Biophysical Assays for Investigating Modulators of Macromolecular Complexes: An Overview.

Drug discovery is a lengthy and intricate process, and in its early stage, crucial steps are the selection of the therapeutic target and the identification of novel ligands. Most targets are dysregulated in pathogenic cells; typically, their activation or deactivation leads to the desired effect, while in other cases, interfering with the target-natural binder complex achieves the therapeutic results. Biophysical assays are a suitable strategy for finding new ligands or interferent agents, being able to evaluate ligand-protein interactions and assessing the effect of small molecules (SMols) on macromolecular complexes. This mini-review provides a detailed analysis of widely used biophysical methods, including fluorescence-based approaches, circular dichroism, isothermal titration calorimetry, microscale thermophoresis, and NMR spectroscopy. After a brief description of the methodologies, examples of interaction and competition experiments are described, together with an analysis of the advantages and disadvantages of each technique. This mini-review provides an overview of the most relevant biophysical technologies that can help in identifying SMols able not only to bind proteins but also to interfere with macromolecular complexes.

Sigma receptor and aquaporin modulators: chiral resolution, configurational assignment, and preliminary biological profile of RC752 enantiomers.

The key role of chiral small molecules in drug discovery programs has been deeply investigated throughout last decades. In this context, our previous studies highlighted the influence of the absolute configuration of different stereocenters on the pharmacokinetic, pharmacodynamic and functional properties of promising Sigma receptor (SR) modulators. Thus, starting from the racemic SR ligand RC752, we report herein the isolation of the enantiomers via enantioselective separation with both HPLC and SFC. After optimization of the eco-sustainable chiral SFC method, both enantiomers were obtained in sufficient amount (tens of mg) and purity (ee up to 95%) to allow their characterization and initial biological investigation. Both enantiomers a) displayed a high affinity for the S1R subtype (Ki = 15.0 ± 1.7 and 6.0 ± 1.2 nM for the (S)- and (R)-enantiomer, respectively), but only negligible affinity toward the S2R (> 350 nM), and b) were rapidly metabolized when incubated with mouse and human hepatic microsomes. Furthermore, the activity on AQP-mediated water permeability indicated a different functional profile for the enantiomers in terms of modulatory effect on the peroxiporins gating.

Identification of HuR-RNA Interfering Compounds by Dynamic Combinatorial Chemistry and Fluorescence Polarization.

The RNA binding protein HuR regulates the post-transcriptional process of different oncogenes and tumor suppressor genes, and its dysregulation is linked with cancer. Thus, modulating the complex HuR-RNA represents a promising anticancer strategy. To search for novel HuR ligands able to interfere with the HuR-RNA complex, the protein-templated dynamic combinatorial chemistry (pt-DCC) method was utilized. The recombinant RRM1+2 protein construct, which contains essential domains for ligand-HuR binding and exhibits enhanced solubility and stability compared to the native protein, was used for pt-DCC. Seven acylhydrazones with over 80% amplification were identified. The binding of the fragments to HuR extracted from DCC was validated using STD-NMR, and molecular modeling studies revealed the ability of the compounds to bind HuR at the mRNA binding pocket. Notably, three compounds effectively interfered with HuR-RNA binding in fluorescence polarization studies, suggesting their potential as foundational compounds for developing anticancer HuR-RNA interfering agents.

Discovery of RC-752, a Novel Sigma-1 Receptor Antagonist with Antinociceptive Activity: A Promising Tool for Fighting Neuropathic Pain.

