A Fast-Binding, Functionally Reversible, COX-2 Radiotracer for CNS PET Imaging.

Cyclooxygenase-2 (COX-2) is an enzyme that plays a pivotal role in peripheral inflammation and pain via the prostaglandin pathway. In the central nervous system (CNS), COX-2 is implicated in neurodegenerative and psychiatric disorders as a potential therapeutic target and biomarker. However, clinical studies with COX-2 have yielded inconsistent results, partly due to limited mechanistic understanding of how COX-2 activity relates to CNS pathology. Therefore, developing COX-2 positron emission tomography (PET) radiotracers for human neuroimaging is of interest. This study introduces [11C]BRD1158, which is a potent and uniquely fast-binding, selective COX-2 PET radiotracer. [11C]BRD1158 was developed by prioritizing potency at COX-2, isoform selectivity over COX-1, fast binding kinetics, and free fraction in the brain. Evaluated through in vivo PET neuroimaging in rodent models with human COX-2 overexpression, [11C]BRD1158 demonstrated high brain uptake, fast target-engagement, functional reversibility, and excellent specific binding, which is advantageous for human imaging applications. Lastly, post-mortem samples from Huntington's disease (HD) patients and preclinical HD mouse models showed that COX-2 levels were elevated specifically in disease-affected brain regions, primarily from increased expression in microglia. These findings indicate that COX-2 holds promise as a novel clinical marker of HD onset and progression, one of many potential applications of [11C]BRD1158 human PET.

Novel Fluorescence-Based High-Throughput FLIPR Assay Utilizing Membrane-Tethered Genetic Calcium Sensors to Identify T-Type Calcium Channel Modulators.

T-type voltage-gated Ca2+ channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca2+ channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca2+ channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca2+ channels are largely inactivated and unable to open to allow Ca2+ influx at -25 mV, the typical resting membrane potential of the cell lines commonly used in cellular screening assays. To address this issue, we developed cell lines that express Kir2.3 channels to hyperpolarize the membrane potential to -70 mV, thus allowing T-type channels to return to their resting state where they can be subsequently activated by membrane depolarization in the presence of extracellular KCl. Furthermore, to simplify the HTS assay and to reduce reagent cost, we stably expressed a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, that displays superior signal to the background compared to the untethered GCaMP6s or the synthetic Ca2+ sensor Fluo-4AM. Here, we describe a novel GCaMP6s-CAAX-based calcium assay utilizing a high-throughput fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) format that can identify both activators and inhibitors of T-type Ca2+ channels. Lastly, we demonstrate the utility of this novel fluorescence-based assay to evaluate the activities of two distinct G-protein-coupled receptors, thus expanding the use of GCaMP6s-CAAX to a wide range of applications relevant for developing cellular assays in drug discovery.

Exploiting an Asp-Glu "switch" in glycogen synthase kinase 3 to design paralog-selective inhibitors for use in acute myeloid leukemia.

Glycogen synthase kinase 3 (GSK3), a key regulatory kinase in the wingless-type MMTV integration site family (WNT) pathway, is a therapeutic target of interest in many diseases. Although dual GSK3α/ß inhibitors have entered clinical trials, none has successfully translated to clinical application. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent ß-catenin stabilization, are a concern in the translation of this target class because mutations and overexpression of ß-catenin are associated with many cancers. Knockdown of GSK3α or GSK3ß individually does not increase ß-catenin and offers a conceptual resolution to targeting GSK3: paralog-selective inhibition. However, inadequate chemical tools exist. The design of selective adenosine triphosphate (ATP)-competitive inhibitors poses a drug discovery challenge due to the high homology (95% identity and 100% similarity) in this binding domain. Taking advantage of an Asp133→Glu196 "switch" in their kinase hinge, we present a rational design strategy toward the discovery of paralog-selective GSK3 inhibitors. These GSK3α- and GSK3ß-selective inhibitors provide insights into GSK3 targeting in acute myeloid leukemia (AML), where GSK3α was identified as a therapeutic target using genetic approaches. The GSK3α-selective compound BRD0705 inhibits kinase function and does not stabilize ß-catenin, mitigating potential neoplastic concerns. BRD0705 induces myeloid differentiation and impairs colony formation in AML cells, with no apparent effect on normal hematopoietic cells. Moreover, BRD0705 impairs leukemia initiation and prolongs survival in AML mouse models. These studies demonstrate feasibility of paralog-selective GSK3α inhibition, offering a promising therapeutic approach in AML.

