Utilization of an optimized AlphaFold protein model for structure-based design of a selective HDAC11 inhibitor with anti-neuroblastoma activity.

AlphaFold is an artificial intelligence approach for predicting the three-dimensional (3D) structures of proteins with atomic accuracy. One challenge that limits the use of AlphaFold models for drug discovery is the correct prediction of folding in the absence of ligands and cofactors, which compromises their direct use. We have previously described the optimization and use of the histone deacetylase 11 (HDAC11) AlphaFold model for the docking of selective inhibitors such as FT895 and SIS17. Based on the predicted binding mode of FT895 in the optimized HDAC11 AlphaFold model, a new scaffold for HDAC11 inhibitors was designed, and the resulting compounds were tested in vitro against various HDAC isoforms. Compound 5a proved to be the most active compound with an IC50 of 365 nM and was able to selectively inhibit HDAC11. Furthermore, docking of 5a showed a binding mode comparable to FT895 but could not adopt any reasonable poses in other HDAC isoforms. We further supported the docking results with molecular dynamics simulations that confirmed the predicted binding mode. 5a also showed promising activity with an EC50 of 3.6 µM on neuroblastoma cells.

Direct GPCR-EGFR interaction enables synergistic membrane-to-nucleus information transfer.

We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.

Structure-Activity Studies of 1,2,4-Oxadiazoles for the Inhibition of the NAD+-Dependent Lysine Deacylase Sirtuin 2.

The NAD+-dependent lysine deacylase sirtuin 2 (Sirt2) is involved in multiple pathological conditions such as cancer. Targeting Sirt2 has thus received an increased interest for therapeutic purposes. Furthermore, the orthologue from Schistosoma mansoni (SmSirt2) has been considered for the potential treatment of the neglected tropical disease schistosomiasis. We previously identified a 1,2,4-oxadiazole-based scaffold from the screening of the "Kinetobox" library as a dual inhibitor of human Sirt2 (hSirt2) and SmSirt2. Herein, we describe the structure-activity studies on 1,2,4-oxadiazole-based analogues, which are potent inhibitors of human Sirt2 deacetylation. As proposed by docking studies, a substrate-competitive and cofactor-noncompetitive binding mode of inhibition could be determined in vitro via binding assays and kinetic analysis and further confirmed by a crystal structure of an oxadiazole inhibitor in complex with hSirt2. Optimized analogues reduced cell viability and inhibited prostate cancer cell migration, in correlation with Sirt2 deacetylase inhibition both in vitro and in cells.

A Novel AMPK Inhibitor Sensitizes Pancreatic Cancer Cells to Ferroptosis Induction.

Cancer cells must develop strategies to adapt to the dynamically changing stresses caused by intrinsic or extrinsic processes, or therapeutic agents. Metabolic adaptability is crucial to mitigate such challenges. Considering metabolism as a central node of adaptability, it is focused on an energy sensor, the AMP-activated protein kinase (AMPK). In a subtype of pancreatic ductal adenocarcinoma (PDAC) elevated AMPK expression and phosphorylation is identified. Using drug repurposing that combined screening experiments and chemoproteomic affinity profiling, it is identified and characterized PF-3758309, initially developed as an inhibitor of PAK4, as an AMPK inhibitor. PF-3758309 shows activity in pre-clinical PDAC models, including primary patient-derived organoids. Genetic loss-of-function experiments showed that AMPK limits the induction of ferroptosis, and consequently, PF-3758309 treatment restores the sensitivity toward ferroptosis inducers. The work established a chemical scaffold for the development of specific AMPK-targeting compounds and deciphered the framework for the development of AMPK inhibitor-based combination therapies tailored for PDAC.

Discovery and Anticancer Screening of Novel Oxindole-Based Derivative Bearing Pyridyl Group as Potent and Selective Dual FLT3/CDK2 Kinase Inhibitor.

