DOTA-based plectin-1 targeted contrast agent enables detection of pancreatic cancer in human tissue.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal malignancy with extremely poor patient survival rates. A key reason for the poor prognosis is the lack of effective diagnostic tools to detect the disease at curable, premetastatic stages. Tumor surgical resection is PDAC first-line treatment, however distinguishing between cancerous and healthy tissue with current imaging tools remains a challenge. In this work, we report a DOTA-based fluorescent probe targeting plectin-1 for imaging PDAC with high specificity. To enable heterogeneous functionalization of the DOTA-core with multiple targeting peptide units and the fluorophore, a novel, fully clickable synthetic route that proceeds in one-pot was developed. Extensive validation of the probe set the stage for PDAC detection in mice and human tissue. Altogether, these findings may pave the way for improved clinical understanding and early detection of PDAC progression as well as more accurate resection criteria.

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.

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.

CLK2 and CLK4 are regulators of DNA damage-induced NF-κB targeted by novel small molecule inhibitors.

Transcription factor NF-κB potently activates anti-apoptotic genes, and its inactivation significantly reduces tumor cell survival following genotoxic stresses. We identified two structurally distinct lead compounds that selectively inhibit NF-κB activation by DNA double-strand breaks, but not by other stimuli, such as TNFα. Our compounds do not directly inhibit previously identified regulators of this pathway, most critically including IκB kinase (IKK), but inhibit signal transmission in-between ATM, PARP1, and IKKγ. Deconvolution strategies, including derivatization and in vitro testing in multi-kinase panels, yielded shared targets, cdc-like kinase (CLK) 2 and 4, as essential regulators of DNA damage-induced IKK and NF-κB activity. Both leads sensitize to DNA damaging agents by increasing p53-induced apoptosis, thereby reducing cancer cell viability. We propose that our lead compounds and derivatives can be used in context of genotoxic therapy-induced or ongoing DNA damage to increase tumor cell apoptosis, which may be beneficial in cancer treatment.

Isoform-selective targeting of PI3K: time to consider new opportunities?

Phosphoinositide-3-kinases (PI3Ks) are central to several cellular signaling pathways in human physiology and are potential pharmacological targets for many pathologies including cancer, thrombosis, and pulmonary diseases. Tremendous efforts to develop isoform-selective inhibitors have culminated in the approval of several drugs, validating PI3K as a tractable and therapeutically relevant target. Although successful therapeutic validation has focused on isoform-selective class I orthosteric inhibitors, recent clinical findings have indicated challenges regarding poor drug tolerance owing to sustained on-target inhibition. Hence, additional approaches are warranted to increase the clinical benefits of specific clinical treatment options, which may involve the employment of so far underexploited targeting modalities or the development of inhibitors for currently underexplored PI3K class II isoforms. We review recent key discoveries in the development of isoform-selective inhibitors, focusing particularly on PI3K class II isoforms, and highlight the emerging importance of developing a broader arsenal of pharmacological tools.

Selective and pH-Independent Detection of Ba2+ in Water by a Benzo-21-crown-7-Functionalized BODIPY.

Herein, we report on highly Ba2+ selective fluorescence sensing in water by a fluorescent probe consisting of a benzo-21-crown-7 as a Ba2+ binding unit (ionophore) and a tetramethylated BODIPY fluorophore as a fluorescence reporter. This fluorescent probe showed a Ba2+ induced fluorescence enhancement (FE) by a factor of 12±1 independently of the pH value and a high Ba2+ sensitivity with a limit of detection (LOD) of (17.2±0.3) µM. Moreover, a second fluorescent probe consisting of the same BODIPY fluorophore, but a benzo-18-crown-6 as a cation-responsive binding moiety, showed an even higher FE upon Ba2+ complexation by a factor of 85±3 and a lower LOD of (13±3) µM albeit a lower Ba2+ selectivity. The fluorescence sensing mechanism of Ba2+ was further investigated by time-resolved fluorescence as well as transient absorption spectroscopy (TAS) and it turned out that within these probes a blocking of a photoinduced electron transfer (PET) by Ba2+ is very likely responsible for the FE.