Functionally Biased D2R Antagonists: Targeting the ß-Arrestin Pathway to Improve Antipsychotic Treatment.

Schizophrenia is a severe neuropsychiatric disease that lacks completely effective and safe therapies. As a polygenic disorder, genetic studies have only started to shed light on its complex etiology. To date, the positive symptoms of schizophrenia are well-managed by antipsychotic drugs, which primarily target the dopamine D2 receptor (D2R). However, these antipsychotics are often accompanied by severe side effects, including motoric symptoms. At D2R, antipsychotic drugs antagonize both G-protein dependent (Gαi/o) signaling and G-protein independent (ß-arrestin) signaling. However, the relevant contributions of the distinct D2R signaling pathways to antipsychotic efficacy and on-target side effects (motoric) are still incompletely understood. Recent evidence from mouse genetic and pharmacological studies point to ß-arrestin signaling as the major driver of antipsychotic efficacy and suggest that a ß-arrestin biased D2R antagonist could achieve an additional level of selectivity at D2R, increasing the therapeutic index of next generation antipsychotics. Here, we characterize BRD5814, a highly brain penetrant ß-arrestin biased D2R antagonist. BRD5814 demonstrated good target engagement via PET imaging, achieving efficacy in an amphetamine-induced hyperlocomotion mouse model with strongly reduced motoric side effects in a rotarod performance test. This proof of concept study opens the possibility for the development of a new generation of pathway selective antipsychotics at D2R with reduced side effect profiles for the treatment of schizophrenia.

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors.

The structure-activity and structure-kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11Å channel leading to the Zn(2+) catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.

Inhibitors of Glycogen Synthase Kinase 3 with Exquisite Kinome-Wide Selectivity and Their Functional Effects.

The mood stabilizer lithium, the first-line treatment for bipolar disorder, is hypothesized to exert its effects through direct inhibition of glycogen synthase kinase 3 (GSK3) and indirectly by increasing GSK3's inhibitory serine phosphorylation. GSK3 comprises two highly similar paralogs, GSK3α and GSK3ß, which are key regulatory kinases in the canonical Wnt pathway. GSK3 stands as a nodal target within this pathway and is an attractive therapeutic target for multiple indications. Despite being an active field of research for the past 20 years, many GSK3 inhibitors demonstrate either poor to moderate selectivity versus the broader human kinome or physicochemical properties unsuitable for use in in vitro systems or in vivo models. A nonconventional analysis of data from a GSK3ß inhibitor high-throughput screening campaign, which excluded known GSK3 inhibitor chemotypes, led to the discovery of a novel pyrazolo-tetrahydroquinolinone scaffold with unparalleled kinome-wide selectivity for the GSK3 kinases. Taking advantage of an uncommon tridentate interaction with the hinge region of GSK3, we developed highly selective and potent GSK3 inhibitors, BRD1652 and BRD0209, which demonstrated in vivo efficacy in a dopaminergic signaling paradigm modeling mood-related disorders. These new chemical probes open the way for exclusive analyses of the function of GSK3 kinases in multiple signaling pathways involved in many prevalent disorders.

Therapeutic potential of isoform selective HDAC inhibitors for the treatment of schizophrenia.

Increasing evidence supports a role for epigenetic involvement in some of the neurobiological alterations observed in neurodegenerative and psychiatric disorders including schizophrenia. In particular, there is mounting evidence implicating dysfunction in acetylation status, a chromatin modification mediated in part by HDACs, as a possible contributing factor to certain facets of this debilitating disease. Additional data support the notion that small molecule inhibition of HDACs may provide therapeutic alternatives to treating many of the symptoms associated with schizophrenia, particularly cognitive deficits. However, the development of highly potent and selective inhibitors of the individual HDAC isoforms will be necessary to delineate the associated biological effects and test the feasibility of such an approach for this complex and chronically treated disease. Here, we summarize current evidence for the role of HDAC isoforms in schizophrenia and highlight the state of the art in developing selective inhibitors of these isoforms as a potential treatment for schizophrenia.

Discovery of the first histone deacetylase 6/8 dual inhibitors.

