Efficacy of combined tumor irradiation and KCa3.1-targeting with TRAM-34 in a syngeneic glioma mouse model.

The intermediate-conductance calcium-activated potassium channel KCa3.1 has been proposed to be a new potential target for glioblastoma treatment. This study analyzed the effect of combined irradiation and KCa3.1-targeting with TRAM-34 in the syngeneic, immune-competent orthotopic SMA-560/VM/Dk glioma mouse model. Whereas neither irradiation nor TRAM-34 treatment alone meaningfully prolonged the survival of the animals, the combination significantly prolonged the survival of the mice. We found an irradiation-induced hyperinvasion of glioma cells into the brain, which was inhibited by concomitant TRAM-34 treatment. Interestingly, TRAM-34 did neither radiosensitize nor impair SMA-560's intrinsic migratory capacities in vitro. Exploratory findings hint at increased TGF-ß1 signaling after irradiation. On top, we found a marginal upregulation of MMP9 mRNA, which was inhibited by TRAM-34. Last, infiltration of CD3+, CD8+ or FoxP3+ T cells was not impacted by either irradiation or KCa3.1 targeting and we found no evidence of adverse events of the combined treatment. We conclude that concomitant irradiation and TRAM-34 treatment is efficacious in this preclinical glioma model.

Photocaging of Pyridinylimidazole-Based Covalent JNK3 Inhibitors Affords Spatiotemporal Control of the Binding Affinity in Live Cells.

The concept of photocaging represents a promising approach to acquire spatiotemporal control over molecular bioactivity. To apply this strategy to pyridinylimidazole-based covalent JNK3 inhibitors, we used acrylamido-N-(4-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)benzamide (1) as a lead compound to design novel covalent inhibitors of JNK3 by modifying the amide bond moiety in the linker. The newly synthesized inhibitors demonstrated IC50 values in the low double-digit nanomolar range in a radiometric kinase assay. They were further characterized in a NanoBRETTM intracellular JNK3 assay, where covalent engagement of the target enzyme was confirmed by compound washout experiments and a loss in binding affinity for a newly generated JNK3(C154A)-NLuc mutant. The most potent compound of the series, N-(3-acrylamidophenyl)-4-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)benzamide (13), was equipped with a photolabile protecting group leading to a nearly 10-fold decrease in intracellular JNK3 binding affinity, which was fully recovered by UV irradiation at a wavelength of 365 nm within 8 min. Our results highlight that photocaged covalent inhibitors can serve as a pharmacological tool to control JNK3 activity in live cells with light.

Structure-Based Design of High-Affinity Fluorescent Probes for the Neuropeptide Y Y1 Receptor.

The recent crystallization of the neuropeptide Y Y1 receptor (Y1R) in complex with the argininamide-type Y1R selective antagonist UR-MK299 (2) opened up a new approach toward structure-based design of nonpeptidic Y1R ligands. We designed novel fluorescent probes showing excellent Y1R selectivity and, in contrast to previously described fluorescent Y1R ligands, considerably higher (∼100-fold) binding affinity. This was achieved through the attachment of different fluorescent dyes to the diphenylacetyl moiety in 2 via an amine-functionalized linker. The fluorescent ligands exhibited picomolar Y1R binding affinities (pKi values of 9.36-9.95) and proved to be Y1R antagonists, as validated in a Fura-2 calcium assay. The versatile applicability of the probes as tool compounds was demonstrated by flow cytometry- and fluorescence anisotropy-based Y1R binding studies (saturation and competition binding and association and dissociation kinetics) as well as by widefield and total internal reflection fluorescence (TIRF) microscopy of live tumor cells, revealing that fluorescence was mainly localized at the plasma membrane.

Design and Synthesis of Highly Selective Brain Penetrant p38α Mitogen-Activated Protein Kinase Inhibitors.