Aß Chronic Exposure Promotes an Activation State of Microglia through Endocannabinoid Signalling Imbalance.

Dysfunctional phenotype of microglia, the primary brain immune cells, may aggravate Alzheimer's disease (AD) pathogenesis by releasing proinflammatory factors, such as nitric oxide (NO). The endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) are bioactive lipids increasingly recognised for their essential roles in regulating microglial activity both under normal and AD-driven pathological conditions. To investigate the possible impact of chronic exposure to ß-amyloid peptides (Aß) on the microglial endocannabinoid signalling, we characterised the functional expression of the endocannabinoid system on neonatal microglia isolated from wild-type and Tg2576 mice, an AD-like model, which overexpresses Aß peptides in the developing brain. We found that Aß-exposed microglia produced 2-fold more 2-AG than normal microglia. Accordingly, the expression levels of diacylglycerol lipase-α (DAGLα) and monoacylglycerol lipase (MAGL), the main enzymes responsible for synthesising and hydrolysing 2-AG, respectively, were consistently modified in Tg2576 microglia. Furthermore, compared to wild-type cells, transgenic microglia basally showed increased expression of the cannabinoid 2 receptor, typically upregulated in an activated proinflammatory phenotype. Indeed, following inflammatory stimulus, Aß-exposed microglia displayed an enhanced production of NO, which was abolished by pharmacological inhibition of DAGLα. These findings suggested that exposure to Aß polarises microglial cells towards a pro-AD phenotype, possibly by enhancing 2-AG signalling.

First in vivo fluorine-19 magnetic resonance imaging of the multiple sclerosis drug siponimod.

Theranostic imaging methods could greatly enhance our understanding of the distribution of CNS-acting drugs in individual patients. Fluorine-19 magnetic resonance imaging (19F MRI) offers the opportunity to localize and quantify fluorinated drugs non-invasively, without modifications and without the application of ionizing or other harmful radiation. Here we investigated siponimod, a sphingosine 1-phosphate (S1P) receptor antagonist indicated for secondary progressive multiple sclerosis (SPMS), to determine the feasibility of in vivo 19F MR imaging of a disease modifying drug. Methods: The 19F MR properties of siponimod were characterized using spectroscopic techniques. Four MRI methods were investigated to determine which was the most sensitive for 19F MR imaging of siponimod under biological conditions. We subsequently administered siponimod orally to 6 mice and acquired 19F MR spectra and images in vivo directly after administration, and in ex vivo tissues. Results: The 19F transverse relaxation time of siponimod was 381 ms when dissolved in dimethyl sulfoxide, and substantially reduced to 5 ms when combined with serum, and to 20 ms in ex vivo liver tissue. Ultrashort echo time (UTE) imaging was determined to be the most sensitive MRI technique for imaging siponimod in a biological context and was used to map the drug in vivo in the stomach and liver. Ex vivo images in the liver and brain showed an inhomogeneous distribution of siponimod in both organs. In the brain, siponimod accumulated predominantly in the cerebrum but not the cerebellum. No secondary 19F signals were detected from metabolites. From a translational perspective, we found that acquisitions done on a 3.0 T clinical MR scanner were 2.75 times more sensitive than acquisitions performed on a preclinical 9.4 T MR setup when taking changes in brain size across species into consideration and using equivalent relative spatial resolution. Conclusion: Siponimod can be imaged non-invasively using 19F UTE MRI in the form administered to MS patients, without modification. This study lays the groundwork for more extensive preclinical and clinical investigations. With the necessary technical development, 19F MRI has the potential to become a powerful theranostic tool for studying the time-course and distribution of CNS-acting drugs within the brain, especially during pathology.

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.

Identification of the Allosteric Binding Site for Thiazolopyrimidine on the C-Type Lectin Langerin.

Langerin is a mammalian C-type lectin expressed on Langerhans cells in the skin. As an innate immune cell receptor, Langerin is involved in coordinating innate and adaptive immune responses against various incoming threats. We have previously reported a series of thiazolopyrimidines as murine Langerin ligands. Prompted by the observation that its human homologue exhibits different binding specificities for these small molecules, we report here our investigations to define their exact binding site. By using structural comparison and molecular dynamics simulations, we showed that the nonconserved short loops have a high degree of conformational flexibility between the human and murine homologues. Sequence analysis and mutational studies indicated that a pair of residues are essential for the recognition of the thiazolopyrimidines. Taking solvent paramagnetic relaxation enhancement NMR studies together with a series of peptides occupying the same site, we could define the cleft between the short and long loops as the allosteric binding site for these aromatic heterocycles.

