Parasitic diseases continue to have a devastating impact on human populations worldwide. Lack of effective treatments, the high cost of existing ones, and frequent emergence of resistance to these agents provide a strong argument for the development of novel therapies. Here we report the results of a hybrid approach designed to obtain a dual acting molecule that would demonstrate activity against a variety of parasitic targets. The antimalarial drug amodiaquine has been covalently joined with a nitric oxide-releasing furoxan to achieve multiple mechanisms of action. Using in vitro and ex vivo assays, the hybrid molecule shows activity against three parasites – Plasmodium falciparum, Schistosoma mansoni, and Ancylostoma ceylanicum.
A total synthesis of LL-Z1640-2 (2), a potent and selective kinase inhibitor, has been completed. The key step of the convergent synthesis utilized a late-stage intramolecular Nozaki-Hiyama-Kishi (NHK) reaction to close the macrocycle at the C6′-C7′ bond.
Small molecule kinase inhibitors are important tools for studying cellular signaling pathways, phenotypes and are, occasionally, useful clinical agents. With stereochemistry pervasive throughout the molecules of life it is no surprise that a single stereocenter can bestow a ligand with distinct binding affinities to various protein targets. While the majority of small molecule kinase inhibitors reported to date are achiral, a number of asymmetric compounds show great utility as tools for probing kinase-associated biomolecular events as well as promising therapeutic leads. The mechanism by which chirality is introduced varies but includes screening of chiral libraries, incorporation of chiral centers during optimization efforts and the rational installation of a chiral moiety as guided by structural and modeling efforts. Here we discuss several advanced chiral small molecule kinase inhibitors where stereochemistry plays an important role in terms of potency and selectivity.
GPR40, free fatty acid receptor 1 (FFAR1), is a member of the GPCR superfamily and a possible target for the treatment of type 2 diabetes. In this work we conducted a bi-directional iterative investigation, including computational modeling and site-directed mutagenesis, aimed at delineating amino acid residues forming the functional “chemoprint” of GPR40 for agonist recognition. The computational and experimental studies revolved around the recognition of the potent synthetic agonist GW9508. Our experimentally supported model suggested that H137(4.56), R183(5.39), N244(6.55), and R258(7.35) are directly involved in interactions with the ligand. We have proposed a polarized NH - π interaction between H137(4.56) and GW9508 as one of the contributing forces leading to the high potency of GW9508. The modeling approach presented in this work provides a general strategy for the exploration of receptor-ligand interactions in GPCRs beginning prior to acquisition of experimental data.
Herein we examine the potential of a nitrile-containing proprionic acid moiety as an electrophile for covalent attack by the active site cysteine residue of caspase 1. The syntheses of several cyanopropanate containing small molecules based upon the optimized peptidic scaffold of the prodrug VX-765 were accomplished and found to be potent inhibitors of caspase 1 (IC50s ≤ 1 nM). Examination of these novel small molecules versus a caspase panel demonstrated an impressive degree of selectivity for caspase 1 inhibition. Assessment of hydrolytic stability and selected ADME properties highlighted these agents as potentially useful tools for studying caspase 1 down-regulation in various settings including in vivo analyses.
Inhibitor; enzymes; prodrugs; peptides; caspase 1 inhibitor; Cysteine proteases; Caspase 1; VX-765; VRT-043198; covalent modifiers,; nitrile caspase inhibitors
The NIH Chemical Genomics Center (NCGC) was the inaugural center of the Molecular Libraries and Screening Center Network (MLSCN). Along with the nine other research centers of the MLSCN, the NCGC was established with a primary goal of bringing industrial technology and experience to empower the scientific community with small molecule compounds for use in their research. We intend this review to serve as 1) an introduction to the NCGC standard operating procedures, 2) an overview of several of the lessons learned during the pilot phase and 3) a review of several of the innovative discoveries reported during the pilot phase of the MLSCN.
Diketopiperazines (DKPs) are a common motif in various biologically active natural products, and hence they may be useful scaffolds for the rational design of receptor probes and therapeutic agents. We constructed a new bicyclic scaffold that combines a DKP bridged with a 10-membered ring. In this way we obtained a three-dimensional molecular skeleton, with several amendable sites that provide a starting point to design a new combinatorial library having diverse substituent groups. Structural variation is based upon the flexibility of alkylation of the nitrogen atoms of the DKP and on the side-chain olefin. We obtained a 10-membered secondary ring through a ring-closure metathesis reaction using the second generation Grubbs catalyst. Rings containing both O-ethers and S-ethers were compared. N-Alkyl or arylalkyl groups were introduced optionally at the two Nα-atoms. This is a general scheme that will allow us to test rings of varying sizes, linkages, and stereochemical parameters. The DKP derivatives were tested for activity in astrocytoma cells expressing receptors coupled to phospholipase C. Inhibitory effects were observed for signaling elicited by activation of human nucleotide P2Y receptors but not m3 muscarinic receptors. Compound 20 selectively inhibited calcium mobilization (IC50 value of 486 ± 16 nM) and phosphoinositide turnover elicited by a selective P2Y1 receptor agonist, but this compound did not compete for binding of a radiolabeled nucleotide-competitive receptor antagonist. Therefore, the new class of DKP derivatives shows utility as pharmacological tools for P2Y receptors.
