A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer.

AIM: Chemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches. METHODS: Gene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipitation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses. RESULTS: We identified IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was significantly associated with resistance to selumetinib, gefitinib, and regorafenib in PDOs and to 5-fluorouracil and oxaliplatin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabilization of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confirmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells. CONCLUSIONS: IGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mitochondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance.

Investigating 3R In Vivo Approaches for Bio-Distribution and Efficacy Evaluation of Nucleic Acid Nanocarriers: Studies on Peptide-Mimicking Ionizable Lipid.

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.

Design, Synthesis and Biological Characterization of Histone Deacetylase 8 (HDAC8) Proteolysis Targeting Chimeras (PROTACs) with Anti-Neuroblastoma Activity.

In addition to involvement in epigenetic gene regulation, histone deacetylases (HDACs) regulate multiple cellular processes through mediating the activity of non-histone protein substrates. The knockdown of HDAC8 isozyme is associated with the inhibition of cell proliferation and apoptosis enhancement in several cancer cell lines. As shown in several studies, HDAC8 can be considered a potential target in the treatment of cancer forms such as childhood neuroblastoma. The present work describes the development of proteolysis targeting chimeras (PROTACs) of HDAC8 based on substituted benzhydroxamic acids previously reported as potent and selective HDAC8 inhibitors. Within this study, we investigated the HDAC8-degrading profiles of the synthesized PROTACs and their effect on the proliferation of neuroblastoma cells. The combination of in vitro screening and cellular testing demonstrated selective HDAC8 PROTACs that show anti-neuroblastoma activity in cells.

Synthesis, Molecular Docking and Biological Characterization of Pyrazine Linked 2-Aminobenzamides as New Class I Selective Histone Deacetylase (HDAC) Inhibitors with Anti-Leukemic Activity.

Class I histone deacetylases (HDACs) are key regulators of cell proliferation and they are frequently dysregulated in cancer cells. We report here the synthesis of a novel series of class-I selective HDAC inhibitors (HDACi) containing a 2-aminobenzamide moiety as a zinc-binding group connected with a central (piperazin-1-yl)pyrazine or (piperazin-1-yl)pyrimidine moiety. Some of the compounds were additionally substituted with an aromatic capping group. Compounds were tested in vitro against human HDAC1, 2, 3, and 8 enzymes and compared to reference class I HDACi (Entinostat (MS-275), Mocetinostat, CI994 and RGFP-966). The most promising compounds were found to be highly selective against HDAC1, 2 and 3 over the remaining HDAC subtypes from other classes. Molecular docking studies and MD simulations were performed to rationalize the in vitro data and to deduce a complete structure activity relationship (SAR) analysis of this novel series of class-I HDACi. The most potent compounds, including 19f, which blocks HDAC1, HDAC2, and HDAC3, as well as the selective HDAC1/HDAC2 inhibitors 21a and 29b, were selected for further cellular testing against human acute myeloid leukemia (AML) and erythroleukemic cancer (HEL) cells, taking into consideration their low toxicity against human embryonic HEK293 cells. We found that 19f is superior to the clinically tested class-I HDACi Entinostat (MS-275). Thus, 19f is a new and specific HDACi with the potential to eliminate blood cancer cells of various origins.

Studies on the Mechanisms of Anti-Inflammatory Activity of Heparin- and Hyaluronan-Containing Multilayer Coatings-Targeting NF-κB Signalling Pathway.

The use of implants can be hampered by chronic inflammatory reactions, which may result in failure of the implanted device. To prevent such an outcome, the present study examines the anti-inflammatory properties of surface coatings made of either hyaluronic acid (HA) or heparin (Hep) in combination with chitosan (Chi) prepared as multilayers through the layer-by-layer (LbL) technique. The properties of glycosaminoglycan (GAG)-modified surfaces were characterized in terms of surface topography, thickness and wettability. Results showed a higher thickness and hydrophilicity after multilayer formation compared to poly (ethylene imine) control samples. Moreover, multilayers containing either HA or Hep dampened the inflammatory response visible by reduced adhesion, formation of multinucleated giant cells (MNGCs) and IL-1ß release, which was studied using THP-1 derived macrophages. Furthermore, investigations regarding the mechanism of anti-inflammatory activity of GAG were focused on nuclear transcription factor-кB (NF-κB)-related signal transduction. Immunofluorescence staining of the p65 subunit of NF-κB and immunoblotting were performed that showed a significant decrease in NF-κB level in macrophages on GAG-based multilayers. Additionally, the association of FITC-labelled GAG was evaluated by confocal laser scanning microscopy and flow cytometry showing that macrophages were able to associate with and take up HA and Hep. Overall, the Hep-based multilayers demonstrated the most suppressive effect making this system most promising to control macrophage activation after implantation of medical devices. The results provide an insight on the anti-inflammatory effects of GAG not only based on their physicochemical properties, but also related to their mechanism of action toward NF-κB signal transduction.

