Rational Design Problematics of Peptide Nucleic Acids as SARS-CoV-2 Inhibitors.

The use of viral protein inhibitors has shown to be insufficiently effective in the case of highly variable SARS-CoV-2. In this work, we examined the possibility of designing agents that bind to a highly conserved region of coronavirus (+)RNA. We demonstrated that while the design of antisense RNAs is based on the complementary interaction of nitrogenous bases, it is possible to use semirigid docking methods in the case of unnatural peptide nucleic acids. The transition from N-(2-aminoethyl)glycine chain to a more conformationally rigid piperidine-containing backbone allowed us to significantly increase the affinity of structures to the target RNA.

Ubiquitin recruiting chimera: more than just a PROTAC.

Ubiquitinylation of protein substrates results in various but distinct biological consequences, among which ubiquitin-mediated degradation is most well studied for its therapeutic application. Accordingly, artificially targeted ubiquitin-dependent degradation of various proteins has evolved into the therapeutically relevant PROTAC technology. This tethered ubiquitinylation of various targets coupled with a broad assortment of modifying E3 ubiquitin ligases has been made possible by rational design of bi-specific chimeric molecules that bring these proteins in proximity. However, forced ubiquitinylation inflicted by the binary warheads of a chimeric PROTAC molecule should not necessarily result in protein degradation but can be used to modulate other cellular functions. In this respect it should be noted that the ubiquitinylation of a diverse set of proteins is known to control their transport, transcriptional activity, and protein-protein interactions. This review provides examples of potential PROTAC usage based on non-degradable ubiquitinylation.

New Insights into Chemoresistance Mediated by Mdm2 Inhibitors: The Benefits of Targeted Therapy over Common Cytostatics.

The inhibition of the Mdm2-p53 protein-protein interaction is a promising strategy for anticancer therapy. However, the problem of developing secondary chemoresistance in tumors treated with such drugs has not yet been sufficiently studied. In this work, we compared the properties of a drug-resistant cell line obtained during long-term cultivation in the presence of an Mdm2 inhibitor, Nutlin-3a, with a similarly obtained line insensitive to the cytostatic drug paclitaxel. We first confirmed the higher safety levels of Mdm2 inhibitors when compared with cytostatics in terms of the development of secondary chemoresistance. We showed that Nutlin-3a affects both the targeted p53-mediated cellular machinery and the universal ABC-mediated efflux mechanism. While both targeted and general defense mechanisms are activated by the Mdm2 inhibitor, it still increases the susceptibility of tumor cells to other drugs. The results obtained indicate that the risks of developing chemoresistance under the therapy with a targeted agent are fundamentally lower than during cytotoxic therapy.

NMR-Verified Dearomatization of 5,7-Substituted Pyrazolo[1,5-a]pyrimidines.

Tetrahydropyrazolo[1,5-a]pyrimidine (THPP) is an attractive scaffold for designing biologically active compounds. The most obvious way to obtain such compounds is to reduce pyrazolopyrimidines with complex hydrides, because the pyrimidine ring is reduced in the preference over the pyrazole ring. The presence of substituents at positions five and seven of pyrazolo[1,5-a]pyrimidines complicates the set of reaction products but makes it more attractive for medicinal chemistry because four possible stereoisomers can be formed during reduction. However, the formation of only syn-isomers has been described in the literature. This article is the first report on the formation of anti-configured isomers along with syn-isomers in the reduction of model 5,7-dimethylpyrazolo[1,5-a]pyrimidine, which was confirmed by NMR. The bicyclic core in the syn-configuration was shown to be conformationally stable, which was used to estimate the long-range interproton distances using NOESY data. At the same time, long-range dipole-dipole interactions corresponding to a distance between protons of more than 6 Å were first registered and quantified. In turn, the bicyclic core in the trans-configuration represents a conformationally labile system. For these structures, an analysis of conformations observed in solutions was carried out. Our results indicate the significant potential of trans-configured tetrahydropyrazolo[1,5-a]pyrimidines for the development of active small molecules. While possessing structural lability due to the low energy of the conformational transition, they have the ability to adjust to the active site of the desired target.

Is It Possible to Obtain a Product of the Desired Configuration from a Single Knoevenagel Condensation? Isomerization vs. Stereodefined Synthesis.

The biological activity of compounds directly depends on the three-dimensional arrangement of affinity fragments since a high degree of pharmacophore compliance with the binding site is required. 3-Benzylidene oxindoles are privileged structures due to their wide spectrum of biological activity, synthetic availability, and ease of modification. In particular, both kinase inhibitors and kinase activators can be found among 3-benzylidene oxindoles. In this work, we studied model compounds obtained via oxindole condensation with aldehydes and alkylphenones. These condensation products can exist in the form of E- and Z-isomers and also undergo isomerization in solutions. The factors causing isomeric transformation of these compounds were established. Comparative kinetic studies to obtain quantitative characteristics of UV-driven isomerization were first performed. The results obtained indicate dramatic differences in two subclasses, which should be considered when developing biologically active molecules.