Protein kinases regulate cellular activities and make up over 60% of oncoproteins and proto-oncoproteins. Among these kinases, FLT3 is a member of class III receptor tyrosine kinase family which is abundantly expressed in individuals with acute leukemia. Our previous oxindole-based hit has a particular affinity toward FLT3 (IC50 = 2.49 µM) and has demonstrated selectivity towards FLT3 ITD-mutated MV4-11 AML cells, with an IC50 of 4.3 µM. By utilizing the scaffold of the previous hit, sixteen new compounds were synthesized and screened against NCI-60 human cancer cell lines. This leads to the discovery of a potent antiproliferative compound, namely 5l, with an average GI50 value against leukemia and colon cancer subpanels equalling 3.39 and 5.97 µM, respectively. Screening against a specific set of 10 kinases that are associated with carcinogenesis indicates that compound 5l has a potent FLT3 inhibition (IC50 = 36.21 ± 1.07 nM). Remarkably, compound 5l was three times more effective as a CDK2 inhibitor (IC50 = 8.17 ± 0.32 nM) compared to sunitinib (IC50 = 27.90 ± 1.80 nM). Compound 5l was further analyzed by means of docking and molecular dynamics simulation for CDK2 and FLT3 active sites which provided a rational for the observed strong inhibition of kinases. These results suggest a novel structural scaffold candidate that simultaneously inhibits CDK2 and FLT3 and gives encouragement for further development as a potential therapeutic for leukemia and colon cancer.

Exploring Aromatic Cage Flexibility Using Cosolvent Molecular Dynamics Simulations─An In-Silico Case Study of Tudor Domains.

Cosolvent molecular dynamics (MD) simulations have proven to be powerful in silico tools to predict hotspots for binding regions on protein surfaces. In the current study, the method was adapted and applied to two Tudor domain-containing proteins, namely Spindlin1 (SPIN1) and survival motor neuron protein (SMN). Tudor domains are characterized by so-called aromatic cages that recognize methylated lysine residues of protein targets. In the study, the conformational transitions from closed to open aromatic cage conformations were investigated by performing MD simulations with cosolvents using six different probe molecules. It is shown that a trajectory clustering approach in combination with volume and atomic distance tracking allows a reasonable discrimination between open and closed aromatic cage conformations and the docking of inhibitors yields very good reproducibility with crystal structures. Cosolvent MDs are suitable to capture the flexibility of aromatic cages and thus represent a promising tool for the optimization of inhibitors.

Key structural requirements of benzamide derivatives for histone deacetylase inhibition: design, synthesis and biological evaluation.

Background: Histone deacetylase inhibitors (HDACIs) are important as anticancer agents. Objective: This study aimed to investigate some key structural features of HDACIs via the design, synthesis and biological evaluation of novel benzamide-based derivatives. Methods: Novel structures, designed using a molecular modification approach, were synthesized and biologically evaluated. Results: The results indicated that a subset of molecules with CH3/NH2 at R2 position possess selective antiproliferative activity. However, only those with an NH2 group showed HDACI activity. Importantly, the shorter the molecule length, the stronger HDACI. Among all, 7j was the most potent HDAC1-3 inhibitor and antiproliferative compound. Conclusion: The results of the present investigation could provide valuable structural knowledge applicable for the development of the HDACIs and benzamide-based antiproliferative agents in the future.

[Box: see text].

Pharmacological degradation of ATR induces antiproliferative DNA replication stress in leukemic cells.

Mammalian cells replicate ~ 3 × 109 base pairs per cell cycle. One of the key molecules that slows down the cell cycle and prevents excessive DNA damage upon DNA replication stress is the checkpoint kinase ataxia-telangiectasia-and-RAD3-related (ATR). Proteolysis-targeting-chimeras (PROTACs) are an innovative pharmacological invention to molecularly dissect, biologically understand, and therapeutically assess catalytic and non-catalytic functions of enzymes. This work defines the first-in-class ATR PROTAC, Abd110/Ramotac-1. It is derived from the ATR inhibitor VE-821 and recruits the E3 ubiquitin-ligase component cereblon to ATR. Abd110 eliminates ATR rapidly in human leukemic cells. This mechanism provokes DNA replication catastrophe and augments anti-leukemic effects of the clinically used ribonucleotide reductase-2 inhibitor hydroxyurea. Moreover, Abd110 is more effective than VE-821 against human primary leukemic cells but spares normal primary immune cells. CRISPR-Cas9 screens show that ATR is a dependency factor in 116 myeloid and lymphoid leukemia cells. Treatment of wild-type but not of cereblon knockout cells with Abd110 stalls their proliferation which verifies that ATR elimination is the primary mechanism of Abd110. Altogether, our findings demonstrate specific anti-leukemic effects of an ATR PROTAC.

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.

