCC, F, and NC) with the aromatic system of benzene. It was found that double hybrid functionals explicitly including dispersion, that is, B2PLYPD and mPW2PLYPD, provide the better agreement with the CCSD(T)/CBS results on both energies and equilibrium geometry, indicating the importance of dispersive contributions in determining this interaction. Among the less expensive functionals, the better performance is provided by the ωB97X and M062X functionals, while the ωB97XD and B97D functionals are shown to work very well for bromine complexes but not so well for chlorine complexes.

Total Synthesis of an Epothilone Analogue based on the Amide‒Triazole Bioisosterism.

Epothilones are 16-membered macrolides that can act as microtubule-targeting agents to tackle cancer. Many synthetic analogues have been investigated for their activity, yet often based on macrolide structures. A notable exception is Ixabepilone, an azalide representing the only epothilone-like molecule approved by the FDA as a chemotherapeutic. Exploiting the amide-triazole bioisosterism, in this work we report the synthesis of the first generation of epothilones lacking the macrolide or azalide structure, with the ester or amide linkage replaced by a triazole unit. Together with the synthesis of this new analogue, computational and biological evaluations have been performed too.

Quinazolinecarboline alkaloid evodiamine as scaffold for targeting topoisomerase I and sirtuins.

This paper reports the synthesis of a series of evodiamine derivatives. We assayed the ability to inhibit cell growth on three human tumour cell lines (H460, MCF-7 and HepG2) and we evaluated the capacity to interfere with the catalytic activity of topoisomerase I both by the relaxation assay and the occurrence of the cleavable complex. Moreover, whose effect on sirtuins 1, 2 and 3 was investigated. Finally, molecular docking analyses were performed in an attempt to rationalize the biological results.

Computational Approaches to the Rational Design of Tubulin-Targeting Agents.

Microtubules are highly dynamic polymers of α,ß-tubulin dimers which play an essential role in numerous cellular processes such as cell proliferation and intracellular transport, making them an attractive target for cancer and neurodegeneration research. To date, a large number of known tubulin binders were derived from natural products, while only one was developed by rational structure-based drug design. Several of these tubulin binders show promising in vitro profiles while presenting unacceptable off-target effects when tested in patients. Therefore, there is a continuing demand for the discovery of safer and more efficient tubulin-targeting agents. Since tubulin structural data is readily available, the employment of computer-aided design techniques can be a key element to focus on the relevant chemical space and guide the design process. Due to the high diversity and quantity of structural data available, we compiled here a guide to the accessible tubulin-ligand structures. Furthermore, we review different ligand and structure-based methods recently used for the successful selection and design of new tubulin-targeting agents.

Halogen-bonding interactions with π systems: CCSD(T), MP2, and DFT calculations.

Halogen bonding is a noncovalent interaction between a halogen atom and a nucleophilic site. Interactions involving the π electrons of aromatic rings have received, up to now, little attention, despite the large number of systems in which they are present. We report binding energies of the interaction between either NCX or PhX (X = F, Cl, Br, I) and the aromatic benzene system as determined with the coupled cluster with perturbative triple excitations method [CCSD(T)] extrapolated at the complete basis set limit. Results are compared with those obtained by Møller-Plesset perturbation theory to second order (MP2) and density functional theory (DFT) calculations by using some of the most common functionals. Results show the important role of DFT in studying this interaction.

Conformational switch and multiple supramolecular structures of a newly identified self-assembling protein-mimetic peptide from Pseudomonas aeruginosa YeaZ protein.