Neuropathic pain (NP) is a chronic condition resulting from damaged pain-signaling pathways. It is a debilitating disorder that affects up to 10% of the world's population. Although opioid analgesics are effective in reducing pain, they present severe risks; so, there is a pressing need for non-opioid pain-relieving drugs. One potential alternative is represented by sigma-1 receptor (S1R) antagonists due to their promising analgesic effects. Here, we report the synthesis and biological evaluation of a series of S1R antagonists based on a 2-aryl-4-aminobutanol scaffold. After assessing affinity toward the S1R and selectivity over the sigma-2 receptor (S2R), we evaluated the agonist/antagonist profile of the compounds by investigating their effects on nerve growth factor-induced neurite outgrowth and aquaporin-mediated water permeability in the presence and absence of oxidative stress. (R/S)-RC-752 emerged as the most interesting compound for S1R affinity (Ki S1R = 6.2 ± 0.9) and functional antagonist activity. Furthermore, it showed no cytotoxic effect in two normal human cell lines or in an in vivo zebrafish model and was stable after incubation in mouse plasma. (R/S)-RC-752 was then evaluated in two animal models of NP: the formalin test and the spinal nerve ligation model. The results clearly demonstrated that compound (R/S)-RC-752 effectively alleviated pain in both animal models, thus providing the proof of concept of its efficacy as an antinociceptive agent.

Identification of a novel extracellular inhibitor of FGF2/FGFR signaling axis by combined virtual screening and NMR spectroscopy approach.

The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signalling pathway drives severe pathologies, including cancer development and angiogenesis-driven pathologies. The perturbation of the FGF2/FGFR axis via extracellular allosteric small inhibitors is a promising strategy for developing FGFR inhibitors with improved safety and efficacy for cancer treatment. We have previously investigated the role of new extracellular inhibitors, such as rosmarinic acid (RA), which bind the FGFR-D2 domain and directly compete with FGF2 for the same binding site, enabling the disruption of the functional FGF2/FGFR interaction. To select ligands for the previously identified FGF2/FGFR RA binding site, NMR data-driven virtual screening has been performed on an in-house library of non-commercial small molecules and metabolites. A novel drug-like compound, a resorcinol derivative named RBA4 has been identified. NMR interaction studies demonstrate that RBA4 binds the FGF2/FGFR complex, in agreement with docking prediction. Residue-level NMR perturbations analysis highlights that the mode of action of RBA4 is similar to RA in terms of its ability to target the FGF2/FGFR-D2 complex, inducing perturbations on both proteins and triggering complex dissociation. Biological assays proved that RBA4 inhibited FGF2 proliferative activity at a level comparable to the previously reported natural product, RA. Identification of RBA4 chemical groups involved in direct interactions represents a starting point for further optimization of drug-like extracellular inhibitors with improved activity.

Structural Insights into the Ligand-LsrK Kinase Binding Mode: A Step Forward in the Discovery of Novel Antimicrobial Agents.

LsrK is a bacterial kinase that triggers the quorum sensing, and it represents a druggable target for the identification of new agents for fighting antimicrobial resistance. Herein, we exploited tryptophan fluorescence spectroscopy (TFS) as a suitable technique for the identification of potential LsrK ligands from an in-house library of chemicals comprising synthetic compounds as well as secondary metabolites. Three secondary metabolites (Hib-ester, Hib-carbaldehyde and (R)-ASME) showed effective binding to LsrK, with KD values in the sub-micromolar range. The conformational changes were confirmed via circular dichroism and molecular docking results further validated the findings and displayed the specific mode of interaction. The activity of the identified compounds on the biofilm formation by some Staphylococcus spp. was investigated. Hib-carbaldehyde and (R)-ASME were able to reduce the production of biofilm, with (R)-ASME resulting in the most effective compound with an EC50 of 14 mg/well. The successful application of TFS highlights its usefulness in searching for promising LsrK inhibitor candidates with inhibitor efficacy against biofilm formation.

From Nature to Synthetic Compounds: Novel 1(N),2,3 Trisubstituted-5-oxopyrrolidines Targeting Multiple Myeloma Cells.

