Design, synthesis, and evaluation of BCL-2 targeting PROTACs.

BCL-2, a member of the BCL-2 protein family, is an antiapoptotic factor that regulates the intrinsic pathway of apoptosis. Due to its aberrant activity, it is frequently implicated in haematopoietic cancers and represents an attractive target for the development of therapeutics that antagonize its activity. A selective BCL-2 inhibitor, venetoclax, was approved for treating chronic lymphocytic leukaemia, acute myeloid leukemia, and other hematologic malignancies, validating BCL-2 as an anticancer target. Since then, alternative therapeutic approaches to modulate the activity of BCL-2 have been explored, such as antibody-drug conjugates and proteolysis-targeting chimeras. Despite numerous research groups focusing on developing degraders of BCL-2 family member proteins, selective BCL-2 PROTACs remain elusive, as disclosed compounds only show dual BCL-xL/BCL-2 degradation. Herein, we report our efforts to develop BCL-2 degraders by incorporating two BCL-2 binding moieties into chimeric compounds that aim to hijack one of three E3 ligases: CRBN, VHL, and IAPs. Even though our project did not result in obtaining a potent and selective BCL-2 PROTAC, our research will aid in understanding the narrow chemical space of BCL-2 degraders.

Optimisation of pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as novel Hsp90 C-terminal domain inhibitors against Ewing sarcoma.

Ewing sarcoma is the second most prevalent paediatric malignant bone tumour. In most cases, it is driven by the fusion oncoprotein EWS::FLI1, which acts as an aberrant transcription factor and dysregulates gene expression. EWS::FLI1 and a large number of downstream dysregulated proteins are Hsp90 client proteins, making Hsp90 an attractive target for the treatment of Ewing sarcoma. In this article, we report a new structural class of allosteric Hsp90 C-terminal domain (CTD) inhibitors based on the virtual screening hit TVS24, which showed antiproliferative activity in the SK-N-MC Ewing sarcoma cell line with an IC50 value of 15.9 ± 0.7 µM. The optimised compounds showed enhanced anticancer activity in the SK-N-MC cell line. Exposure of Ewing sarcoma cells to the most potent analogue 11c resulted in depletion of critical Hsp90 client proteins involved in cancer pathways such as EWS::FLI1, CDK4, RAF-1 and IGF1R, without inducing a heat shock response. The results of this study highlight Hsp90 CTD inhibitors as promising new agents for the treatment of Ewing sarcoma.

Discovery of a New Drug-like Series of OGT Inhibitors by Virtual Screening.

O-GlcNAcylation is an essential post-translational modification installed by the enzyme O-ß-N-acetyl-d-glucosaminyl transferase (OGT). Modulating this enzyme would be extremely valuable to better understand its role in the development of serious human pathologies, such as diabetes and cancer. However, the limited availability of potent and selective inhibitors hinders the validation of this potential therapeutic target. To explore new chemotypes that target the active site of OGT, we performed virtual screening of a large library of commercially available compounds with drug-like properties. We purchased samples of the most promising virtual hits and used enzyme assays to identify authentic leads. Structure-activity relationships of the best identified OGT inhibitor were explored by generating a small library of derivatives. Our best hit displays a novel uridine mimetic scaffold and inhibited the recombinant enzyme with an IC50 value of 7 µM. The current hit represents an excellent starting point for designing and developing a new set of OGT inhibitors that may prove useful for exploring the biology of OGT.

Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments.

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.

Pyridylethanol(phenylethyl)amines are non-azole, highly selective Candida albicans sterol 14α-demethylase inhibitors.

Sterol 14α-demethylase (CYP51) is the main drug target for the treatment of fungal infections. The worldwide increase in the incidence of opportunistic fungal infections and the emerging resistance to available azole-based antifungal drugs, raise the need to develop structurally distinct and selective fungal CYP51 inhibitors. In this work we have, for the first time, investigated the binding of pyridylethanol(phenylethyl)amines to any fungal CYP51. The comparison of the binding to Candida albicans and human CYP51 studied by spectroscopic and modeling methods revealed moieties decisive for selectivity and potency and resulted in the development of highly selective derivatives with significantly increased inhibitory potency. The structure-based insight into the selectivity requirements of this new chemical class of fungal CYP51 inhibitors, their unique binding properties and the low molecular weight of lead derivatives offer novel directions for the targeted development of antifungal clinical candidates.