Probing Factor Xa Protein-Ligand Interactions: Accurate Free Energy Calculations and Experimental Validations of Two Series of High-Affinity Ligands.

The accurate prediction of protein-ligand binding affinity belongs to one of the central goals in computer-based drug design. Molecular dynamics (MD)-based free energy calculations have become increasingly popular in this respect due to their accuracy and solid theoretical basis. Here, we present a combined study which encompasses experimental and computational studies on two series of factor Xa ligands, which enclose a broad chemical space including large modifications of the central scaffold. Using this integrated approach, we identified several new ligands with different heterocyclic scaffolds different from the previously identified indole-2-carboxamides that show superior or similar affinity. Furthermore, the so far underexplored terminal alkyne moiety proved to be a suitable non-classical bioisosteric replacement for the higher halogen-π aryl interactions. With this challenging example, we demonstrated the ability of the MD-based non-equilibrium free energy calculation approach for guiding crucial modifications in the lead optimization process, such as scaffold replacement and single-site modifications at molecular interaction hot spots.

Antagonistic control of active surface integrins by myotubularin and phosphatidylinositol 3-kinase C2ß in a myotubular myopathy model.

X-linked centronuclear myopathy (XLCNM) is a severe human disease without existing therapies caused by mutations in the phosphoinositide 3-phosphatase MTM1. Loss of MTM1 function is associated with muscle fiber defects characterized by impaired localization of ß-integrins and other components of focal adhesions. Here we show that defective focal adhesions and reduced active ß-integrin surface levels in a cellular model of XLCNM are rescued by loss of phosphatidylinositiol 3-kinase C2ß (PI3KC2ß) function. Inactivation of the Mtm1 gene impaired myoblast differentiation into myotubes and resulted in reduced surface levels of active ß1-integrins as well as corresponding defects in focal adhesions. These phenotypes were rescued by concomitant genetic loss of Pik3c2b or pharmacological inhibition of PI3KC2ß activity. We further demonstrate that a hitherto unknown role of PI3KC2ß in the endocytic trafficking of active ß1-integrins rather than rescue of phosphatidylinositol 3-phosphate levels underlies the ability of Pik3c2b to act as a genetic modifier of cellular XLCNM phenotypes. Our findings reveal a crucial antagonistic function of MTM1 and PI3KC2ß in the control of active ß-integrin surface levels, thereby providing a molecular mechanism for the adhesion and myofiber defects observed in XLCNM. They further suggest specific pharmacological inhibition of PI3KC2ß catalysis as a viable treatment option for XLCNM patients.

An LPAR 5 -antagonist that reduces nociception and increases pruriception.

Introduction: The G-protein coupled receptor LPAR5 plays a prominent role in LPA-mediated pain and itch signaling. In this study we focus on the LPAR5-antagonist compound 3 (cpd3) and its ability to affect pain and itch signaling, both in vitro and in vivo. Methods: Nociceptive behavior in wild type mice was induced by formalin, carrageenan or prostaglandin E2 (PGE2) injection in the hind paw, and the effect of oral cpd3 administration was measured. Scratch activity was measured after oral administration of cpd3, in mice overexpressing phospholipase A2 ( sPLA 2 tg ), in wild type mice (WT) and in TRPA1-deficient mice (Trpa1 KO). In vitro effects of cpd3 were assessed by measuring intracellular calcium release in HMC-1 and HEK-TRPA1 cells. Results: As expected, nociceptive behavior (induced by formalin, carrageenan or PGE2) was reduced after treatment with cpd3. Unexpectedly, cpd3 induced scratch activity in mice. In vitro addition of cpd3 to HEK-TRPA1 cells induced an intracellular calcium wave that could be inhibited by the TRPA1-antagonist A-967079. In Trpa1 KO mice, however, the increase in scratch activity after cpd3 administration was not reduced. Conclusions: Cpd3 has in vivo antinociceptive effects but induces scratch activity in mice, probably by activation of multiple pruriceptors, including TRPA1. These results urge screening of antinociceptive candidate drugs for activity with pruriceptors.