The insurgence of drug resistance in treating Multiple Myeloma (MM) still represents a major hamper in finding effective treatments, although over the past decades new classes of drugs, such as proteasome inhibitors and immunomodulatory drugs, have been discovered. Recently, our research team, within a Nature-Aided Drug Discovery project, isolated from Hibiscus Sabdariffa L. calyces the secondary metabolite called Hib-ester which possesses antiproliferative properties against human multiple myeloma RPMI 8226 cells, reduces migration and cell invasion and inhibits proteasome without neurotoxic effects. In the present study, we explored the chemical spaces of the hit compound Hib-ester. We explored the structure-activity relationships (SAR), and we optimized the scaffold through sequentially modifying Hib-ester subunits. Compound screening was performed based on cytotoxicity against the RPMI 8226 cells to assess the potential efficacy toward human MM. The ability of the most effective molecules to inhibit the proteasome was evaluated and the binding mode of the most promising compounds in the proteasome chymotrypsin binding pocket was deciphered through molecular modeling simulations. Compounds 13 and 14 are more potent than Hib-ester, demonstrating that our strategy was suitable for the identification of a novel chemotype for developing possible drug candidates and hopefully widening the drug armamentarium against MM.

Urea-based anticancer agents. Exploring 100-years of research with an eye to the future.

Suramin was the first urea-based drug to be approved in clinic, and in the following century a number of milestone drugs based on this scaffold were developed. Indeed, urea soon became a privileged scaffold in medicinal chemistry for its capability to establish a peculiar network of drug-target interactions, for its physicochemical properties that are useful for tuning the druggability of the new chemical entities, and for its structural and synthetic versatility that opened the door to numerous drug design possibilities. In this review, we highlight the relevance of the urea moiety in the medicinal chemistry scenario of anticancer drugs with a special focus on the kinase inhibitors for which this scaffold represented and still represents a pivotal pharmacophoric feature. A general outlook on the approved drugs, recent patents, and current research in this field is herein provided, and the role of the urea moiety in the drug discovery process is discussed form a medicinal chemistry standpoint. We believe that the present review can benefit both academia and pharmaceutical companies' medicinal chemists to prompt research towards new urea derivatives as anticancer agents.

Repurposing the Antiplatelet Agent Ticlopidine to Counteract the Acute Phase of ER Stress Condition: An Opportunity for Fighting Coronavirus Infections and Cancer.

Different pathological conditions, including viral infections and cancer, can have a massive impact on the endoplasmic reticulum (ER), causing severe damage to the cell and exacerbating the disease. In particular, coronavirus infections, including SARS coronavirus-2 (SARS-CoV-2), responsible for COVID-19, cause ER stress as a consequence of the enormous amounts of viral glycoproteins synthesized, the perturbation of ER homeostasis and the modification of ER membranes. Therefore, ER has a central role in the viral life cycle, thus representing one of the Achilles' heels on which to focus therapeutic intervention. On the other hand, prolonged ER stress has been demonstrated to promote many pro-tumoral attributes in cancer cells, having a key role in tumor growth, metastasis and response to therapies. In this report, adopting a repurposing approach of approved drugs, we identified the antiplatelet agent ticlopidine as an interferent of the unfolded protein response (UPR) via sigma receptors (SRs) modulation. The promising results obtained suggest the potential use of ticlopidine to counteract ER stress induced by viral infections, such as COVID-19, and cancer.

Chiral 2-phenyl-3-hydroxypropyl esters as PKC-alpha modulators: HPLC enantioseparation, NMR absolute configuration assignment, and molecular docking studies.

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. In our previous work, we identified in racemate 1-2, based on the 2-benzyl-3-hydroxypropyl ester scaffold, two new potent and promising PKCα and PKCδ ligands, targeting the C1 domain of these two kinases. Herein, we report the resolution of the racemates by enantioselective semi-preparative HPLC. The attribution of the absolute configuration (AC) of homochirals 1 was performed by NMR, via methoxy-α-trifluoromethyl-α-phenylacetic acid derivatization (MTPA or Mosher's acid). Moreover, the match between the experimental and predicted electronic circular dichroism (ECD) spectra confirmed the assigned AC. These results proved that Mosher's esters can be properly exploited for the determination of the AC also for chiral primary alcohols. Lastly, homochiral 1 and 2 were assessed for binding affinity and functional activity against PKCα. No significative differences in the Ki of the enantiopure compounds was observed, thus suggesting that chirality does not seem to play a significant role in targeting PKC C1 domain. These results are in accordance with the molecular docking studies performed using a new homology model for the human PKCαC1B domain.