Another structural correction for 1-oxo-1H-phenalene-2,3-dicarbonitriles: Synthesis of a potent BCL-2 inhibiting 7-phenoxy derivative.

Aromatic scaffolds are an important part of biologically active compounds and molecular probes used to study biochemical pathways and the involved targeted proteins of interest. 1-Oxo-1H-phenalene-2,3-dicarbonitrile-based compounds have been described as inhibitors of the BCL-2 family of proteins, and this core structure represents numerous possibilities for modifications that could lead to improved inhibitory potencies. Many studies demonstrated intriguing characteristics of these compounds in terms of reactivity and, interestingly, some contradictory literature reports appeared about reaction outcomes to synthesize them. Here, we initially provide a condensed overview of transformations performed on the phenalene scaffold, followed by the resynthesis of a 6-phenoxy-substituted derivative. We show that the initial determination of this particular structure was wrong and provide two-dimensional nuclear magnetic resonance (NMR) evidence to assign the structure properly. When preparing new derivatives using the same synthetic route, we observed 6- and 7-substituted regioisomers. After confirming their structures by NMR experiments, the ability of these compounds to inhibit BCL-2 was evaluated. The most potent 1-oxo-1H-phenalene-2,3-dicarbonitrile derivatives inhibited BCL-2 in the nanomolar range and showed double-digit micromolar cytotoxicity against four different cancer cell lines.

Synthesis and Biochemical Evaluation of Warhead-Decorated Psoralens as (Immuno)Proteasome Inhibitors.

The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. Selective inhibition of its catalytic activities is therefore a viable approach for the treatment of these diseases. However, the development of immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task. We previously reported 7H-furo[3,2-g]chromen-7-one (psoralen)-based compounds with an oxathiazolone warhead as selective inhibitors of the chymotrypsin-like (ß5i) subunit of immunoproteasome. Here, we describe the influence of the electrophilic warhead variations at position 3 of the psoralen core on the inhibitory potencies. Despite mapping the chemical space with different warheads, all compounds showed decreased inhibition of the ß5i subunit of immunoproteasome in comparison to the parent oxathiazolone-based compound. Although suboptimal, these results provide crucial information about structure-activity relationships that will serve as guidance for the further design of (immuno)proteasome inhibitors.

Intracellular Hydrolysis of Small-Molecule O-Linked N-Acetylglucosamine Transferase Inhibitors Differs among Cells and Is Not Required for Its Inhibition.

O-GlcNAcylation is an essential post-translational modification that occurs on nuclear and cytoplasmic proteins, regulating their function in response to cellular stress and altered nutrient availability. O-GlcNAc transferase (OGT) is the enzyme that catalyzes this reaction and represents a potential therapeutic target, whose biological role is still not fully understood. To support this research field, a series of cell-permeable, low-nanomolar OGT inhibitors were recently reported. In this study, we resynthesized the most potent OGT inhibitor of the library, OSMI-4, and we used it to investigate OGT inhibition in different human cell lines. The compound features an ethyl ester moiety that is supposed to be cleaved by carboxylesterases to generate its active metabolite. Our LC-HRMS analysis of the cell lysates shows that this is not always the case and that, even in the cell lines where hydrolysis does not occur, OGT activity is inhibited.

Glycosaminoglycans as Tools to Decipher the Platelet Tumor Cell Interaction: A Focus on P-Selectin.

Tumor cell-platelet interactions are regarded as an initial crucial step in hematogenous metastasis. Platelets protect tumor cells from immune surveillance in the blood, mediate vascular arrest, facilitate tumor extravasation, growth, and finally angiogenesis in the metastatic foci. Tumor cells aggregate platelets in the bloodstream by activation of the plasmatic coagulation cascade and by direct contact formation. Antimetastatic activities of unfractionated or low molecular weight heparin (UFH/LMWH) can undoubtedly be related to attenuated platelet activation, but molecular mechanisms and contribution of contact formation vs. coagulation remain to be elucidated. Using a set of non-anticoagulant heparin derivatives varying in size or degree of sulfation as compared with UFH, we provide insight into the relevance of contact formation for platelet activation. Light transmission aggregometry and ATP release assays confirmed that only those heparin derivatives with P-selectin blocking capacities were able to attenuate breast cancer cell-induced platelet activation, while pentasaccharide fondaparinux was without effects. Furthermore, a role of P-selectin in platelet activation and signaling could be confirmed by proteome profiler arrays detecting platelet kinases. In this study, we demonstrate that heparin blocks tumor cell-induced coagulation. Moreover, we identify platelet P-selectin, which obviously acts as molecular switch and controls aggregation and secretion of procoagulant platelets.