Screening for Selective Protein Inhibitors by Using the IANUS Peptide Array.

Finding new road blacks: A peptidic inhibitor of calcineurin (CaN)-mediated nuclear factor of activated T cells (NFAT) dephosphorylation, which is developed through a template-assisted IANUS (Induced orgANisation of strUcture by matrix-assisted togethernesS) peptide array, is cell permeable and able to block the translocation of green fluorescent protein-NFAT fusion protein (GFP-NFAT) into the nucleus after stimulation.

Identification of PKMYT1 inhibitors by screening the GSK published protein kinase inhibitor set I and II.

As a member of the Wee-kinase family protein kinase PKMYT1 is involved in G2/M checkpoint regulation of the cell cycle. Recently, a peptide microarray approach led to the identification of a small peptide; EFS247-259 as substrate of PKMYT1, which allowed for subsequent development of an activity assay. The developed activity assay was used to characterize the PKMYT1 catalyzed phosphorylation of EFS247-259. For the first time kinetic parameters for PKMYT1, namely Km, Km, ATP and vmax were determined. The optimized assay was used to screen the published protein kinase inhibitor sets (PKIS I and II), two sets of small molecule ATP-competitive kinase inhibitors reported by GlaxoSmithKline. We identified ten inhibitors, providing different scaffolds. The inhibitors were further characterized by using binding assay, activity and functional assay. In addition, docking studies were carried out in order to rationalize the observed biological activities. The derived results provide the basis for further chemical optimization of PKMYT1 inhibitors and for further analysis of PKMYT1 as target for anti-cancer therapy.

Regulation of G2/M Transition by Inhibition of WEE1 and PKMYT1 Kinases.

In the cell cycle, there are two checkpoint arrests that allow cells to repair damaged DNA in order to maintain genomic integrity. Many cancer cells have defective G1 checkpoint mechanisms, thus depending on the G2 checkpoint far more than normal cells. G2 checkpoint abrogation is therefore a promising concept to preferably damage cancerous cells over normal cells. The main factor influencing the decision to enter mitosis is a complex composed of Cdk1 and cyclin B. Cdk1/CycB is regulated by various feedback mechanisms, in particular inhibitory phosphorylations at Thr14 and Tyr15 of Cdk1. In fact, Cdk1/CycB activity is restricted by the balance between WEE family kinases and Cdc25 phosphatases. The WEE kinase family consists of three proteins: WEE1, PKMYT1, and the less important WEE1B. WEE1 exclusively mediates phosphorylation at Tyr15, whereas PKMYT1 is dual-specific for Tyr15 as well as Thr14. Inhibition by a small molecule inhibitor is therefore proposed to be a promising option since WEE kinases bind Cdk1, altering equilibria and thus affecting G2/M transition.

Thioxylated cyclosporin A for studying protein-drug interactions.

Single atom substitution of cyclosporin A (CsA) through thioxylation has been used to study the structure-activity relationship of the immunosuppressive complex, involving the CsA receptor protein cyclophilin 18 (Cyp18) and the immunological target protein phosphatase calcineurin (CaN), illustrating the contributions of peptide backbone in protein-drug interaction. Moreover, the subtle difference between thioxylation positions in CsA has led to a remarkable change in the quenching effect on Cyp18 intrinsic fluorescence. Using the thioxylated compound Cs7 as an isosteric derivative of CsA in competition assay, the experiment has led to the determination of koff value in solution. Whereas the conformational heterogeneity of CsA has been found to be associated with its two-phase binding kinetics to Cyp18, the dissociation rate of CsA from complex is independent from the initial ligand structure.

Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum.

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.

Identification of peptidic substrates for the human kinase Myt1 using peptide microarrays.

Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the cell cycle. So far, no peptide substrate suitable for activity-based screening has been reported, hampering systematic development of Myt1 kinase inhibitors. Myt1 inhibitors had to be identified by using either binding assays or activity assays with expensive proteinous substrates. Here, a peptide microarray approach was used to identify peptidic Myt1 substrates. Wee1 kinase was profiled for comparison using the same technology. Myt1 hits from peptide microarray experiments were verified in solution by a fluorescence polarization assay and several peptide substrates derived from cellular proteins were identified. Subsequently, phosphorylation site determination was carried out by MS fragmentation studies and identified substrates were validated by kinase inhibitor profiling.