ATP Mimetic Attack on the Nucleotide-Binding Domain to Overcome ABC Transporter Mediated Chemoresistance.

Since the problem of transporter-mediated multidrug resistance of tumor cells is becoming increasingly important in cancer therapy, it is necessary to modulate the activity of efflux transporters of the ABC family, among which P-glycoprotein is the best known. We consider the nucleotide binding domain, a universal fragment of these transporters, as a target for the rational design of small molecule compounds capable of preventing ATP-dependent drug efflux. Using various ATP mimetics, we showed that they suppress the efflux of fluorescent substrates and paclitaxel from the cells due to suppressing the ATPase activity of the transporters. The combined use of paclitaxel and ATP mimetics significantly increases its antitumor efficacy, including in cells with the multidrug resistance phenotype. The considered compounds are promising agents for the development of therapeutic efflux modulators, since they are not toxic at the given concentrations and do not induce the transporter overexpression. Moreover, the compounds overcome not only P-gp-mediated but also BCRP-mediated resistance of tumor cells.

Analysis of P-Glycoprotein Transport Cycle Reveals a New Way to Identify Efflux Inhibitors.

P-glycoprotein (P-gp) is found to be of considerable interest for the design of drugs capable of treating chemoresistant tumors. This transporter is an interesting target for which an efficient approach has not yet been developed in terms of computer simulation. In this work, we use a combination of docking, molecular dynamics, and metadynamics to fully explore the states that occur during the capture of a ligand and subsequent efflux by P-gp. The proposed approach allowed us to substantiate a number of experimentally established facts, as well as to develop a new criterion for identifying potential P-gp inhibitors.

Implication of ABC transporters in non-proliferative diseases.

ABC transporters play an essential role in the development of multidrug resistance and thus are of interest in the context of anticancer therapy. However, MDR1, BCRP and MRP1 are involved in a number of key processes that maintain the viability of the body as a whole, as well as individual organs and cells. These transporters support protective properties of anatomical and histohematic barriers, determining the entry of both toxins and drugs into organs and tissues, as well as facilitating their elimination. This review discusses the main areas in which the use of modulators of the ABC exporter activity may be relevant due to either an initial imbalance in their activity or the need for the temporary change in the efflux rate for therapeutic purposes. Controlled modulation of the activity of the ABC family efflux transporters opens up broad prospects in the treatment of various diseases associated both with universal difficulties in the delivery of drugs that are transporter substrates and with the characteristics of individual patients caused by single nucleotide polymorphisms. Both activators and inhibitors of the transporters will find their application.

Establishment of drug-resistant cell lines under the treatment with chemicals acting through different mechanisms.

The problem of chemoresistance development is an inescapable flipside of modern oncotherapy, in particular for сolorectal cancer patients. The search for or development of drugs effective against resistant tumors involves the use of model resistant cell lines in vitro. To obtain such lines, we reproduced the development of chemoresistance of human colon adenocarcinoma cells under the treatment with drugs of different mechanisms, a cytostatic (paclitaxel) and a targeted agent (Nutlin-3a, an inhibitor of p53-Mdm2 protein-protein interaction). In each case, we gradually increased the content of the substance in the medium, starting from effective concentrations that do not cause total cell death. When studying the lines resistant to the corresponding drug, we noted a reduced sensitivity to the drug of another mechanism of action. Analysis of the original and resistant lines showed that the cells use the universal efflux defense mechanism. The observed effect can be partially neutralized using inhibitors of the ABC transport proteins, including P-glycoprotein, known for its oncoprotective function. The role of the latter was confirmed by real-time RT-PCR and Western blotting.

Activating Effect of 3-Benzylidene Oxindoles on AMPK: From Computer Simulation to High-Content Screening.

AMP-activated protein kinase (AMPK) is currently the subject of intensive study and active discussions. AMPK performs its functions both at the cellular level, providing the switch between energy-consuming and energy-producing processes, and at the whole body level, particularly, regulating certain aspects of higher nervous activity and behavior. Control of such a 'main switch' compensates dysfunctions and associated diseases. In the present paper, we studied the binding of 3-benzylidene oxindoles to the kinase domain of the AMPK α-subunit, which is thought to prevent its interaction with the autoinhibitory domain and thus result in the AMPK activation. For this purpose, we developed the cellular test system based on the AMPKAR plasmid, which implements the FRET effect, synthesized a number of 3-benzylidene oxindole compounds and simulated their binding to various sites of the kinase domain. The most probable binding site for the studied compounds was established by the correlation of calculated and experimental data. The obtained results allow to analyze various classes of AMPK activators using virtual and high-content screening.

Simulation of MDM2 N-terminal domain conformational lability in the presence of imidazoline based inhibitors of MDM2-p53 protein-protein interaction.