Discovery of Immunoproteasome Inhibitors Using Large-Scale Covalent Virtual Screening.

Large-scale virtual screening of boronic acid derivatives was performed to identify nonpeptidic covalent inhibitors of the ß5i subunit of the immunoproteasome. A hierarchical virtual screening cascade including noncovalent and covalent docking steps was applied to a virtual library of over 104,000 compounds. Then, 32 virtual hits were selected, out of which five were experimentally confirmed. Biophysical and biochemical tests showed micromolar binding affinity and time-dependent inhibitory potency for two compounds. These results validate the computational protocol that allows the screening of large compound collections. One of the lead-like boronic acid derivatives identified as a covalent immunoproteasome inhibitor is a suitable starting point for chemical optimization.

Identification of indole scaffold-based dual inhibitors of NOD1 and NOD2.

NOD1 and NOD2 are important members of the pattern recognition receptor family and play a crucial role within the context of innate immunity. However, overactivation of NODs, especially of NOD1, has also been implicated in a number of diseases. Surprisingly, NOD1 remains a virtually unexploited target in this respect. To gain additional insight into the structure-activity relationships of NOD1 inhibitors, a series of novel analogs has been designed and synthesized and then screened for their NOD1-inhibitory activity. Selected compounds were also investigated for their NOD2-inhibitory activity. Two compounds 4 and 15, were identified as potent mixed inhibitors of NOD1 and NOD2, displaying a balanced inhibitory activity on both targets in the low micromolar range. The results obtained have enabled a deeper understanding of the structural requirements for NOD1 and NOD2 inhibition.

Nonpeptidic Selective Inhibitors of the Chymotrypsin-Like (ß5 i) Subunit of the Immunoproteasome.

Elevated expression of the immunoproteasome has been associated with autoimmune diseases, inflammatory diseases, and various types of cancer. Selective inhibitors of the immunoproteasome are not only scarce, but also almost entirely restricted to peptide-based compounds. Herein, we describe nonpeptidic reversible inhibitors that selectively block the chymotrypsin-like (ß5i) subunit of the human immunoproteasome in the low micromolar range. The most potent of the reversibly acting compounds were then converted into covalent, irreversible, nonpeptidic inhibitors that retained selectivity for the ß5i subunit. In addition, these inhibitors discriminate between the immunoproteasome and the constitutive proteasome in cell-based assays. Along with their lack of cytotoxicity, these data point to these nonpeptidic compounds being suitable for further investigation as ß5i-selective probes for possible application in noncancer diseases related to the immunoproteasome.

Novel N-amidinopiperidine-based proteasome inhibitor preserves dendritic cell functionality and rescues their Th1-polarizing capacity in Ramos-conditioned tumor environment.

The tumor microenvironment represents a burden that hampers the proper activation of immune cells, including the dendritic cells (DCs). It is, therefore, desired that the important characteristics of a given anticancer drug candidate be seen as consisting not solely of its antitumor properties, but that it also lacks potential side effects that could additionally constrain the development and function of immune cells associated with tumor immunity. We have previously identified compounds with a N-amidinopiperidine scaffold that selectively induce apoptosis in Burkitt's lymphoma cells through proteasome inhibition. Here, we demonstrate that SPI-15 affected neither the viability of DCs nor their differentiation. In addition, the compound had no significant effect on their cytokine secretion or allostimulatory capacity. Moreover, DC functionality in the context of tumor microenvironment was also unaffected, as demonstrated by experiments performed on DCs differentiated in Ramos-conditioned media in the presence or absence of SPI-15. The cytokine profile and functional assays revealed that SPI-15 rescues DC differentiation from the immunosuppressive environment produced by Ramos cells; this was seen by their reacquired ability to induce IFN-γ-secretion from naïve CD4(+)CD45RA(+) T cells and the consequently induced Th1-effector differentiation. Herein, we present novel characteristics of an N-amidinopiperidine-based protease inhibitor whose anticancer properties are not associated with the immunosuppression of DCs. We propose future studies toward the design of structurally similar compounds with the aim of developing potent anticancer drugs with minimal negative effects on crucial factors involved in tumor immunity.