The glycoglycerolipid 1,2-dipalmitoyl-3-(N-palmitoyl-6'-amino-6'-deoxy-α-d-glucosyl)-sn-glycerol is no inhibitor of the human Myt1 kinase.

Previously, a glycoglycerolipid isolated from marine algae was reported to be a potent and selective inhibitor of the human Myt1 kinase, an enzyme involved in cell cycle regulation with great potential as an anti-cancer target. Based on that report, a lot of research effort has been invested by several working groups to synthesize and derivatize this compound. However, reliable assay data confirming the inhibitory potential and the mechanism of action of these glycoglycerolipids are missing so far. Here, based on experimental data and theoretical considerations, we show that the aforesaid glycoglycerolipid 1,2-dipalmitoyl-3-(N-palmitoyl-6'-amino-6'-deoxy-α-d-glucosyl)-sn-glycerol is not an inhibitor of the human Myt1 kinase.

Application of docking and QM/MM-GBSA rescoring to screen for novel Myt1 kinase inhibitors.

Identification of compounds that can bind to a target protein with high affinity is a nontrivial task in structure-based drug design. Several approaches ranging from simple scoring methods to more computationally demanding methods are usually applied for this purpose. In the current work, we used ligand docking in combination with QM/MM-GBSA, MM-GBSA, and MM-PBSA rescoring to discriminate between active and inactive Myt1 kinase inhibitors. Results show that QM/MM-GBSA rescoring performs better than normal docking scores or MM-GBSA rescoring in classifying active and inactive inhibitors. We also applied QM/MM-GBSA rescoring to estimate the binding affinities of compounds from different virtual screening runs. To prove our approach and to confirm its predictive power, a few compounds which were predicted to be active were purchased and experimentally tested. Among the five selected compounds, three showed significant inhibition of recombinant Myt1. PD-173952, which yielded a favorable QM/MM-GBSA binding free energy, showed a K(i) value of 8.1 nM. In addition, two compounds, PD-180970 and saracatinib, showed inhibition at the low micromolar level. Thus, the developed protocol might be useful for further virtual screening experiments to better discriminate between active and inactive compounds and to further optimize the identified hits.

Design, synthesis, and biological characterization of proteolysis targeting chimera (PROTACs) for the ataxia telangiectasia and RAD3-related (ATR) kinase.

The Ataxia telangiectasia and RAD3-related (ATR) kinase is a key regulator of DNA replication stress responses and DNA-damage checkpoints. Several potent and selective ATR inhibitors are reported and four of them are currently in clinical trials in combination with radio- or chemotherapy. Based on the idea of degrading target proteins rather than inhibiting them, we designed, synthesized and biologically characterized a library of ATR-targeted proteolysis targeting chimera (PROTACs). Among the synthesized compounds, the lenalidomide-based PROTAC 42i was the most promising. In pancreatic and cervix cancer cells cancer cells, it reduced ATR to 40 % of the levels in untreated cells. 42i selectively degraded ATR through the proteasome, dependent on the E3 ubiquitin ligase component cereblon, and without affecting the associated kinases ATM and DNA-PKcs. 42i may be a promising candidate for further optimization and biological characterization in various cancer cells.

Synthesis and in†vitro Metabolic Stability of Sterically Shielded Antimycobacterial Phenylalanine Amides.

Nα-aroyl-N-aryl-phenylalanine amides (AAPs) are RNA polymerase inhibitors with activity against Mycobacterium tuberculosis and non-tuberculous mycobacteria. We observed that AAPs rapidly degrade in microsomal suspensions, suggesting that avoiding hepatic metabolism is critical for their effectiveness in vivo. As both amide bonds are potential metabolic weak points of the molecule, we synthesized 16 novel AAP analogs in which the amide bonds are shielded by methyl or fluoro substituents in close proximity. Some derivatives show improved microsomal stability, while being plasma-stable and non-cytotoxic. In parallel with the metabolic stability studies, the antimycobacterial activity of the AAPs against Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium avium and Mycobacterium intracellulare was determined. The stability data are discussed in relation to the antimycobacterial activity of the panel of compounds and reveal that the concept of steric shielding of the anilide groups by a fluoro substituent has the potential to improve the stability and bioavailability of AAPs.

In vitro and in silico studies on substrate recognition and acceptance of human PKMYT1, a Cdk1 inhibitory kinase.