We disclose the first small molecule histone deacetylase (HDAC) inhibitor (3, BRD73954) capable of potently and selectively inhibiting both HDAC6 and HDAC8 despite the fact that these isoforms belong to distinct phylogenetic classes within the HDAC family of enzymes. Our data demonstrate that meta substituents of phenyl hydroxamic acids are readily accommodated upon binding to HDAC6 and, furthermore, are necessary for the potent inhibition of HDAC8.

Potent and selective inhibition of histone deacetylase 6 (HDAC6) does not require a surface-binding motif.

Hydroxamic acids were designed, synthesized, and evaluated for their ability to selectively inhibit human histone deacetylase 6 (HDAC6). Several inhibitors, including compound 14 (BRD9757), exhibited excellent potency and selectivity despite the absence of a surface-binding motif. The binding of these highly efficient ligands for HDAC6 is rationalized via structure-activity relationships. These results demonstrate that high selectivity and potent inhibition of HDAC6 can be achieved through careful choice of linker element only.

A Potent and Selective Quinoxalinone-Based STK33 Inhibitor Does Not Show Synthetic Lethality in KRAS-Dependent Cells.

The KRAS oncogene is found in up to 30% of all human tumors. In 2009, RNAi experiments revealed that lowering mRNA levels of a transcript encoding the serine/threonine kinase STK33 was selectively toxic to KRAS-dependent cancer cell lines, suggesting that small-molecule inhibitors of STK33 might selectively target KRAS-dependent cancers. To test this hypothesis, we initiated a high-throughput screen using compounds in the Molecular Libraries Small Molecule Repository (MLSMR). Several hits were identified, and one of these, a quinoxalinone derivative, was optimized. Extensive SAR studies were performed and led to the chemical probe ML281 that showed low nanomolar inhibition of purified recombinant STK33 and a distinct selectivity profile as compared to other STK33 inhibitors that were reported in the course of these studies. Even at the highest concentration tested (10 µM), ML281 had no effect on the viability of KRAS-dependent cancer cells. These results are consistent with other recent reports using small-molecule STK33 inhibitors. Small molecules having different chemical structures and kinase-selectivity profiles are needed to fully understand the role of STK33 in KRAS-dependent cancers. In this regard, ML281 is a valuable addition to small-molecule probes of STK33.

Macrocyclic Hedgehog Pathway Inhibitors: Optimization of Cellular Activity and Mode of Action Studies.

Macrocyclic Hedgehog (Hh) pathway inhibitors have been discovered with improved potency and maximal inhibition relative to the previously reported macrocycle robotnikinin. Analogues were prepared using a modular and efficient build-couple-pair (BCP) approach, with a ring-closing metathesis step to form the macrocyclic ring. Varying the position of the macrocycle nitrogen and oxygen atoms provided inhibitors with improved activity in cellular assays; the most potent analogue was 29 (BRD-6851), with an IC(50) of 0.4 µM against C3H10T1/2 cells undergoing Hh-induced activation, as measured by Gli1 transcription and alkaline phosphatase induction. Studies with Patched knockout (Ptch(-/-)) cells and competition studies with the Smoothened (Smo) agonists SAG and purmorphamine demonstrate that in contrast to robotnikinin, select analogues are Smo antagonists.

Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria.

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure-activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.

Development of small-molecule probes that selectively kill cells induced to express mutant RAS.

Synthetic lethal screening is a chemical biology approach to identify small molecules that selectively kill oncogene-expressing cell lines with the goal of identifying pathways that provide specific targets against cancer cells. We performed a high-throughput screen of 303,282 compounds from the National Institutes of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) against immortalized BJ fibroblasts expressing HRAS(G12V) followed by a counterscreen of lethal compounds in a series of isogenic cells lacking the HRAS(G12V) oncogene. This effort led to the identification of two novel molecular probes (PubChem CID 3689413, ML162 and CID 49766530, ML210) with nanomolar potencies and 4-23-fold selectivities, which can potentially be used for identifying oncogene-specific pathways and targets in cancer cells.

Unbiased binding assays for discovering small-molecule probes and drugs.

2011 marks the 10-year anniversary of milestone manuscripts describing drafts of the human genome sequence. Over the past decade, a number of new proteins have been linked to disease-many of which fall into classes that have been historically considered challenging from the perspective of drug discovery. Several of these newly associated proteins lack structural information or strong annotation with regard to function, making development of conventional in vitro functional assays difficult. A recent resurgence in the popularity of simple small molecule binding assays has led to new approaches that do not require knowledge of protein structure or function in advance. Here we briefly review selected methods for executing binding assays that have been used successfully to discover small-molecule probes or drug candidates.