Discovery of novel small-molecule compounds with selective cytotoxicity for Burkitt's lymphoma cells using 3D ligand-based virtual screening.

We describe a ligand-based approach towards compounds with more specific targeting for Burkitt's lymphoma. Using three-dimensional ligand-based similarity searches and a previously described hit compound, we have identified six compounds that are chemically different but with similar spatial conformations. Biological evaluation revealed that one compound has better growth inhibition and improved selectivity towards Burkitt's lymphoma cells than the query compound. However, initial mechanism-of-action studies show a different target profile in comparison with the previous hit compound, which does not involve the inhibition of the proteasome or the NFκB pathway. The data from this study provide a solid basis for further efforts in the search for selective agents against Burkitt's lymphoma.

Halogenation of common phenolic household and personal care product ingredients enhances their AhR-modulating capacity.

The Aryl Hydrocarbon Receptor (AhR), a ligand-activated transcription factor, orchestrates responses to numerous structurally diverse endogenous and exogenous ligands. In addition to binding various xenobiotics, AhR also recognizes endocrine disruptors, particularly those featuring chlorinated or brominated aromatic structures. There is limited data available on the impact of common household and personal care product ingredients let alone their halogenated transformation products. Herein we bridge this knowledge gap by preparing a library of chlorinated and brominated parabens, bisphenols, UV filters, and nonylphenols. An evaluation of total of 125 compounds for agonistic and antagonistic activity on AhR unveiled a low micromolar agonist, Cl2BPAF with an EC50 of 13 µM. Moreover, our study identified several AhR antagonists, with BrBzP emerging as the most potent with an IC50 of 8.9 µM. To further investigate the functional implications of these compounds, we subjected the most potent agonist and antagonist to a functional assay involving cytokine secretion from peripheral blood mononuclear cells and compared their activity with the commercially available AhR agonist and antagonist. Cl2BPAF exhibited an overall immunosuppressive effect by reducing the secretion of proinflammatory cytokines, including IL-6, IFN-γ, and TNF-α, while BrBzP displayed opposite effects, leading to an increase of those cytokines. Notably, the immunomodulatory effects of Cl2BPAF surpassed those of ITE, a bona fide AhR agonist, while the impact of BrBzP exceeded that of CH223191, a bona fide AhR antagonist. In summary, our study underscores the potential influence of halogenated transformation products on the AhR pathway and, consequently, their role in shaping the immune responses.

Discovery of 2,3-Diaminoindole Derivatives as a Novel Class of NOD Antagonists.

NOD1 and NOD2 are members of the pattern recognition receptors involved in the innate immune response. Overactivation of NOD1 is implicated in inflammatory disorders, multiple sclerosis, and cancer cell metastases. NOD1 antagonists would represent valuable pharmacological tools to gain further insight into protein roles, potentially leading to new therapeutic strategies. We herein report the expansion of the chemical space of NOD1 antagonists via a multicomponent synthetic approach affording a novel chemotype, namely, 2,3-diaminoindoles. These efforts resulted in compound 37, endowed with low micromolar affinity toward NOD1. Importantly, a proof-of-evidence of direct binding to NOD1 of Noditinib-1 and derivative 37 is provided here for the first time. Additionally, the combination of computational studies and NMR-based displacement assays enabled the characterization of the binding modality of 37 to NOD1, thus providing key unprecedented knowledge for the design of potent and selective NOD1 antagonists.

Antimicrobial activity and cytotoxicity of some 2-amino-5-alkylidene-thiazol-4-ones.

We report a small, focused library of 30 diverse 2-amino-5-alkylidene-thiazol-4-ones that was assayed in a whole-cell antibacterial screen against a panel of several bacterial strains and a yeast. Most of the compounds exhibited modest to significant antibacterial activity against Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus, and no activity against Salmonella typhimurium and Escherichia coli. The antibacterial activity depends markedly upon substituents on the thiazol-4-one core, and the most potent compound assayed ([Formula: see text]-4-((2-(4-methylpiperidin-1-yl)-4-oxothiazol-5(4H)-ylidene)methyl)benzonitrile) reached a minimal inhibitory concentration (MIC) value of [Formula: see text] on P. aeruginosa strain. An important feature of the tested compounds is their low influence on cell viability, as determined in a HEK-293 metabolic activity assay. In light of the encouraging in vitro antimicrobial assay results against several bacterial strains, we have generated a pharmacophore model using the Discovery studio 3.0 package (Accelrys Inc., San Diego, USA), which exposed the spatial arrangement of key molecular properties responsible for our observed MIC results. Considering the absence of a defined target and the limitation of the described approach to pool different scaffolds, the calculated pharmacophore model could be used for library enrichment to identify compounds with a thiazolidinone scaffold with improved antimicrobial potency and physico-chemical properties.

