Structural Basis for Highly Selective Class II Alpha Phosphoinositide-3-Kinase Inhibition.

Class II phosphoinositide-3-kinases (PI3Ks) play central roles in cell signaling, division, migration, and survival. Despite evidence that all PI3K class II isoforms serve unique cellular functions, the lack of isoform-selective inhibitors severely hampers the systematic investigation of their potential relevance as pharmacological targets. Here, we report the structural evaluation and molecular determinants for selective PI3K-C2α inhibition by a structure-activity relationship study based on a pteridinone scaffold, leading to the discovery of selective PI3K-C2α inhibitors called PITCOINs. Cocrystal structures and docking experiments supported the rationalization of the structural determinants essential for inhibitor activity and high selectivity. Profiling of PITCOINs in a panel of more than 118 diverse kinases showed no off-target kinase inhibition. Notably, by addressing a selectivity pocket, PITCOIN4 showed nanomolar inhibition of PI3K-C2α and >100-fold selectivity in a general kinase panel. Our study paves the way for the development of novel therapies for diseases related to PI3K-C2α function.

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.

An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor.

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the αC helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases.

Discovery of tetrazolo-pyridazine-based small molecules as inhibitors of MACC1-driven cancer metastasis.

Metastasis is directly linked to poor prognosis of cancer patients and warrants search for effective anti-metastatic drugs. MACC1 is a causal key molecule for metastasis. High MACC1 expression is prognostic for metastasis and poor survival. Here, we developed novel small molecule inhibitors targeting MACC1 expression to impede metastasis formation. We performed a human MACC1 promoter-driven luciferase reporter-based high-throughput screen (HTS; 118.500 compound library) to identify MACC1 transcriptional inhibitors. HTS revealed 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds as efficient transcriptional inhibitors of MACC1 expression, able to decrease MACC1-induced cancer cell motility in vitro. Structure-activity relationships identified the essential inhibitory core structure. Best candidates were evaluated for metastasis inhibition in xenografted mouse models demonstrating metastasis restriction. ADMET showed high drug-likeness of these new candidates for cancer therapy. The NFκB pathway was identified as one mode of action targeted by these compounds. Taken together, 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds are effective MACC1 inhibitors and pose promising candidates for anti-metastatic therapies particularly for patients with MACC1-overexpressing cancers, that are at high risk to develop metastases. Although further preclinical and clinical development is necessary, these compounds represent important building blocks for an individualized anti-metastatic therapy for solid cancers.

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.

Role of endothelial cells and angiotensin converting enzyme-II in COVID-19 and brain damages post-infection.

Severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) causes coronavirus disease 2019 (COVID-19), which became a pandemic in late 2019 and early 2020. Apart from many other symptoms of this infection, such as loss of smell and taste, rashes, body aches, fatigue, and psychological and cardiac symptoms, it also causes vasodilation in response to inflammation via nitric oxide release. SARS CoV-2 affects microcirculation, resulting in the swelling and damage of endothelial cells, micro thrombosis, constriction of capillaries, and damage to pericytes that are vital for the integrity of capillaries, angiogenesis, and the healing process. Cytokine storming has been associated with COVID-19 illness. Capillary damage and congestion may cause limited diffusion exchange of oxygen in the lungs and hence hypoxemia and tissue hypoxia occur. This perspective study will explore the involvement of capillary damage and inflammation by their interference with blood and tissue oxygenation as well as brain function in the persistent symptoms and severity of COVID-19. The overall effects of capillary damage due to COVID-19, microvascular damage, and hypoxia in vital organs are also discussed in this perspective. Once initiated, this vicious cycle causes inflammation due to hypoxia, resulting in limited capillary function, which in turn causes inflammation and tissue damage. Low oxygen levels and high cytokines in brain tissue may lead to brain damage. The after-effects may be in the form of psychological symptoms such as mood changes, anxiety, depression, and many others that need to be investigated.

Large-scale microRNA functional high-throughput screening identifies miR-515-3p and miR-519e-3p as inducers of human cardiomyocyte proliferation.

Ischemic cardiomyopathy, driven by loss of cardiomyocytes and inadequate proliferative response, persists to be a major global health problem. Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-CM. Whereas miR-inhibitors failed to enhance EdU uptake, overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the primate-specific C19MC-cluster. Two of these miRNAs, miR-515-3p and miR-519e-3p, increased markers of early and late mitosis, indicative of cell division, and substantially alter signaling pathways relevant for cardiomyocyte proliferation in hiPSC-CM.

Small molecule inhibiting microglial nitric oxide release could become a potential treatment for neuroinflammation.

