Structure-Based Design of Xanthine-Imidazopyridines and -Imidazothiazoles as Highly Potent and In Vivo Efficacious Tryptophan Hydroxylase Inhibitors.

Tryptophan hydroxylases catalyze the first and rate-limiting step in the biosynthesis of serotonin, a well-known neurotransmitter that plays an important role in multiple physiological functions. A reduction of serotonin levels, especially in the brain, can cause dysregulation leading to depression or insomnia. In contrast, overproduction of peripheral serotonin is associated with symptoms like carcinoid syndrome and pulmonary arterial hypertension. Recently, we developed a class of TPH inhibitors based on xanthine-benzimidazoles, characterized by a tripartite-binding mode spanning the binding sites of the cosubstrate pterin and the substrate tryptophan and by chelation of the catalytic iron ion. Herein, we describe the structure-based development of a second generation of xanthine-imidiazopyridines and -imidazothiazoles designed to inhibit TPH1 in the periphery while preventing the interaction with TPH2 in the brain. Lead compound 32 (TPT-004) shows superior pharmacokinetic and pharmacodynamic properties as well as efficacy in preclinical models of peripheral serotonin attenuation and colorectal tumor growth.

Small Molecules Targeting Human UDP-GlcNAc 2-Epimerase.

Uridine diphosphate N-acetylglucosamine 2-epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high-throughput screening campaign of 68,640 drug-like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real-time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion, and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems.

Disruptors of AKAP-Dependent Protein-Protein Interactions.

A-kinase anchoring proteins (AKAPs) are a family of multivalent scaffolding proteins. They engage in direct protein-protein interactions with protein kinases, kinase substrates and further signaling molecules. Each AKAP interacts with a specific set of protein interaction partners and such sets can vary between different cellular compartments and cells. Thus, AKAPs can coordinate signal transduction processes spatially and temporally in defined cellular environments. AKAP-dependent protein-protein interactions are involved in a plethora of physiological processes, including processes in the cardiovascular, nervous, and immune system. Dysregulation of AKAPs and their interactions is associated with or causes widespread diseases, for example, cardiac diseases such as heart failure. However, there are profound shortcomings in understanding functions of specific AKAP-dependent protein-protein interactions. In part, this is due to the lack of agents for specifically targeting defined protein-protein interactions. Peptidic and non-peptidic inhibitors are invaluable molecular tools for elucidating the functions of AKAP-dependent protein-protein interactions. In addition, such interaction disruptors may pave the way to new concepts for the treatment of diseases where AKAP-dependent protein-protein interactions constitute potential drug targets.Here we describe screening approaches for the identification of small molecule disruptors of AKAP-dependent protein-protein interactions. Examples include interactions of AKAP18 and protein kinase A (PKA) and of AKAP-Lbc and RhoA. We discuss a homogenous time-resolved fluorescence (HTRF) and an AlphaScreen® assay for small molecule library screening and human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) as a cell system for the characterization of identified hits.

Aurora Kinase A Is Involved in Controlling the Localization of Aquaporin-2 in Renal Principal Cells.

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown.

Pentafluorosulfanyl (SF5) as a Superior 19F Magnetic Resonance Reporter Group: Signal Detection and Biological Activity of Teriflunomide Derivatives.

Fluorine (19F) magnetic resonance imaging (MRI) is severely limited by a low signal-to noise ratio (SNR), and tapping it for 19F drug detection in vivo still poses a significant challenge. However, it bears the potential for label-free theranostic imaging. Recently, we detected the fluorinated dihydroorotate dehydrogenase (DHODH) inhibitor teriflunomide (TF) noninvasively in an animal model of multiple sclerosis (MS) using 19F MR spectroscopy (MRS). In the present study, we probed distinct modifications to the CF3 group of TF to improve its SNR. This revealed SF5 as a superior alternative to the CF3 group. The value of the SF5 bioisostere as a 19F MRI reporter group within a biological or pharmacological context is by far underexplored. Here, we compared the biological and pharmacological activities of different TF derivatives and their 19F MR properties (chemical shift and relaxation times). The 19F MR SNR efficiency of three MRI methods revealed that SF5-substituted TF has the highest 19F MR SNR efficiency in combination with an ultrashort echo-time (UTE) MRI method. Chemical modifications did not reduce pharmacological or biological activity as shown in the in vitro dihydroorotate dehydrogenase enzyme and T cell proliferation assays. Instead, SF5-substituted TF showed an improved capacity to inhibit T cell proliferation, indicating better anti-inflammatory activity and its suitability as a viable bioisostere in this context. This study proposes SF5 as a novel superior 19F MR reporter group for the MS drug teriflunomide.

Design and synthesis of novel fluorescently labeled analogs of vemurafenib targeting MKK4.

The mitogen-activated protein kinase kinase 4 (MKK4) plays a key role in liver regeneration and is under investigation as a target for stimulating hepatocytes to increased proliferation. Therefore, new small molecules inhibiting MKK4 may represent a promising approach for treating acute and chronic liver diseases. Fluorescently labeled compounds are useful tools for high-throughput screenings of large compound libraries. Here we utilized the azaindole-based scaffold of FDA-approved BRAF inhibitor vemurafenib 1, which displays off-target activity on MKK4, as a starting point in our fluorescent compound design. Chemical variation of the scaffold and optimization led to a selection of fluorescent 5-TAMRA derivatives which possess high binding affinities on MKK4. Compound 45 represents a suitable tool compound for Fluorescence polarization assays to identify new small-molecule inhibitors of MKK4.

Vinylphosphonites for Staudinger-induced chemoselective peptide cyclization and functionalization.

In this paper, we introduce vinylphosphonites for chemoselective Staudinger-phosphonite reactions (SPhR) with azides to form vinylphosphonamidates for the subsequent modification of cysteine residues in peptides and proteins. An electron-rich alkene is turned into an electron-deficient vinylphosphonamidate, thereby inducing electrophilic reactivity for a following thiol addition. We show that by varying the phosphonamidate ester substituent we can fine-tune the reactivity of the thiol addition and even control the functional properties of the final conjugate. Furthermore, we observed a drastic increase in thiol addition efficiency when the SPhR is carried out in the presence of a thiol substrate in a one-pot reaction. Hence, we utilize vinylphosphonites for the chemoselective intramolecular cyclization of peptides carrying an azide-containing amino acid and a cysteine in high yields. Our concept was demonstrated for the stapling of a cell-permeable peptidic inhibitor for protein-protein interaction (PPI) between BCL9 and beta-catenin, which is known to create a transcription factor complex playing a role in embryonic development and cancer origin, and for macrocyclization of cell-penetrating peptides (CPPs) to enhance the cellular uptake of proteins.

An AKAP-Lbc-RhoA interaction inhibitor promotes the translocation of aquaporin-2 to the plasma membrane of renal collecting duct principal cells.

Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking.

Small Molecules Targeting Human N-Acetylmannosamine Kinase.

N-Acetylmannosamine kinase (MNK) plays a key role in the biosynthesis of sialic acids and glycosylation of proteins. Sialylated glycoconjugates affect a large number of biological processes, including immune modulation and cancer transformation. In search of effective inhibitors of MNK we applied high-throughput screening of drug-like small molecules. By applying different orthogonal assays for their validation we identified four potential MNK-specific inhibitors with IC50 values in the low-micromolar range. Molecular modelling of the inhibitors into the active site of MNK supports their binding to the sugar or the ATP-binding pocket of the enzyme or both. These compounds are promising for downregulation of the sialic acid content of glycoconjugates and for studying the functional contribution of sialic acids to disease development.