Specific Inhibitors of Mitochondrial Deacylase Sirtuin 4 Endowed with Cellular Activity.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in aging-related diseases. Mammalian Sirtuin 4 (Sirt4) is located in mitochondria and a potential therapeutic target for cancer and metabolic diseases, but no potent and selective Sirt4 inhibitors have been reported. Here, we describe the identification of potent Sirt4-specific small-molecule inhibitors. Testing hits from a target-based virtual screen revealed 12 active compounds. A focused screen based on two top compounds, followed by structure-assisted design of derivatives, yielded four first-in-class potent Sirt4 inhibitors. Kinetic analyses indicate compound competition with the acyl peptide substrate, consistent with the docking models and implicating Sirt4's unique acyl binding site. The compounds indeed show preference for Sirt4 over other isoforms, with one of them (69) being highly isoform selective, and they are active in cells. Our results provide first lead compounds and mechanistic insights for optimization toward Sirt4-specific inhibitors useful as experimental tools and potential therapeutics.

Comparative Structure-Based Virtual Screening Utilizing Optimized AlphaFold Model Identifies Selective HDAC11 Inhibitor.

HDAC11 is a class IV histone deacylase with no crystal structure reported so far. The catalytic domain of HDAC11 shares low sequence identity with other HDAC isoforms, which makes conventional homology modeling less reliable. AlphaFold is a machine learning approach that can predict the 3D structure of proteins with high accuracy even in absence of similar structures. However, the fact that AlphaFold models are predicted in the absence of small molecules and ions/cofactors complicates their utilization for drug design. Previously, we optimized an HDAC11 AlphaFold model by adding the catalytic zinc ion and minimization in the presence of reported HDAC11 inhibitors. In the current study, we implement a comparative structure-based virtual screening approach utilizing the previously optimized HDAC11 AlphaFold model to identify novel and selective HDAC11 inhibitors. The stepwise virtual screening approach was successful in identifying a hit that was subsequently tested using an in vitro enzymatic assay. The hit compound showed an IC50 value of 3.5 µM for HDAC11 and could selectively inhibit HDAC11 over other HDAC subtypes at 10 µM concentration. In addition, we carried out molecular dynamics simulations to further confirm the binding hypothesis obtained by the docking study. These results reinforce the previously presented AlphaFold optimization approach and confirm the applicability of AlphaFold models in the search for novel inhibitors for drug discovery.

Synthesis and Evaluation of Novel Substituted N-Aryl 1,4-Dihydropyridines as Antituberculostatic Agents.

BACKGROUND: Tuberculosis has been the main cause of mortality of infectious diseases worldwide, with strongly limited therapeutic options. With increasing resistance and missing suitable drugs in those cases, there is a strong need for novel antituberculostatic drugs. We developed novel N-aryl 1,4-dihydropyridines with various substitution patterns to evaluate them as antituberculostatic agents. METHODS: 1,4-Dihydropyridine derivatives were synthesized and purified by column chromatography or recrystallization. The mycobacterial growth inhibition was determined in a fluorescent mycobacterial growth assay. RESULTS: The compounds were prepared in a simple one-pot reaction under acidic conditions with structurally varied components. The substituent effects on the determined mycobacterial growth inhibitory properties are discussed. CONCLUSION: Lipophilic diester substituted derivatives show promising activities that were additionally affected by the aromatic substituent functions. Thus, we identified compounds with activities almost reaching that of the used antimycobacterial drug as control.

Design and synthesis of bioreductive prodrugs of class I histone deacetylase inhibitors and their biological evaluation in virally transfected acute myeloid leukemia cells.

Although histone deacetylase (HDAC) inhibitors show promise in treating various types of hematologic malignancies, they have some limitations, including poor pharmacokinetics and off-target side effects. Prodrug design has shown promise as an approach to improve pharmacokinetic properties and to improve target tissue specificity. In this work, several bioreductive prodrugs for class I HDACs were designed based on known selective HDAC inhibitors. The zinc-binding group of the HDAC inhibitors was masked with various nitroarylmethyl residues to make them substrates of nitroreductase (NTR). The developed prodrugs showed weak HDAC inhibitory activity compared to their parent inhibitors. The prodrugs were tested against wild-type and NTR-transfected THP1 cells. Cellular assays showed that both 2-nitroimidazole-based prodrugs 5 and 6 were best activated by the NTR and exhibited potent activity against NTR-THP1 cells. Compound 6 showed the highest cellular activity (GI50 = 77 nM) and exhibited moderate selectivity. Moreover, activation of prodrug 6 by NTR was confirmed by liquid chromatography-mass spectrometry analysis, which showed the release of the parent inhibitor after incubation with Escherichia coli NTR. Thus, compound 6 can be considered a novel prodrug selective for class I HDACs, which could be used as a good starting point for increasing selectivity and for further optimization.