A Composite Nanosystem as a Potential Tool for the Local Treatment of Glioblastoma: Chitosan-Coated Solid Lipid Nanoparticles Embedded in Electrospun Nanofibers.

Glioblastoma multiforme (GBM) is one of the most prevalent and aggressive brain tumors for which there is currently no cure. A novel composite nanosystem (CN), consisting of chitosan-coated Solid Lipid Nanoparticles (c-SLN) embedded in O-carboxymethyl chitosan (O-CMCS)-containing nanofibers (NFs), was proposed as a potential tool for the local delivery of lipophilic anti-proliferative drugs. Coacervation was selected as a solvent-free method for the preparation of stearic acid (SA) and behenic acid (BA)-based SLN (SA-SLN and BA-SLN respectively). BA-SLN, containing 0.75% w/w BA sodium salt and 3% w/w poly(vinyl alcohol) (PVA), were selected for the prosecution of the work since they are characterized by the lowest size functional to their subsequent coating and incorporation in nanofibers. BA-SLN were coated with chitosan (CS) by means of a two-step coating method based on the physical absorption of positively charged CS chains on the SLN negative surface. Nile Red (NR), chosen as the hydrophobic model dye, was dissolved in a micellar solution of BA sodium salt and then added with a coacervating solution until pH ≅ 2.5 was reached. Immunocytochemistry analyses highlighted that CS-coated BA-SLN (c-BA-SLN) exhibited a higher accumulation in human glioblastoma cells (U-373) after 6 h than CS-free BA-SLN. Finally, the c-BA-SLN dispersion was blended with a solution consisting of freely soluble polymers (O-CMCS, poly(ethylene oxide) and poloxamer) and then electrospun to obtain NFs with a mean diameter equal to 850 nm. After the NFs dissolution in an aqueous media, c-BA-SLN maintained their physicochemical properties and zeta potential.

Enantiomeric Resolution and Absolute Configuration of a Chiral δ-Lactam, Useful Intermediate for the Synthesis of Bioactive Compounds.

During the past several years, the frequency of discovery of new molecular entities based on γ- or δ-lactam scaffolds has increased continuously. Most of them are characterized by the presence of at least one chiral center. Herein, we present the preparation, isolation and the absolute configuration assignment of enantiomeric 2-(4-bromophenyl)-1-isobutyl-6-oxopiperidin-3-carboxylic acid (trans-1). For the preparation of racemic trans-1, the Castagnoli-Cushman reaction was employed. (Semi)-preparative enantioselective HPLC allowed to obtain enantiomerically pure trans-1 whose absolute configuration was assigned by X-ray diffractometry. Compound (+)-(2R,3R)-1 represents a reference compound for the configurational study of structurally related lactams.

Tackling Antimicrobial Resistance with Small Molecules Targeting LsrK: Challenges and Opportunities.

Antimicrobial resistance (AMR) is a growing threat with severe health and economic consequences. The available antibiotics are losing efficacy, and the hunt for alternative strategies is a priority. Quorum sensing (QS) controls biofilm and virulence factors production. Thus, the quenching of QS to prevent pathogenicity and to increase bacterial susceptibility to antibiotics is an appealing therapeutic strategy. The phosphorylation of autoinducer-2 (a mediator in QS) by LsrK is a crucial step in triggering the QS cascade. Thus, LsrK represents a valuable target in fighting AMR. Few LsrK inhibitors have been reported so far, allowing ample room for further exploration. This perspective aims to provide a comprehensive analysis of the current knowledge about the structural and biological properties of LsrK and the state-of-the-art technology for LsrK inhibitor design. We elaborate on the challenges in developing novel LsrK inhibitors and point out promising avenues for further research.