Stress-induced p38α mitogen-activated protein (MAP) kinase activation modulates cytokine overproduction and is associated with neuroinflammation and neurodegeneration. As a potential therapeutic approach, novel Skepinone-based p38α MAP kinase inhibitors were optimized to cross the blood-brain barrier via either amino acid transporters or hydrophobic diffusion. To enhance absorption from the oral route, we used methyl ester prodrugs of the active carboxy analogs. Of these, 3-(8-((2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoic acid (43; p38α, IC50 = 5.5 nM) and 4-(8-((2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)butanoic acid (44; p38α, IC50 = 12 nM) had brain-to-plasma ratios of 1.4 and 4.4, respectively. Compound 70, 3-(8-((2-aminophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoic acid (p38α, IC50 = 1.0 nM), the Skepinone-N counterpart of 43, was most present in the mouse brain (brain-to-plasma ratio of 4.7; 0.4 mg/kg p.o., 2 h, 580 nmol/kg). Compounds 43, 44, and 70 were p38α-MAP-kinase-selective, metabolically stable, hERG nonbinding, and able to modulate IL-6 and TNF-α production in cell-based assays.

Addressing a Trapped High-Energy Water: Design and Synthesis of Highly Potent Pyrimidoindole-Based Glycogen Synthase Kinase-3ß Inhibitors.

In small molecule binding, water is not a passive bystander but rather takes an active role in the binding site, which may be decisive for the potency of the inhibitor. Here, by addressing a high-energy water, we improved the IC50 value of our co-crystallized glycogen synthase kinase-3ß (GSK-3ß) inhibitor by nearly two orders of magnitude. Surprisingly, our results demonstrate that this high-energy water was not displaced by our potent inhibitor (S)-3-(3-((7-ethynyl-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile ((S)-15, IC50 value of 6 nM). Instead, only a subtle shift in the location of this water molecule resulted in a dramatic decrease in the energy of this high-energy hydration site, as shown by the WaterMap analysis combined with microsecond timescale molecular dynamics simulations. (S)-15 demonstrated both a favorable kinome selectivity profile and target engagement in a cellular environment and reduced GSK-3 autophosphorylation in neuronal SH-SY5Y cells. Overall, our findings highlight that even a slight adjustment in the location of a high-energy water can be decisive for ligand binding.

Controlling the Covalent Reactivity of a Kinase Inhibitor with Light.

Covalent kinase inhibitors account for some of the most successful drugs that have recently entered the clinic and many others are in preclinical development. A common strategy is to target cysteines in the vicinity of the ATP binding site using an acrylamide electrophile. To increase the tissue selectivity of kinase inhibitors, it could be advantageous to control the reactivity of these electrophiles with light. Here, we introduce covalent inhibitors of the kinase JNK3 that function as photoswitchable affinity labels (PALs). Our lead compounds contain a diazocine photoswitch, are poor non-covalent inhibitors in the dark, and become effective covalent inhibitors after irradiation with visible light. Our proposed mode of action is supported by X-ray structures that explain why these compounds are unreactive in the dark and undergo proximity-based covalent attachment following exposure to light.

Selective mono-de-O-acetylation of the per-O-acetylated brasilicardin carbohydrate side chain.

Methanol dried over powdered 4 Å molecular sieves can be used for a selective mono-de-O-acetylation of the phenolic acetyl group of the per-O-acetyl protected brasilicardin A carbohydrate side chain. This reaction opens a practical procedure for a synthetic access to derivates of the immunosuppressive and cytotoxic natural product brasilicardin A.

Genetic Engineering in Combination with Semi-Synthesis Leads to a New Route for Gram-Scale Production of the Immunosuppressive Natural Product Brasilicardin†A.

Brasilicardin A (1) consists of an unusual anti/syn/anti-perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non-pathogenic bacterial strains producing brasilicardin A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A (1 a) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A.

A novel LysR-type regulator negatively affects biosynthesis of the immunosuppressant brasilicardin.