Characterization of (R)- and (S)-[18F]OF-NB1 in Rodents as Positron Emission Tomography Probes for Imaging GluN2B Subunit-Containing N-Methyl-d-Aspartate Receptors.

The N-methyl-d-aspartate receptor (NMDAR) subtype 2B (GluN1/2B) is implicated in various neuropathologies. Given the lack of a validated radiofluorinated positron emission tomography (PET) probe for the imaging of GluN1/2B receptors, we comprehensively investigated the enantiomers of [18F]OF-NB1 in rodents. Particularly, the (R)- and (S)- enantiomers were evaluated using in silico docking, in vitro autoradiography, in vivo PET imaging, and ex vivo biodistribution studies. A select panel of GluN1/2B antagonists (CP-101,606, CERC-301, and eliprodil) and the off-target sigma-1 receptor ligands (fluspidine and SA4503) were used to determine the specificity and selectivity of the tested enantiomers. Additionally, a nonmetal-mediated radiofluorination strategy was devised that harnesses the potential of diaryliodoniums in the nucleophilic radiofluorination of nonactivated aromatic compounds. Both enantiomers exhibited known GluN1/2B binding patterns; however, the R-enantiomer showed higher GluN1/2B-specific accumulation in rodent autoradiography and higher brain uptake in PET imaging experiments compared to the S-enantiomer. Molecular simulation studies provided further insights with respect to the difference in binding, whereby a reduced ligand-receptor interaction was observed for the S-enantiomer. Nonetheless, both enantiomers showed dose dependency when two different doses (1 and 5 mg/kg) of the GluN1/2B antagonist, CP-101,606, were used in the PET imaging study. Taken together, (R)-[18F]OF-NB1 appears to exhibit the characteristics of a suitable PET probe for imaging of GluN2B-containing NMDARs in clinical studies.

Histone Acetyltransferase and Deacetylase Inhibitors-New Aspects and Developments.

Epigenetic processes modulate gene transcription and genomic stability, ensuring proper cell development and differentiation [...].

Utilization of AlphaFold models for drug discovery: Feasibility and challenges. Histone deacetylase 11 as a case study.

Histone deacetylase 11 (HDAC11), an enzyme that cleaves acyl groups from acylated lysine residues, is the sole member of class IV of HDAC family with no reported crystal structure so far. The catalytic domain of HDAC11 shares low sequence identity with other HDAC isoforms which complicates the conventional template-based homology modeling. AlphaFold is a neural network machine learning approach for predicting the 3D structures of proteins with atomic accuracy even in absence of similar structures. However, the structures predicted by AlphaFold are missing small molecules as ligands and cofactors. In our study, we first optimized the HDAC11 AlphaFold model by adding the catalytic zinc ion followed by assessment of the usability of the model by docking of the selective inhibitor FT895. Minimization of the optimized model in presence of transplanted inhibitors, which have been described as HDAC11 inhibitors, was performed. Four complexes were generated and proved to be stable using three replicas of 50 ns MD simulations and were successfully utilized for docking of the selective inhibitors FT895, MIR002 and SIS17. For SIS17, The most reasonable pose was selected based on structural comparison between HDAC6, HDAC8 and the HDAC11 optimized AlphaFold model. The manually optimized HDAC11 model is thus able to explain the binding behavior of known HDAC11 inhibitors and can be used for further structure-based optimization.

Challenges in natural product-based drug discovery assisted with in silico-based methods.