The human Myt1 kinase (PKMYT1) is an important regulator of the G2/M transition in the cell cycle. Presently, limited knowledge about its substrate recognition is available. Here, various potential substrates were investigated by different antibody based techniques including fluorescence polarization immunoassays and immunoblotting. Regarding both Thr and Tyr kinase activity, only protein substrates were found to be phosphorylated by Myt1, whereas any tested peptide was not recognized. In silico molecular dynamics studies were used to compare the stability of the Myt1 peptide complex with Wee1 peptide complex and support the biochemical findings. Furthermore, a Myt1 kinase binding assay suggests Myt1 being insensitive to staurosporine.

Development of Alkylated Hydrazides as Highly Potent and Selective Class I Histone Deacetylase Inhibitors with T cell Modulatory Properties.

Histone deacetylases (HDACs) are epigenetic regulators and additionally control the activity of non-histone substrates. We recently demonstrated that inhibition of HDAC8 overexpressed in various of cancers reduces hepatocellular carcinoma tumorigenicity in a T cell-dependent manner. Here, we present alkylated hydrazide-based class I HDAC inhibitors in which the n-hexyl side chain attached to the hydrazide moiety shows HDAC8 selectivity in vitro. Analysis of the mode of inhibition of the most promising compound 7d against HDAC8 revealed a substrate-competitive binding mode. 7d marked induced acetylation of the HDAC8 substrates H3K27 and SMC3 but not tubulin in CD4+ T lymphocytes, and significantly upregulated gene expressions for memory and effector functions. Furthermore, intraperitoneal injection of 7d (10 mg/kg) in C57BL/6 mice increased interleukin-2 expression in CD4+ T cells and CD8+ T cell proportion with no apparent toxicity. This study expands a novel chemotype of HDAC8 inhibitors with T cell modulatory properties for future therapeutic applications.

Identification of histone deacetylase 10 (HDAC10) inhibitors that modulate autophagy in transformed cells.

Histone deacetylases (HDACs) are a family of 18 epigenetic modifiers that fall into 4 classes. Histone deacetylase inhibitors (HDACi) are valid tools to assess HDAC functions. HDAC6 and HDAC10 belong to the class IIb subgroup of the HDAC family. The targets and biological functions of HDAC10 are ill-defined. This lack of knowledge is due to a lack of specific and potent HDAC10 inhibitors with cellular activity. Here, we have synthesized and characterized piperidine-4-acrylhydroxamates as potent and highly selective inhibitors of HDAC10. This was achieved by targeting the acidic gatekeeper residue Glu274 of HDAC10 with a basic piperidine moiety that mimics the interaction of the polyamine substrate of HDAC10. We have confirmed the binding modes of selected inhibitors using X-ray crystallography. Promising candidates were selected based on their specificity by in vitro profiling using recombinant HDACs. The most promising HDAC10 inhibitors 10c and 13b were tested for specificity in acute myeloid leukemia (AML) cells with the FLT3-ITD oncogene. By immunoblot experiments we assessed the hyperacetylation of histones and tubulin-α, which are class I and HDAC6 substrates, respectively. As validated test for HDAC10 inhibition we used flow cytometry assessing autolysosome formation in neuroblastoma and AML cells. We demonstrate that 10c and 13b inhibit HDAC10 with high specificity over HDAC6 and with no significant impact on class I HDACs. The accumulation of autolysosomes is not a consequence of apoptosis and 10c and 13b are not toxic for normal human kidney cells. These data show that 10c and 13b are nanomolar inhibitors of HDAC10 with high specificity. Thus, our new HDAC10 inhibitors are tools to identify the downstream targets and functions of HDAC10 in cells.

The novel calcineurin inhibitor CN585 has potent immunosuppressive properties in stimulated human T cells.

The Ca2+/calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6- diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library. By rational design of the parent compound we have attained the derivative 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine (CN585) that noncompetitively and reversibly inhibits calcineurin activity with a K(i) value of 3.8 mum. This derivative specifically inhibits calcineurin without affecting other Ser/Thr protein phosphatases or peptidyl prolyl cis/trans isomerases. CN585 shows potent immunosuppressive effects by inhibiting NFAT nuclear translocation and transactivation, cytokine production, and T cell proliferation. Moreover, the calcineurin inhibitor exhibits no cytotoxicity in the effective concentration range. Therefore, calcineurin inhibition by CN585 may represent a novel promising strategy for immune intervention.

Augmented photoswitching modulates immune signaling.

Reversible and non-invasive photoswitching of the immunosuppressive effect of a drug would be a very valuable tool for precisely regulating the immune system. Using a combination of protein borrowing and two-photon photoisomerization, we designed and synthesized derivatives of cyclosporin A. Here we demonstrate photoswitching of the local conformation within small molecules, which we used to modulate inhibitory potencies for cyclophilin, influence ternary and quaternary complex formations and regulate T-cell transcriptional activation in situ.