Brasilicardin A (BraA) is a promising immunosuppressive compound produced naturally by the pathogenic bacterium Nocardia terpenica IFM 0406. Heterologous host expression of brasilicardin gene cluster showed to be efficient to bypass the safety issues, low production levels and lack of genetic tools related with the use of native producer. Further improvement of production yields requires better understanding of gene expression regulation within the BraA biosynthetic gene cluster (Bra-BGC); however, the only so far known regulator of this gene cluster is Bra12. In this study, we discovered the protein LysRNt, a novel member of the LysR-type transcriptional regulator family, as a regulator of the Bra-BGC. Using in vitro approaches, we identified the gene promoters which are controlled by LysRNt within the Bra-BGC. Corresponding genes encode enzymes involved in BraA biosynthesis as well as the key Bra-BGC regulator Bra12. Importantly, we provide in vivo evidence that LysRNt negatively affects production of brasilicardin congeners in the heterologous host Amycolatopsis japonicum. Finally, we demonstrate that some of the pathway related metabolites, and their chemical analogs, can interact with LysRNt which in turn affects its DNA-binding activity.

1-{3-[(7-Fluoro-9H-pyrimido[4,5-b]indol-4-yl)(meth-yl)amino]-piperidin-1-yl}propan-1-one.

The title compound, C19H22FN5O, has been synthesized as an inhibitor of glycogen synthase kinase-3ß. Two mol-ecules inter-act via two N-H⋯N hydrogen bonds, forming centrosymmetric dimers.

Discovery and Evaluation of Enantiopure 9H-pyrimido[4,5-b]indoles as Nanomolar GSK-3ß Inhibitors with Improved Metabolic Stability.

Glycogen synthase kinase-3ß (GSK-3ß) is a potential target in the field of Alzheimer's disease drug discovery. We recently reported a new class of 9H-pyrimido[4,5-b]indole-based GSK-3ß inhibitors, of which 3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile (1) demonstrated promising inhibitory potency. However, this compound underwent rapid degradation by human liver microsomes. Starting from 1, we prepared a series of amide-based derivatives and studied their structure-activity relationships against GSK-3ß supported by 1 µs molecular dynamics simulations. The biological potency of this series was substantially enhanced by identifying the eutomer configuration at the stereocenter. Moreover, the introduction of an amide bond proved to be an effective strategy to eliminate the metabolic hotspot. The most potent compounds, (R)-3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)-3-oxopropanenitrile ((R)-2) and (R)-1-(3-((7-bromo-9Hpyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propan-1-one ((R)-28), exhibited IC50 values of 480 nM and 360 nM, respectively, and displayed improved metabolic stability. Their favorable biological profile is complemented by minimal cytotoxicity and neuroprotective properties.

(2S,3'S,3a'R,5'R,7a'R)-5'-[(E)-5-(Furan-3-yl)-2-methyl-pent-1-en-1-yl]-3-hy-droxy-3',4,7'-trimethyl-1',2',3',3a',5',7a'-hexa-hydro-5H-spiro-[furan-2,4'-inden]-5-one.

The title compound, ircinianin, C25H32O4, belongs to the sesterterpene tetronic acid compound family and was isolated from the marine sponge Ircinia wistarii. These chemical scaffolds are pharmacologically relevant, since they represent a new class of glycine receptor modulators. The furan ring makes a dihedral angle of 35.14 (12)° to the 4-hy-droxy-3-methyl-furan-2(5H)-one ring. The crystal packing is characterized by inter-molecular O-H⋯O hydrogen bonds, which generate [010] chains.

Pyridinylimidazoles as GSK3ß Inhibitors: The Impact of Tautomerism on Compound Activity via Water Networks.

Glycogen synthase kinase-3ß (GSK3ß) is involved in many pathological conditions and represents an attractive drug target. We previously reported dual GSK3ß/p38α mitogen-activated protein kinase inhibitors and identified N-(4-(4-(4-fluorophenyl)-2-methyl-1H-imidazol-5-yl)pyridin-2-yl)cyclopropanecarboxamide (1) as a potent dual inhibitor of both target kinases. In this study, we aimed to design selective GSK3ß inhibitors based on our pyridinylimidazole scaffold. Our efforts resulted in several novel and potent GSK3ß inhibitors with IC50 values in the low nanomolar range. 5-(2-(Cyclopropanecarboxamido)pyridin-4-yl)-4-cyclopropyl-1H-imidazole-2-carboxamide (6g) displayed very good kinase selectivity as well as metabolical stability and inhibited GSK3ß activity in neuronal SH-SY5Y cells. Interestingly, we observed the importance of the 2-methylimidazole's tautomeric state for the compound activity. Finally, we reveal how this crucial tautomerism effect is surmounted by imidazole-2-carboxamides, which are able to stabilize the binding via enhanced water network interactions, regardless of their tautomeric state.