The application of traditional medicine by humans for the treatment of ailments as well as improving the quality of life far outdates recorded history. To date, a significant percentage of humans, especially those living in developing/underprivileged communities still rely on traditional medicine for primary healthcare needs. In silico-based methods have been shown to play a pivotal role in modern pharmaceutical drug discovery processes. The application of these methods in identifying natural product (NP)-based hits has been successful. This is very much observed in many research set-ups that use rationally in silico-based methods in combination with experimental validation techniques. The combination has rendered the use of in silico-based approaches even more popular and successful in the investigation of NPs. However, identifying and proposing novel NP-based hits for experimental validation comes with several challenges such as the availability of compounds by suppliers, the huge task of separating pure compounds from complex mixtures, the quantity of samples available from the natural source to be tested, not to mention the potential ecological impact if the natural source is exhausted. Because most peer-reviewed publications are biased towards "positive results", these challenges are generally not discussed in publications. In this review, we highlight and discuss these challenges. The idea is to give interested scientists in this field of research an idea of what they can come across or should be expecting as well as prompting them on how to avoid or fix these issues.

Evaluation of thiadiazine-based PET radioligands for imaging the AMPA receptor.

As a subclass of ionotropic glutamate receptors (iGluRs), α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been implicated in various neurological disorders and neurodegenerative diseases. To further our understanding of AMPA receptor-related disorders in the central nervous system (CNS), it is important to be able to image and quantify AMPA receptors in vivo. In this study, we identified a novel F-containing AMPA positive allosteric modulator (PAM) 6 as a potential lead compound. Molecular docking studies and CNS PET multi-parameter optimization (MPO) analysis were used to predict the absorption, distribution, metabolism, and excretion (ADME) characteristics of 6 as a PET probe. The resulting PET probe, [18F]6 (codename [18F]AMPA-2109), was successfully radiolabeled and demonstrated excellent blood-brain barrier (BBB) permeability and high brain uptake in rodents and non-human primates. However, [18F]6 did not show substantial specific binding in the rodent or non-human primate brain. Further medicinal chemistry efforts are necessary to improve specific binding, and our work may serve as a starting point for the design of novel 18F-labeled AMPA receptor-targeted PET radioligands aimed for clinical translation.

1,3-Diphenylureido hydroxamate as a promising scaffold for generation of potent antimalarial histone deacetylase inhibitors.

We report a series of 1,3-diphenylureido hydroxamate HDAC inhibitors evaluated against sensitive and drug-resistant P. falciparum strains. Compounds 8a-d show potent antiplasmodial activity, indicating that a phenyl spacer allows improved potency relative to cinnamyl and di-hydrocinnamyl linkers. In vitro, mechanistic studies demonstrated target activity for PfHDAC1 on a recombinant level, which agreed with cell quantification of the acetylated histone levels. Compounds 6c, 7c, and 8c, identified as the most active in phenotypic assays and PfHDAC1 enzymatic inhibition. Compound 8c stands out as a remarkable inhibitor, displaying an impressive 85% inhibition of PfHDAC1, with an IC50 value of 0.74 µM in the phenotypic screening on Pf3D7 and 0.8 µM against multidrug-resistant PfDd2 parasites. Despite its potent inhibition of PfHDAC1, 8c remains the least active on human HDAC1, displaying remarkable selectivity. In silico studies suggest that the phenyl linker has an ideal length in the series for permitting effective interactions of the hydroxamate with PfHDAC1 and that this compound series could bind as well as in HsHDAC1. Taken together, these results highlight the potential of diphenylurea hydroxamates as a privileged scaffold for the generation of potent antimalarial HDAC inhibitors with improved selectivity over human HDACs.

Synthesis and Biological Evaluation of Enantiomerically Pure (R)- and (S)-[18F]OF-NB1 for Imaging the GluN2B Subunit-Containing NMDA Receptors.

GluN2B subunit-containing N-methyl-d-aspartate (NMDA) receptors have been implicated in various neurological disorders. Nonetheless, a validated fluorine-18 labeled positron emission tomography (PET) ligand for GluN2B imaging in the living human brain is currently lacking. The aim of this study was to develop a novel synthetic approach that allows an enantiomerically pure radiosynthesis of the previously reported PET radioligands (R)-[18F]OF-NB1 and (S)-[18F]OF-NB1 as well as to assess their in vitro and in vivo performance characteristics for imaging the GluN2B subunit-containing NMDA receptor in rodents. A novel synthetic approach was successfully developed, which allows for the enantiomerically pure radiosynthesis of (R)-[18F]OF-NB1 and (S)-[18F]OF-NB1 and the translation of the probe to the clinic. While both enantiomers were selective over sigma2 receptors in vitro and in vivo, (R)-[18F]OF-NB1 showed superior GluN2B subunit specificity by in vitro autoradiography and higher volumes of distribution in the rodent brain by small animal PET studies.