Exploring the RC-106 Chemical Space: Design and Synthesis of Novel (E)-1-(3-Arylbut-2-en-1-yl)-4-(Substituted) Piperazine Derivatives as Potential Anticancer Agents.

Despite the fact that significant advances in treatment of common cancers have been achieved over the years, orphan tumors still represent an important unmet medical need. Due to their complex multifactorial origin and limited number of cases, such pathologies often have very limited treatment options and poor prognosis. In the search for new anticancer agents, our group recently identified RC-106, a Sigma receptor modulator endowed with proteasome inhibition activity. This compound showed antiproliferative activity toward different cancer cell lines, among them glioblastoma (GB) and multiple myeloma (MM), two currently unmet medical conditions. In this work, we directed our efforts toward the exploration of chemical space around RC-106 to identify new active compounds potentially useful in cancer treatment. Thanks to a combinatorial approach, we prepared 41 derivatives of the compound and evaluated their cytotoxic potential against MM and GB. Three novel potential anticancer agents have been identified.

Sigma-1 receptor antagonists: promising players in fighting neuropathic pain.

Sigma-1 receptors (S1Rs) are strongly correlated to neuropathic pain (NP), since their inactivation may decrease allodynia or dysesthesia, promoting analgesic effects. In the recent patent landscape, S1R antagonists endowed with nanomolar S1Rs affinity emerged as potent antinociceptive agents. So far, three patented compounds have been proposed for counteracting NP. Particularly PV-752 and AV1066, disclosed by the University of Pavia (Italy) and Anavex, respectively, showed good analgesic activity in preclinical studies. Moreover, E-52862 developed by Esteve (Spain) has been proved to be effective, both in preclinical and Phase II clinical trials, against several symptoms of NP. These patents ascertain S1R antagonists as potential drugs, alone or in combination with other analgesic drugs, for managing NP in humans.

BOPC1 Enantiomers Preparation and HuR Interaction Study. From Molecular Modeling to a Curious DEEP-STD NMR Application.

The Hu family of RNA-binding proteins plays a crucial role in post-transcriptional processes; indeed, Hu-RNA complexes are involved in various dysfunctions (i.e., inflammation, neurodegeneration, and cancer) and have been recently proposed as promising therapeutic targets. Intrigued by this concept, our research efforts aim at identifying small molecules able to modulate HuR-RNA interactions, with a focus on subtype HuR, upregulated and dysregulated in several cancers. By applying structure-based design, we had already identified racemic trans-BOPC1 as promising HuR binder. In this Letter, we accomplished the enantio-resolution, the assignment of the absolute configuration, and the recognition study with HuR of enantiomerically pure trans-BOPC1. For the first time, we apply DEEP (differential epitope mapping)-STD NMR to study the interaction of BOPC1 with HuR and compare its enantiomers, gaining information on ligand orientation and amino acids involved in the interaction, and thus increasing focus on the in silico binding site model.

Pyrrolomycins as antimicrobial agents. Microwave-assisted organic synthesis and insights into their antimicrobial mechanism of action.

New compounds able to counteract staphylococcal biofilm formation are needed. In this study we investigate the mechanism of action of pyrrolomycins, whose potential as antimicrobial agents has been demonstrated. We performed a new efficient and easy method to use microwave organic synthesis suitable for obtaining pyrrolomycins in good yields and in suitable amount for their in vitro in-depth investigation. We evaluate the inhibitory activity towards Sortase A (SrtA), a transpeptidase responsible for covalent anchoring in Gram-positive peptidoglycan of many surface proteins involved in adhesion and in biofilm formation. All compounds show a good inhibitory activity toward SrtA, having IC50 values ranging from 130 to 300 µM comparable to berberine hydrochloride. Of note compound 1d shows a good affinity in docking experiment to SrtA and exhibits the highest capability to interfere with biofilm formation of S. aureus showing an IC50 of 3.4 nM. This compound is also effective in altering S. aureus murein hydrolase activity that is known to be responsible for degradation, turnover, and maturation of bacterial peptidoglycan and involved in the initial stages of S. aureus biofilm formation.