Protein-mimetic peptides (PMPs) are shorter sequences of self-assembling proteins, that represent remarkable building blocks for the generation of bioinspired functional supramolecular structures with multiple applications. The identification of novel aminoacidic sequences that permit the access to valuable biocompatible materials is an attractive area of research. In this work, in silico analysis of the Pseudomonas aeruginosa YeaZ protein (PaYeaZ) led to the identification of a tetradecapeptide that represents the shortest sequence responsible for the YeaZ-YeaZ dimer formation. Based on its sequence, an innovative 20-meric peptide, called PMP-2, was designed, synthesized, and characterized in terms of secondary structure and self-assembly properties. PMP-2 conserves a helical character and self-assembles into helical nanofibers in non-polar solvents (DMSO and trifluoroethanol), as well as in dilute (0.5 mM) aqueous solutions. In contrast, at higher concentrations (>2 mM) in water, a conformational transition from α-helix to ß-sheet occurs, which is accompanied by the Protein-mimetic peptide aggregation into 2D-sheets and formation supramolecular gel in aqueous environment. Our findings reveal a newly identified Protein-mimetic peptide that could turn as a promising candidate for future material applications.

Metadynamics study of a ß-hairpin stability in mixed solvents.

Understanding the molecular mechanisms that allow some organisms to survive in extremely harsh conditions is an important achievement that might disclose a wide range of applications and that is constantly drawing the attention of many research fields. The high adaptability of these living creatures is related to the presence in their tissues of a high concentration of osmoprotectants, small organic, highly soluble molecules. Despite osmoprotectants having been known for a long time, a full disclosure of the machinery behind their activity is still lacking. Here we describe a computational approach that, taking advantage of the recently developed metadynamics technique, allows one to fully describe the free energy surface of a small ß-hairpin peptide and how it is affected by an osmoprotectant, glycine betaine (GB) and for comparison by urea, a common denaturant. Simulations led to relevant thermodynamic information, including how the free energy difference of denaturation is affected by the two cosolvents; unlike urea, GB caused a considerable increase of the folded basin stability, which transposes into a higher melting temperature. NMR experiments confirmed the picture derived from the theoretical study. Further molecular dynamics simulations of selected conformations allowed investigation into deeper detail the role of GB in folded state protection. Simulations of the protein in GB solutions clearly showed an excess of osmoprotectant in the solvent bulk, rather than in the protein domain, confirming the exclusion from the protein surface, but also highlighted interesting features on its interactions, opening to new scenarios besides the classic "indirect mechanism" hypothesis.

Halogen bonding in ligand-receptor systems in the framework of classical force fields.

Halogen bond is an important non-covalent interaction which is receiving a growing attention in the study of protein-ligand complexes. Many drugs are halogenated molecules and it has been recently shown that many halogenated ligands establish halogen bonds with biomolecules. As the halogen bond nature is due to an anisotropy of the electrostatic potential around halogen atoms, it is not possible to use traditional force fields based on a set of atom-centred charges to study halogen bonds in biomolecules. We show that the introduction of pseudo-atoms on halogens permits us to correctly describe the anisotropy of the electrostatic potential and to perform molecular dynamics simulations on complexes of proteins with halogenated ligands that reproduce experimental values. The results are compared with crystallographic data and with hybrid quantum mechanics/molecular mechanics calculations.

α-Synuclein: An All-Inclusive Trip Around its Structure, Influencing Factors and Applied Techniques.

Alpha-synuclein (αSyn) is a highly expressed and conserved protein, typically found in the presynaptic terminals of neurons. The misfolding and aggregation of αSyn into amyloid fibrils is a pathogenic hallmark of several neurodegenerative diseases called synucleinopathies, such as Parkinson's disease. Since αSyn is an Intrinsically Disordered Protein, the characterization of its structure remains very challenging. Moreover, the mechanisms by which the structural conversion of monomeric αSyn into oligomers and finally into fibrils takes place is still far to be completely understood. Over the years, various studies have provided insights into the possible pathways that αSyn could follow to misfold and acquire oligomeric and fibrillar forms. In addition, it has been observed that αSyn structure can be influenced by different parameters, such as mutations in its sequence, the biological environment (e.g., lipids, endogenous small molecules and proteins), the interaction with exogenous compounds (e.g., drugs, diet components, heavy metals). Herein, we review the structural features of αSyn (wild-type and disease-mutated) that have been elucidated up to present by both experimental and computational techniques in different environmental and biological conditions. We believe that this gathering of current knowledge will further facilitate studies on αSyn, helping the planning of future experiments on the interactions of this protein with targeting molecules especially taking into consideration the environmental conditions.