Novel Benzindoloazecines and Dibenzazecines: synthesis and affinities for the Dopamine receptors

Azecine sind eine neuartige Klasse von Dopaminrezeptor-Antagonisten, gekennzeichnet durch ihre nanomolare Affinitäten an den Dopaminrezeptoren und vor allem ihre Selektivität für die Rezeptoren der D1-Familie. Strukturwirkungsbeziehungen dieser Verbinungsklasse wurde umfassend erforscht. Die Strategie der vorliegenden Arbeit war die Strukturen der Leitverbindungen LE300 und LE404 so zu modifizieren, dass die erforderlichen strukturellen Merkmale weitgehend erhalten werden sollten. Das Benzindoloazecin LE300 wurde vielfältig modifiziert; z.B. durch Veränderung des Annellierungsmusters vom Ringgerüst oder Einfügen eines Substituents an verschiedenen Stellen. Außerdem wurde die phenolische Gruppe der Leitstruktur LE404 vom Dibenzazecin-Typ durch eine Methylendioxygruppe ersetzt. Das Ziel dieser Arbeit war die Strukturwirkungsbeziehungen dieser neuartigen Klasse von Dopaminrezeptor-Antagonisten weiter zu entwickeln, um Derivate mit höheren Affinitäten und besser ausgeprägten Subtypselektivität zu erreichen. Insgesamt wurden 29 Zielverbindungen synthetisiert und die Affinitäten an den verschiedenen Dopaminrezeptoren (human) durch Radioligandbindungsstudien ermittelt. Weiterhin wurde die Funktionalität der Verbindungen (Agonisten oder Antagonisten) mit Hilfe eines Calcium Assays bestimmt

Exploring aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction : an in silico study

Some of the main challenges faced in drug discovery are pocket flexibility and binding mode prediction. In this work, we explored the aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction by means of in silico approaches. We first investigated the Spindlin1 aromatic cage plasticity by analyzing the available crystal structures and through molecular dynamic simulations. Then we assessed the ability of rigid docking and flexible docking to rightly reproduce the binding mode of a known ligand into Spindlin1, as an example of a reader protein displaying flexibility in the binding pocket. The ability of induced fit docking was further probed to test if the right ligand binding mode could be obtained through flexible docking regardless of the initial protein conformation. Finally, the stability of generated docking poses was verified by molecular dynamic simulations. Accurate binding mode prediction was obtained showing that the herein reported approach is a highly promising combination of in silico methods able to rightly predict the binding mode of small molecule ligands in flexible binding pockets, such as those observed in some reader proteins.Publikationsfonds ML

Muscle carnitine palmitoyltransferase II deficiency : a review of enzymatic controversy and clinical features

CPT (carnitine palmitoyltransferase) II muscle deficiency is the most common form of muscle fatty acid metabolism disorders. In contrast to carnitine deficiency, it is clinically characterized by attacks of myalgia and rhabdomyolysis without persistent muscle weakness and lipid accumulation in muscle fibers. The biochemical consequences of the disease-causing mutations are still discussed controversially. CPT activity in muscles of patients with CPT II deficiency ranged from not detectable to reduced to normal. Based on the observation that in patients, total CPT is completely inhibited by malony-CoA, a deficiency of malonyl-CoA-insensitive CPT II has been suggested. In contrast, it has also been shown that in muscle CPT II deficiency, CPT II protein is present in normal concentrations with normal enzymatic activity. However, CPT II in patients is abnormally sensitive to inhibition by malonyl-CoA, Triton X-100 and fatty acid metabolites. A recent study on human recombinant CPT II enzymes (His6-N-hCPT2 and His6-N-hCPT2/S113L) revealed that the wild-type and the S113L variants showed the same enzymatic activity. However, the mutated enzyme showed an abnormal thermal destabilization at 40 and 45 °C and an abnormal sensitivity to inhibition by malony-CoA. The thermolability of the mutant enzyme might explain why symptoms in muscle CPT II deficiency mainly occur during prolonged exercise, infections and exposure to cold. In addition, the abnormally regulated enzyme might be mostly inhibited when the fatty acid metabolism is stressed

Fatty acid binding to the human transport proteins FABP3, FABP4, and FABP5 from a Ligand’s perspective

Fatty acid binding proteins (FABPs) are a family of amphiphilic transport proteins with high diversity in terms of their amino acid sequences and binding preferences. Beyond their main biological role as cytosolic fatty acid transporters, many aspects regarding their binding mechanism and functional specializations in human cells remain unclear. In this work, the binding properties and thermodynamics of FABP3, FABP4, and FABP5 were analyzed under various physical conditions. For this purpose, the FABPs were loaded with fatty acids bearing fluorescence or spin probes as model ligands, comparing their binding affinities via microscale thermophoresis (MST) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy. The CW EPR spectra of non-covalently bound 5- and 16-DOXYL stearic acid (5/16-DSA) deliver in-depth information about the dynamics and chemical environments of ligands inside the binding pockets of the FABPs. EPR spectral simulations allow the construction of binding curves, revealing two different binding states (‘intermediately’ and ‘strongly’ bound). The proportion of bound 5/16-DSA depends strongly on the FABP concentration and the temperature but with remarkable differences between the three isoforms. Additionally, the more dynamic state (‘intermediately bound’) seems to dominate at body temperature with thermodynamic preference. The ligand binding studies were supplemented by aggregation studies via dynamic light scattering and bioinformatic analyses. Beyond the remarkably fine-tuned binding properties exhibited by each FABP, which were discernible with our EPR-centered approach, the results of this work attest to the power of simple spectroscopic experiments to provide new insights into the ligand binding mechanisms of proteins in general on a molecular level