Design, Synthesis and Biological Evaluation of 7-Chloro-9H-pyrimido[4,5-b]indole-based Glycogen Synthase Kinase-3ß Inhibitors.

Glycogen synthase kinase-3ß (GSK-3ß) represents a relevant drug target for the treatment of neurodegenerative pathologies including Alzheimer's disease. We herein report on the optimization of a novel class of GSK-3ß inhibitors based on the tofacitinib-derived screen hit 3-((3R,4R)-3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile (1). We synthesized a series of 19 novel 7-chloro-9H-pyrimido[4,5-b]indole-based derivatives and studied their structure-activity relationships with focus on the cyanoacetyl piperidine moiety. We unveiled the crucial role of the nitrile group and its importance for the activity of this compound series. A successful rigidization approach afforded 3-(3aRS,7aSR)-(1-(7-chloro-9H-pyrimido[4,5-b]indol-4-yl)octahydro-6H-pyrrolo[2,3-c]pyridin-6-yl)-propanenitrile (24), which displayed an IC50 value of 130 nM on GSK-3ß and was further characterized by its metabolic stability. Finally, we disclosed the putative binding modes of the most potent inhibitors within the ATP binding site of GSK-3ß by 1 µs molecular dynamics simulations.

Pyridinylimidazoles as dual glycogen synthase kinase 3ß/p38α mitogen-activated protein kinase inhibitors.

Compounds simultaneously inhibiting two targets that are involved in the progression of the same complex disease may exhibit additive or even synergistic therapeutic effects. Here we unveil 2,4,5-trisubstituted imidazoles as dual inhibitors of p38α mitogen-activated protein kinase and glycogen synthase kinase 3ß (GSK3ß). Both enzymes are potential therapeutic targets for neurodegenerative disorders, like Alzheimer's disease. A set of 39 compounds was synthesized and evaluated in kinase activity assays for their ability to inhibit both target kinases. Among the synthesized compounds, potent dual-target-directed inhibitors showing IC50 values down to the low double-digit nanomolar range, were identified. One of the best balanced dual inhibitors presented in here is N-(4-(2-ethyl-4-(4-fluorophenyl)-1H-imidazol-5-yl)pyridin-2-yl)cyclopropanecarboxamide (20c) (p38α, IC50 = 16 nM; GSK3ß, IC50 = 35 nM) featuring an excellent metabolic stability and an appreciable isoform selectivity over the closely related GSK3α. Our findings were rationalized by computational docking studies based on previously published X-ray structures.

Switching Between Bicyclic and Linear Peptides - The Sulfhydryl-Specific Linker TPSMB Enables Reversible Cyclization of Peptides.

Phage display-selected bicyclic peptides have already shown their great potential for the development as bioactive modulators of therapeutic targets. They can provide enhanced proteolytic stability and improved membrane permeability. Molecular design of new linker molecules has led to a variety of new synthetic approaches for the generation of chemically constrained cyclic peptides. This diversity can be useful for the development of novel peptide-based therapeutic, diagnostic, and scientific tools. Herein, we introduce 1,3,5-tris((pyridin-2-yldisulfanyl)methyl)benzene (TPSMB) as a planar, trivalent, sulfhydryl-specific linker that facilitates reversible cyclization and linearization via disulfide bond formation and cleavage of bicyclic peptides of the format CXnCXnC, where X is any proteinogenic amino acid except cysteine. The rapid and highly sulfhydryl-specific reaction of TPSMB under physiological conditions is demonstrated by selecting bicyclic peptide binders against c-Jun N-terminal kinase 3 (JNK3) as a model target. While model peptides remain stably cyclized for several hours in presence of typical blood levels of glutathione in vitro, high cytosolic concentrations of glutathione linearize these peptides completely within 1 h. We propose that reversible linkers can be useful tools for several technical applications where target affinity depends on the bicyclic structure of the peptide.