Microglia are the immune effector cells of the central nervous system (CNS) and react to pathologic events with a complex process including the release of nitric oxide (NO). NO is a free radical, which is toxic for all cells at high concentrations. To target an exaggerated NO release, we tested a library of 16 544 chemical compounds for their effect on lipopolysaccharide (LPS)-induced NO release in cell line and primary neonatal microglia. We identified a compound (C1) which significantly reduced NO release in a dose-dependent manner, with a low IC50 (252 nM) and no toxic side effects in vitro or in vivo. Target finding strategies such as in silico modelling and mass spectroscopy hint towards a direct interaction between C1 and the nitric oxide synthase making C1 a great candidate for specific intra-cellular interaction with the NO producing machinery.

Development of selective inhibitors of phosphatidylinositol 3-kinase C2α.

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.

Glycolytic flux control by drugging phosphoglycolate phosphatase.

Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.

TSG101 associates with PARP1 and is essential for PARylation and DNA damage-induced NF-κB activation.

In a genome-wide screening for components of the dsDNA-break-induced IKK-NF-κB pathway, we identified scores of regulators, including tumor susceptibility gene TSG101. TSG101 is essential for DNA damage-induced formation of cellular poly(ADP-ribose) (PAR). TSG101 binds to PARP1 and is required for PARP1 activation. This function of TSG101 is independent of its role in the ESCRT-I endosomal sorting complex. In the absence of TSG101, the PAR-dependent formation of a nuclear PARP1-IKKγ signalosome, which triggers IKK activation, is impaired. According to its requirement for PARP1 and NF-κB activation, TSG101-deficient cells are defective in DNA repair and apoptosis protection. Loss of TSG101 results in PARP1 trapping at damage sites and mimics the effect of pharmacological PARP inhibition. We also show that the loss of TSG101 in connection with inactivated tumor suppressors BRCA1/2 in breast cancer cells is lethal. Our results imply TSG101 as a therapeutic target to achieve synthetic lethality in cancer treatment.

Structure-Based Design of Xanthine-Benzimidazole Derivatives as Novel and Potent Tryptophan Hydroxylase Inhibitors.

Tryptophan hydroxylases catalyze the first and rate-limiting step in the synthesis of serotonin. Serotonin is a key neurotransmitter in the central nervous system and, in the periphery, functions as a local hormone with multiple physiological functions. Studies in genetically altered mouse models have shown that dysregulation of peripheral serotonin levels leads to metabolic, inflammatory, and fibrotic diseases. Overproduction of serotonin by tumor cells causes severe symptoms typical for the carcinoid syndrome, and tryptophan hydroxylase inhibitors are already in clinical use for patients suffering from this disease. Here, we describe a novel class of potent tryptophan hydroxylase inhibitors, characterized by spanning all active binding sites important for catalysis, specifically those of the cosubstrate pterin, the substrate tryptophan as well as directly chelating the catalytic iron ion. The inhibitors were designed to efficiently reduce serotonin in the periphery while not passing the blood-brain barrier, thus preserving serotonin levels in the brain.

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.

From Pyrazolones to Azaindoles: Evolution of Active-Site SHP2 Inhibitors Based on Scaffold Hopping and Bioisosteric Replacement.

The tyrosine phosphatase SHP2 controls the activity of pivotal signaling pathways, including MAPK, JAK-STAT, and PI3K-Akt. Aberrant SHP2 activity leads to uncontrolled cell proliferation, tumorigenesis, and metastasis. SHP2 signaling was recently linked to drug resistance against cancer medications such as MEK and BRAF inhibitors. In this work, we present the development of a novel class of azaindole SHP2 inhibitors. We applied scaffold hopping and bioisosteric replacement concepts to eliminate unwanted structural motifs and to improve the inhibitor characteristics of the previously reported pyrazolone SHP2 inhibitors. The most potent azaindole 45 inhibits SHP2 with an IC50 = 0.031 µM in an enzymatic assay and with an IC50 = 2.6 µM in human pancreas cells (HPAF-II). Evaluation in a series of cellular assays for metastasis and drug resistance demonstrated efficient SHP2 blockade. Finally, 45 inhibited proliferation of two cancer cell lines that are resistant to cancer drugs and diminished ERK signaling.

Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP.

Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells through regulation of the water channel aquaporin-2 (AQP2). The hormone binds to vasopressin V2 receptors (V2R) on the surface of the cells and stimulates cAMP synthesis. The cAMP activates protein kinase A (PKA), which initiates signaling that causes an accumulation of AQP2 in the plasma membrane of the cells facilitating water reabsorption from primary urine and fine-tuning of body water homeostasis. AVP-mediated PKA activation also causes an increase in the AQP2 protein abundance through a mechanism that involves dephosphorylation of AQP2 at serine 261 and a decrease in its poly-ubiquitination. However, the signaling downstream of PKA that controls the localization and abundance of AQP2 is incompletely understood. We carried out an siRNA screen targeting 719 kinase-related genes, representing the majority of the kinases of the human genome and analyzed the effect of the knockdown on AQP2 by high-content imaging and biochemical approaches. The screening identified 13 hits whose knockdown inhibited the AQP2 accumulation in the plasma membrane. Amongst the candidates was the so far hardly characterized cyclin-dependent kinase 18 (CDK18). Our further analysis revealed a hitherto unrecognized signalosome comprising CDK18, an E3 ubiquitin ligase, STUB1 (CHIP), PKA and AQP2 that controls the localization and abundance of AQP2. CDK18 controls AQP2 through phosphorylation at serine 261 and STUB1-mediated ubiquitination. STUB1 functions as an A-kinase anchoring protein (AKAP) tethering PKA to the protein complex and bridging AQP2 and CDK18. The modulation of the protein complex may lead to novel concepts for the treatment of disorders which are caused or are associated with dysregulated AQP2 and for which a satisfactory treatment is not available, e.g., hyponatremia, liver cirrhosis, diabetes insipidus, ADPKD or heart failure.

A Semi-automated Organoid Screening Method Demonstrates Epigenetic Control of Intestinal Epithelial Differentiation.

Intestinal organoids are an excellent model to study epithelial biology. Yet, the selection of analytical tools to accurately quantify heterogeneous organoid cultures remains limited. Here, we developed a semi-automated organoid screening method, which we applied to a library of highly specific chemical probes to identify epigenetic regulators of intestinal epithelial biology. The role of epigenetic modifiers in adult stem cell systems, such as the intestinal epithelium, is still undefined. Based on this resource dataset, we identified several targets that affected epithelial cell differentiation, including HDACs, EP300/CREBBP, LSD1, and type I PRMTs, which were verified by complementary methods. For example, we show that inhibiting type I PRMTs, which leads enhanced epithelial differentiation, blocks the growth of adenoma but not normal organoid cultures. Thus, epigenetic probes are powerful tools to study intestinal epithelial biology and may have therapeutic potential.

Identification of TMEM206 proteins as pore of PAORAC/ASOR acid-sensitive chloride channels.

Acid-sensing ion channels have important functions in physiology and pathology, but the molecular composition of acid-activated chloride channels had remained unclear. We now used a genome-wide siRNA screen to molecularly identify the widely expressed acid-sensitive outwardly-rectifying anion channel PAORAC/ASOR. ASOR is formed by TMEM206 proteins which display two transmembrane domains (TMs) and are expressed at the plasma membrane. Ion permeation-changing mutations along the length of TM2 and at the end of TM1 suggest that these segments line ASOR's pore. While not belonging to a gene family, TMEM206 has orthologs in probably all vertebrates. Currents from evolutionarily distant orthologs share activation by protons, a feature essential for ASOR's role in acid-induced cell death. TMEM206 defines a novel class of ion channels. Its identification will help to understand its physiological roles and the diverse ways by which anion-selective pores can be formed.

A New Highly Thyrotropin Receptor-Selective Small-Molecule Antagonist with Potential for the Treatment of Graves' Orbitopathy.

BACKGROUND: The thyrotropin receptor (TSHR) is the target for autoimmune thyroid stimulating antibodies (TSAb) triggering hyperthyroidism. Whereas elevated thyroid hormone synthesis by the thyroid in Graves' disease can be treated by antithyroid agents, for the pathogenic activation of TSHR in retro-orbital fibroblasts of the eye, leading to Graves' orbitopathy (GO), no causal TSHR directed therapy is available. METHODS: Due to the therapeutic gap for severe GO, TSHR inhibitors were identified by high-throughput screening in Chinese hamster ovary cells expressing the TSHR. Stereo-selective synthesis of the screening hits led to the molecule S37, which contains seven chiral centers. Enantiomeric separation of the molecule S37 resulted in the enantiopure molecule S37a-a micro-molar antagonist of thyrotropin-induced cyclic adenosine monophosphate accumulation in HEK 293 cells expressing the TSHR. RESULTS: The unique rigid bent shape of molecule S37a may mediate the observed high TSHR selectivity. Most importantly, the closely related follitropin and lutropin receptors were not affected by this compound. S37a not only inhibits the TSHR activation by thyrotropin itself but also activation by monoclonal TSAb M22 (human), KSAb1 (murine), and the allosteric small-molecule agonist C2. Disease-related ex vivo studies in HEK 293 cells expressing the TSHR showed that S37a also inhibits cyclic adenosine monophosphate formation by oligoclonal TSAb, which are highly enriched in GO patients' sera. Initial in vivo pharmacokinetic studies revealed no toxicity of S37a and a remarkable 53% oral bioavailability in mice. CONCLUSION: In summary, a novel highly selective inhibitor for the TSHR is presented, which has promising potential for further development for the treatment of GO.