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

Ternary complex modeling, induced fit docking and molecular dynamics simulations as a successful approach for the design of VHL-mediated PROTACs targeting the kinase FLT3

Proteolysis targeting chimeras (PROTACs) have proven to be a novel approach for the degradation of disease-causing proteins in drug discovery. One of the E3 ligases for which efficient PROTACs have been described is the Von Hippel-Lindau factor (VHL). However, the development of PROTACs has so far often relied on a minimum of computational tools, so that it is mostly based on a trial-and-error process. Therefore, there is a great need for resource- and time-efficient structure-based or computational approaches to streamline PROTAC design. In this study, we present a combined computational approach that integrates static ternary complex formation, induced-fit docking, and molecular dynamics (MD) simulations. Our methodology was tested using four experimentally derived ternary complex structures of VHL PROTACs, reported for BRD4, SMARCA2, FAK, and WEE1. In addition, we applied the validated approach to model a recently in-house developed FLT3-targeted PROTAC (MA49). The results show that static ternary models generated with a protein–protein docking method implemented in the software MOE have a high predictive power for reproducing the experimental 3D structures. The induced-fit docking of different active PROTACs to their respective models showed the reliability of this model for the development of new VHL-mediated degraders. In particular, the induced-fit docking was sensitive to structural changes in the PROTACs, as evidenced by the failed binding modes of the PROTAC negative controls. Furthermore, MD simulations confirmed the stability of the generated complexes and emphasized the importance of dynamic studies for understanding the relationship between PROTAC structure and function

Synthesis and antimycobacterial assays of some new ethambutol analogs

Ethambutol (EMB) is a first-line anti-tuberculosis drug that is also considered in treatment regimens for infections caused by non-tuberculous mycobacteria (NTM). EMB targets the arabinosyl transferases EmbCAB, which are important for the synthesis of cell wall constituents. To further explore and narrow down the structural variability of EMB, we synthesized three series of new EMB analogs. We tested their activity against Mycobacterium tuberculosis, Mycobacterium smegmatis, Mycobacterium abscessus and Mycobacterium intracellulare. Only analogs that very closely resembled EMB showed comparable antimycobacterial activity

Application of ligand- and structure-based prediction models for the design of alkylhydrazide-based HDAC3 inhibitors as novel anti-cancer compounds

Histone deacetylases (HDAC) represent promising epigenetic targets for several diseases including different cancer types. The HDAC inhibitors approved to date are pan-HDAC inhibitors and most show a poor selectivity profile, side effects, and in particular hydroxamic-acid-based inhibitors lack good pharmacokinetic profiles. Therefore, the development of isoform-selective non-hydroxamic acid HDAC inhibitors is a highly regarded field in medicinal chemistry. In this study, we analyzed different ligand-based and structure-based drug design techniques to predict the binding mode and inhibitory activity of recently developed alkylhydrazide HDAC inhibitors. Alkylhydrazides have recently attracted more attention as they have shown promising effects in various cancer cell lines. In this work, pharmacophore models and atom-based quantitative structure–activity relationship (QSAR) models were generated and evaluated. The binding mode of the studied compounds was determined using molecular docking as well as molecular dynamics simulations and compared with known crystal structures. Calculated free energies of binding were also considered to generate QSAR models. The created models show a good explanation of in vitro data and were used to develop novel HDAC3 inhibitors

Aromatic Amino Acid Hydroxylases as Off-Targets of Histone Deacetylase Inhibitors

The aromatic amino acid hydroxylases (AAAHs) phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylases 1 and 2 are structurally related enzymes that contain an active site iron atom and depend on tetrahydrobiopterin (BH4) as cosubstrate. Due to their important roles in synthesis of serotonin, dopamine, noradrenaline, and adrenaline and their involvement in cardiovascular, neurological, and endocrine disorders, AAAHs have been targeted by substrate analogs, iron chelators, and allosteric ligands. Phenylalanine hydroxylase is also off-target of the histone deacetylase (HDAC) inhibitor panobinostat. To systematically explore the binding of HDAC inhibitors to AAAHs, we screened a library of 307 HDAC inhibitors and structural analogs against tryptophan hydroxylase 1 using a fluorescence-based thermal stability assay, followed by activity assays. Selected hits were enzymatically tested against all four purified human AAAHs. Cellular thermal shift assay was performed for phenylalanine hydroxylase. We show that panobinostat and structurally related compounds such as TB57, which similarly to panobinostat also contains a cinnamoyl hydroxamate, bind to human AAAHs and inhibit these enzymes with high selectivity within the class (panobinostat inhibition (IC50): phenylalanine hydroxylase (18 nM) > tyrosine hydroxylase (450 nM) > tryptophan hydroxylase 1 (1960 nM). This study shows that panobinostat and related hydroxamic acid type HDAC inhibitors inhibit all AAAHs at therapeutically relevant concentrations. Our results warrant further investigations of the off-target relevance of HDAC inhibitors intended for clinical use and provide directions for new dual HDAC/AAAH and selective AAAH inhibitors. These findings may also provide a new mechanistic link between regulation of histone modification, AAAH function, and monoaminergic neurotransmission.publishedVersio