Targeting of MDM2-p53 protein-protein interaction is a current approach for the development of potent anticancer agents. The classical pharmacophore hypothesis for the design of such molecules describes the three point binding of a small molecule inhibitor to the MDM2 protein. However, this hypothesis is not confirmed when considering the activity of a number of known potent MDM2 inhibitors. Here we demonstrate the important role of the flexible N-terminal region of the MDM2 protein in the binding with small molecule compounds, which contributes to the transition from three point binding to four point binding during the development of new anticancer agents. To evaluate the contribution of the MDM2 N-terminal region to the structure-activity relationship of known MDM2 inhibitors, compounds of nutlin series, whose spatial configuration was shown to dramatically affect the target activity, were used as objects of the study. The key amino acid residues within the N-terminal region involved in the interaction with small molecule ligands were determined by means of molecular dynamics. The conformational stability of the flexible MDM2 fragment was simulated under different conditions. The effects of point mutations on the N-terminal region stability were also demonstrated.

Design, in silico prioritization and biological profiling of apoptosis-inducing lactams amenable by the Castagnoli-Cushman reaction.

Five lactam chemotypes amenable by the Castagnoli-Cushman reaction of imines and cyclic anhydrides have been investigated for their ability to activate p53 tumor suppressing transcription factor thus induce apoptosis in p53+ cancer cells. A virtual library of 1.07 million chemically diverse compounds based on these scaffolds was subjected to in silico screening first. The compounds displaying high docking score were visually prioritized to identify the best-fitting compounds, i. e. the ones which adequately mimic the interactions of clinical candidate inhibitor Nutlin-3a. These 38 compounds were synthesized and tested for apoptosis induction in p53+ H116 cancer cells to identify 9 potent apoptosis-inducers (two of them exceeding the activity of Nutlin-3a) which belonged to four different chemotypes. The activation of p53 involved in the proapoptotic activity observed was supported by effective induction of EGFP expression in human osteocarcinoma U2OS-pLV reporter cell line. Moreover, the two most potent apoptosis inducers displayed antiproliferative profile identical to several known advanced p53 activators: they inhibited the growth of p53+/+ HCT116 cells in much lower concentration range compared to p53-/- HCT116 cells.

Amino acids as chiral derivatizing agents for antiproliferative substituted N-benzyl isoindolinones.

The absolute configurations of the diastereomers of novel amino acid ester derivatives of 2,3-substituted isoindolinones, which are known as apoptosis activators due to their ability to inhibit the MDM2-p53 PPI, were assigned using NMR and computational methods. Procedures for diastereomer separation and determining the absolute configuration were developed to perform the study. The high significance of N-benzyl fragment for the determination of the diastereomer absolute configuration by NMR methods was established; it is determined by a number of factors inherent in this fragment and the structural features of the studied substrates. Analysis of the individual isomer activity showed that the target inhibitory effect of S- and R-isoindolinone L-valinates differs by less than 20%. It can be explained by the presence of a flexible linker between the isoindolinone core and amino acid fragment, which provides the optimal arrangement of the molecule in the hydrophobic cavity of MDM2 for both isomers.

Advanced palladium free approach to the synthesis of substituted alkene oxindoles via aluminum-promoted Knoevenagel reaction.

A synthetic route for the synthesis of C24, as well as for the design of focused libraries of direct AMPK activators was developed based on a convergent strategy. The proposed scheme corresponds to the current trends in C-H bond functionalization. The use of aluminum isopropoxide for the Knoevenagel condensation of oxindole with benzophenones is a noticeable point of this work.

Proapoptotic modification of substituted isoindolinones as MDM2-p53 inhibitors.

A series of novel amino acid ester derivatives of 2,3-substituted isoindolinones was synthesized and evaluated for p53-mediated apoptotic activity. The rationale for augmentation of the target activity of 2,3-substituted isoindolinones was based on the introduction of new fragments in the structure of the inhibitor that would provide additional binding sites in the hydrophobic cavity of MDM2. To select for the anticipated modifications we employed molecular docking. Synthesized molecules were evaluated for their ability to induce apoptosis in two cancer cell lines and their derivatives with different status of p53 (colorectal HCT116 and osteosarcoma U2OS cells) by Annexin V staining. The target activity was estimated using high-content imaging system Operetta. Valine and phenylglycine ester derivatives were identified as potentially active MDM2-p53 inhibitors.

The 26S proteasome is a multifaceted target for anti-cancer therapies.

Proteasomes play a critical role in the fate of proteins that are involved in major cellular processes, including signal transduction, gene expression, cell cycle, replication, differentiation, immune response, cellular response to stress, etc. In contrast to non-specific degradation by lysosomes, proteasomes are highly selective and destroy only the proteins that are covalently labelled with small proteins, called ubiquitins. Importantly, many diseases, including neurodegenerative diseases and cancers, are intimately connected to the activity of proteasomes making them an important pharmacological target. Currently, the vast majority of inhibitors are aimed at blunting the proteolytic activities of proteasomes. However, recent achievements in solving structures of proteasomes at very high resolution provided opportunities to design new classes of small molecules that target other physiologically-important enzymatic activities of proteasomes, including the de-ubiquitinating one. This review attempts to catalog the information available to date about novel classes of proteasome inhibitors that may have important pharmacological ramifications.