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.

Weizhouochrones: Gorgonian-Derived Symmetric Dimers and Their Structure Elucidation Using Anisotropic NMR Combined with DP4+ Probability and CASE-3D.

Natural product dimers have intriguing structural features and often have remarkable pharmacological activities. We report here two uncommon marine gorgonian-derived symmetric dimers, weizhouochrones A (1) and B (2), with indenone-derived monomers, that were isolated from the coral Anthogorgia ochracea collected from the South China Sea. These dimers are difficult targets for structure elucidation that solely relies upon conventional NMR data such as NOEs and J-couplings. Here, to explore the application of emerging methods on the structure elucidation of challenging molecules, we explored a number of different anisotropic and computational NMR approaches. The measurements of anisotropic NMR parameters of weizhouochrone A, including residual dipolar couplings (RDCs) and residual chemical shift anisotropy (RCSA), allowed us to successfully determine the planar structure and its relative configuration. This result was corroborated by a computational NMR analysis based on DP4+ probability and computer-assisted 3D structure elucidation (CASE-3D).

Compound Interaction Screen on a Photoactivatable Cellulose Membrane (CISCM) Identifies Drug Targets.

Identifying the protein targets of drugs is an important but tedious process. Existing proteomic approaches enable unbiased target identification but lack the throughput needed to screen larger compound libraries. Here, we present a compound interaction screen on a photoactivatable cellulose membrane (CISCM) that enables target identification of several drugs in parallel. To this end, we use diazirine-based undirected photoaffinity labeling (PAL) to immobilize compounds on cellulose membranes. Functionalized membranes are then incubated with protein extract and specific targets are identified via quantitative affinity purification and mass spectrometry. CISCM reliably identifies known targets of natural products in less than three hours of analysis time per compound. In summary, we show that combining undirected photoimmobilization of compounds on cellulose with quantitative interaction proteomics provides an efficient means to identify the targets of natural products.

Donor manipulation for constructing a pH sensing thermally activated delayed fluorescent probe to detect alkaliphiles.

pH homeostasis is essential for alkaliphiles, given their widespread use in biotechnological applications. However, quantitative monitoring of alkaline pH in alkaliphiles remains challenging. Here, we synthesized for the first time, a thermally activated delayed fluorescent (TADF) pH probe: NI-D-OH. Our probe exhibits a good linear relationship between fluorescence intensity and pH in the neutral to alkaline range (pH 7.0-8.6), as well as long-lived TADF emission. We further show that NI-D-OH can be used to monitor intracellular pH in living organisms, and evaluate the effect of Na+ on the pH homeostasis, demonstrating the potential for alkaline pH monitoring and time-resolved fluorescence imaging.

Phosphoinositide Conversion Inactivates R-RAS and Drives Metastases in Breast Cancer.

Breast cancer is the most prevalent cancer and a major cause of death in women worldwide. Although early diagnosis and therapeutic intervention significantly improve patient survival rate, metastasis still accounts for most deaths. Here it is reported that, in a cohort of more than 2000 patients with breast cancer, overexpression of PI3KC2α occurs in 52% of cases and correlates with high tumor grade as well as increased probability of distant metastatic events, irrespective of the subtype. Mechanistically, it is demonstrated that PI3KC2α synthetizes a pool of PI(3,4)P2 at focal adhesions that lowers their stability and directs breast cancer cell migration, invasion, and metastasis. PI(3,4)P2 locally produced by PI3KC2α at focal adhesions recruits the Ras GTPase activating protein 3 (RASA3), which inactivates R-RAS, leading to increased focal adhesion turnover, migration, and invasion both in vitro and in vivo. Proof-of-concept is eventually provided that inhibiting PI3KC2α or lowering RASA3 activity at focal adhesions significantly reduces the metastatic burden in PI3KC2α-overexpressing breast cancer, thereby suggesting a novel strategy for anti-breast cancer therapy.