In vitro investigation of immunomodulatory activities of selected UV-filters.

Organic UV filters are ubiquitous as they are used in numerous personal care products. Consequently, people constantly come into direct or indirect contact with these chemicals. Albeit studies of the effects of UV filters on human health have been undertaken, their toxicological profiles are not complete. In this work, we investigated the immunomodulatory properties of eight UV filters representing different chemotypes, including benzophenone-1, benzophenone-3, ethylhexyl methoxycinnamate, octyldimethyl-para-aminobenzoic acid, octyl salate, butylmethoxydibenzoylmethane, 3-benzylidenecamphor, and 2,4-di-tert-butyl-6-(5-chlorobenzotriazol-2-yl)phenol. We demonstrated that none of these UV filters were cytotoxic to THP-1 cells at concentrations up to 50 µM. Importantly, our study highlighted the capacity of nontoxic concentrations of avobenzone and 3-benzylidene camphor to increase the secretion of interleukin 8 (IL-8) from both THP-1 cells and THP-1 derived macrophages. Further, they also exhibited a pronounced decrease of IL-6 and IL-10 release from lipopolysaccharide-stimulated peripheral blood mononuclear cells. The observed immune cell alterations suggest that exposure to 3-BC and BMDM could be involved in immune deregulation. Our research thus provided additional insight into UV filter safety profile.

Design and synthesis of amino-substituted N-arylpiperidinyl-based inhibitors of the (immuno)proteasome.

The constitutive proteasome and the immunoproteasome represent validated targets for pharmacological intervention in the context of various diseases, such as cancer, inflammation, and autoimmune diseases. The development of novel chemical scaffolds of non-peptidic nature, capable of inhibiting different catalytically active subunits of both isoforms, is a viable approach against these diseases. Such compounds are also useful as leads for the development of biochemical probes that enable the studies of the roles of both isoforms in various biological contexts. Here, we present a ligand-based computational design of (immuno)proteasome inhibitors, which resulted in the amino-substituted N-arylpiperidine-based compounds that can inhibit different subunits of the (immuno)proteasome in the low micromolar range. The compounds represent a useful starting point for further structure-activity relationship studies that will, hopefully, lead to non-peptidic compounds that could be used in pharmacological and biochemical studies of both proteasomes.

Nanofibers with genotyped Bacillus strains exhibiting antibacterial and immunomodulatory activity.

Biofilm-associated diseases such as periodontitis are widespread and challenging to treat which calls for new strategies for their effective management. Probiotics represent a promising approach for targeted treatment of dysbiosis in biofilm and modulation of host immune response. In this interdisciplinary study, nanofibers with two autochthonous Bacillus strains 27.3.Z and 25.2.M were developed. The strains were isolated from the oral microbiota of healthy individuals, and their genomes were sequenced and screened for genes associated with antimicrobial and immunomodulatory activities, virulence factors, and transferability of resistance to antibiotics. Spores of two Bacillus strains were incorporated individually or in combination into hydrophilic poly(ethylene oxide) (PEO) and composite PEO/alginate nanofibers. The nanofiber mats were characterised by a high loading of viable spores (> 7 log CFU/mg) and they maintained viability during electrospinning and 6 months of storage at room temperature. Spores were rapidly released from PEO nanofibers, while presence of alginate in the nanofibers prolonged their release. All formulations exhibited swelling, followed by transformation of the nanofiber mat into a hydrogel and polymer erosion mediating spore release kinetics. The investigated Bacillus strains released metabolites, which were not cytotoxic to peripheral blood mononuclear cells (PBMCs) in vitro. Moreover, their metabolites exhibited antibacterial activity against two periodontopathogens, an antiproliferative effect on PBMCs, and inhibition of PBMC expression of proinflammatory cytokines. In summary, the developed nanofiber-based delivery system represents a promising therapeutic approach to combat biofilm-associated disease on two fronts, namely via modulation of the local microbiota with probiotic bacteria and host immune response with their metabolites.