The Symmetric Tetravalent Sulfhydryl-Specific Linker NATBA Facilitates a Combinatorial "Tool Kit" Strategy for Phage Display-Based Selection of Functionalized Bicyclic Peptides.

The rigid conformation of constrained bicyclic peptides provides a number of advantages over larger protein-based ligands, including better chemical stability, enhanced tissue penetration, and a wider field of possible applications. Selective chemical modification strategies are able to extend the scope of applications not only in a therapeutic manner but also for the development of novel tools for protein capturing, bioimaging, and targeted drug delivery. Herein, we report the synthesis of an adamantane-based, symmetrical, tetravalent, sulfhydryl-specific peptide linker. We have developed an in vitro two-step modification strategy that allows the generation of differently functionalized bicyclic peptides. This "tool kit" strategy was applied to cyclize and functionalize a phage-encoded peptide library bearing the sequence CX6CX6C. After phage display against a model target, isolated peptides show strong consensus sequences, indicating target-specific binding. The newly developed symmetric tetravalent linker opens new avenues for the combinatorial selection and functionalization of bicyclic peptide ligands with affinity to virtually any target.

Structural Optimization of a Pyridinylimidazole Scaffold: Shifting the Selectivity from p38α Mitogen-Activated Protein Kinase to c-Jun N-Terminal Kinase 3.

Starting from known p38α mitogen-activated protein kinase (MAPK) inhibitors, a series of inhibitors of the c-Jun N-terminal kinase (JNK) 3 was obtained. Altering the substitution pattern of the pyridinylimidazole scaffold proved to be effective in shifting the inhibitory activity from the original target p38α MAPK to the closely related JNK3. In particular, a significant improvement for JNK3 selectivity could be achieved by addressing the hydrophobic region I with a small methyl group. Furthermore, additional structural modifications permitted to explore structure-activity relationships. The most potent inhibitor 4-(4-methyl-2-(methylthio)-1H-imidazol-5-yl)-N-(4-morpholinophenyl)pyridin-2-amine showed an IC50 value for the JNK3 in the low triple digit nanomolar range and its binding mode was confirmed by X-ray crystallography.

Probing Substituents in the 1- and 3-Position: Tetrahydropyrazino-Annelated Water-Soluble Xanthine Derivatives as Multi-Target Drugs With Potent Adenosine Receptor Antagonistic Activity.

Tetrahydropyrazino-annelated theophylline (1,3-dimethylxanthine) derivatives have previously been shown to display increased water-solubility as compared to the parent xanthines due to their basic character. In the present study, we modified this promising scaffold by replacing the 1,3-dimethyl residues by a variety of alkyl groups including combinations of different substituents in both positions. Substituted benzyl or phenethyl residues were attached to the N8 of the resulting 1,3-dialkyl-tetrahydropyrazino[2,1-f ]purinediones with the aim to obtain multi-target drugs that block human A1 and A2A adenosine receptors (ARs) and monoaminoxidase B (MAO-B). 1,3-Diethyl-substituted derivatives showed high affinity for A1 ARs, e.g., 15d (PSB-18339, 8-m-bromobenzyl-substituted) displayed a Ki value of 13.6 nM combined with high selectivity. 1-Ethyl-3-propargyl-substituted derivatives exhibited increased A2A AR affinity. The 8-phenethyl derivative 20h was selective for the A2A AR (Ki 149 nM), while the corresponding 8-benzyl-substituted compound 20e (PSB-1869) blocked A1 and A2A ARs with equal potency (Ki A1, 180 nM; A2A, 282 nM). The 1-ethyl-3-methyl-substituted derivative 16a (PSB-18405) bearing a m,p-dichlorobenzyl residue at N8 blocked all three targets, A1 ARs (Ki 396 nM), A2A ARs (Ki 1,620 nM), and MAO-B (IC50 106 nM) with high selectivity vs. the other subtypes (A2B and A3 ARs, MAO-A), and can thus be considered as a multi-target drug. Our findings were rationalized by molecular docking studies based on previously published X-ray structures of the protein targets. The new drugs have potential for the treatment of neurodegenerative diseases, in particular Parkinson's disease.