Chemoenzymatic synthesis of uridine diphosphate-GlcNAc and uridine diphosphate-GalNAc analogs for the preparation of unnatural glycosaminoglycans.

Eight N-acetylglucosamine-1-phosphate and N-acetylgalactosamine-1-phosphate analogs have been synthesized chemically and were tested for their recognition by the GlmU uridyltransferase enzyme. Among these, only substrates that have an amide linkage to the C-2 nitrogen were transferred by GlmU to afford their corresponding uridine diphosphate(UDP)-sugar nucleotides. Resin-immobilized GlmU showed comparable activity to nonimmobilized GlmU and provides a more facile final step in the synthesis of an unnatural UDP-donor. The synthesized unnatural UDP-donors were tested for their activity as substrates for glycosyltransferases in the preparation of unnatural glycosaminoglycans in vitro. A subset of these analogs was useful as donors, increasing the synthetic repertoire for these medically important polysaccharides.

A novel HDAC inhibitor with a hydroxy-pyrimidine scaffold.

Histone deacetylases (HDACs) are enzymes involved in many important biological functions. They have been linked to a variety of cancers, psychiatric disorders, and other diseases. Since small molecules can serve as probes to study the relevant biological roles of HDACs, novel scaffolds are necessary to develop more efficient, selective drug candidates. Screening libraries of molecules may yield structurally diverse probes that bind these enzymes and modulate their functions in cells. Here we report a small molecule with a novel hydroxy-pyrimidine scaffold that inhibits multiple HDAC enzymes and modulates acetylation levels in cells. Analogs were synthesized in an effort to evaluate structure-activity relationships.

Chemoenzymatic design of heparan sulfate oligosaccharides.

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C(5)-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.

Inhibition of human vascular NADPH oxidase by apocynin derived oligophenols.

Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors, resulting in a trimer hydroxylated quinone (IIIHyQ) that inhibited NADPH oxidase with an IC(50)=31nM. Apocynin itself possessed minimal inhibitory activity. NADPH oxidase is believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47(phox) and p22(phox). To that end, while apocynin was unable to block the interaction of his-tagged p47(phox) with a surface immobilized biotinylated p22(phox) peptide, the IIIHyQ product strongly interfered with this interaction (apparent IC(50)=1.6microM). These results provide evidence that peroxidase-generated AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase.

Synthesis and Biological Evaluation of Non-Hydrolizable 1,2,3-Triazole Linked Sialic Acid Derivatives as Neuraminidase Inhibitors.

α-Sialic acid azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazole derivatives of sialic acid using the copper-catalyzed azide-alkyne Huisgen cycloaddition ("click chemistry"). Our approach is to generate non-natural N-glycosides of sialic acid that are resistant to neuraminidase catalyzed hydrolysis as opposed to the natural O-glycosides. These N-glycosides would act as neuraminidase inhibitors to prevent the release of new virions. As a preliminary study, a small library of 1,2,3-triazole-linked sialic acid derivatives has been synthesized in 71-89% yield. A disaccharide mimic of sialic acid has also been prepared using the α-sialic acid azide 1 and a C-8 propargyl sialic acid acceptor in 68% yield. A model sialic acid coated dendrimer was also synthesized from a per-propargylated pentaerythritol acceptor. These novel sialic acid derivatives were then evaluated as potential neuraminidase inhibitors using a 96-well plate fluorescence assay; micromolar IC50 values were observed, comparable to the known sialidase inhibitor Neu5Ac2en.

Synthesis of uridine 5'-diphosphoiduronic acid: a potential substrate for the chemoenzymatic synthesis of heparin.

An improved understanding of the biological activities of heparin requires structurally defined heparin oligosaccharides. The chemoenzymatic synthesis of heparin oligosaccharides relies on glycosyltransferases that use UDP-sugar nucleotides as donors. Uridine 5'-diphosphoiduronic acid (UDP-IdoA) and uridine 5'-diphosphohexenuronic acid (UDP-HexUA) have been synthesized as potential analogues of uridine 5'-diphosphoglucuronic acid (UDP-GlcA) for enzymatic incorporation into heparin oligosaccharides. Non-natural UDP-IdoA and UDP-HexUA were tested as substrates for various glucuronosyltransferases to better understand enzyme specificity.