The dedifferentiation agent ‘reversine’ (2-(4-morpholinoanilino)-N6-cyclohexyladenine 2) was found to be a moderately potent antagonist for the human A3 adenosine receptor (AR) with a Ki value 0.66 μM. This result prompted an exploration of the structure-activity relationship of related derivatives, synthesized via sequential substitution of 6-chloro-2-fluoropurine with selected nucleophiles. Optimization of substituents at these two positions identified 2-phenylamino-N6-(cyclohexyl)adenine 12, 2-phenylamino-N6-(cycloheptyl)adenine 19, and 2-phenylamino-N6-(endo-norbornyl)adenine 21 as potent A3 AR ligands with Ki values of 51, 42 and 37 nM, respectively, with 30 – 200-fold selectivity in comparison to A1 and A2A ARs. The most selective A3 AR antagonist (>200-fold) was 2-phenyloxy-N6-(cyclohexyl)adenine 22. 9-Methylation of 12, but not 19, was well tolerated in A3 AR binding. Extension of the 2-phenylamino group to 2-benzyl- and 2-(2-phenylethylamino) reduced affinity. In the series of 2-phenylamino, 2-phenyloxy, and 2-phenylthio substitutions, the order of affinity at the A3 AR was oxy ≥ amino > thio. Selected derivatives, including reversine (KB value of 466 nM in Schild analysis), competitively antagonized the functional effects of a selective A3 AR agonist, i.e. inhibition of forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells. These results are in agreement with other studies suggesting the presence of a lipophilic pocket in the AR binding site that is filled by moderately sized cycloalkyl rings at the N6 position of both adenine and adenosine derivatives. Thus, the compound series reported herein comprise an important new series of selective A3 AR antagonists. We were unable to reproduce the dedifferentiation effect of reversine, previously reported, or to demonstrate any connection between A3 AR antagonist effects and dedifferentiation.
Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys358. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys358 to Ser358 oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.
Substitutions on the 2-position of the imidizole ring of histamine have proven useful in a number of biochemical settings. Current art for the synthesis of these constructs relies upon a cumbersome and low-yielding condensation reaction. Here-in we report a new procedure for the synthesis of a series of substituted 2-phenylhistamines utilizing a microwave-promoted Suzuki coupling.
heteroaromatic Suzuki coupling; microwave promoted Suzuki coupling; 2-phenylhistamine
We herein describe the rapid synthesis of a diverse set of dihydroquinazolin-4-ones and quinazolin-4-ones, their biological evaluation as thyroid stimulating hormone receptor (TSHR) agonists, and SAR analysis. Among the compounds screened, 8b was 60-fold more potent than the hit compound 1a, which was identified from a high throughput screen of over 73,000 compounds.
We measured the “druggability” of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo™, Promega). Quantitative high throughput screening (qHTS) was used to determine IC50’s of 198,899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo™). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC50 < 10 μM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the maximum affinity identified in the lucPpy qHTS was 50 nM while for Kinase-Glo this value increased to 600 nM. Compounds with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compound interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quantitative analysis of structure-activity relationships (>100K compounds) in addressing important questions such as a target's druggability.
chemical profiling; enzyme assay; PubChem; luciferase; quantitative high-throughput screening
Tasigna® (Nilotinib) is a recently approved BCR-ABL kinase inhibitor by the Food and Drug Administration, which is indicated for the treatment of drug-resistant chronic myelogenous leukemia (CML). The efflux of tyrosine kinase inhibitors by ATP-binding cassette (ABC) drug transporters, which actively pump these drugs out of cells utilizing ATP as an energy source, has been linked to the development of drug resistance in CML patients. We report here synthesis and characterization of a fluorescent derivative of Tasigna to study its interaction with two major ABC transporters, P-glycoprotein (Pgp) and ABCG2, in in vitro and ex vivo assays. A fluorescent derivative of Tasigna, BODIPY® FL Tasigna, inhibited the BCR-ABL kinase activity in K562 cells and was also effluxed by Pgp- and ABCG2-expressing cells in both cultured cells and rat brain capillaries expressing Pgp and ABCG2. In addition, [3H]-Tasigna was also found to be transported by Pgp-expressing polarized LLC-PK1 cells in a transepithelial transport assay. Consistent with these results, both Tasigna and BODIPY® FL Tasigna were less effective at inhibiting the phosphorylation of Crkl (a substrate of BCR-ABL kinase) in Pgp- and ABCG2-expressing K562 cells due to their reduced intracellular concentration. Taken together, these data provide evidence that BODIPY® FL Tasigna is transported by Pgp and ABCG2, and Tasigna is transported by Pgp. Further, we propose that BODIPY® FL Tasigna can potentially be used as a probe to study Tasigna in imaging Pgp- and/or ABCG2- expressing cancer cells and other preclinical studies.
ABC transporter; ABCG2; BCR-ABL kinase; multidrug resistance; P-glycoprotein; Tasigna
The α-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one) potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating α-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified α-hydroxytropolones exhibit antiviral activity at non-cytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogs can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved α-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use.
An integrin found on platelets, αIIbβ3 mediates platelet aggregation, and αIIbβ3 antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg2+) located in the β subunit metal ion–dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the αIIb subunit and a carboxyl group that coordinates the MIDAS Mg2+ in the β3 subunits. They induce conformational changes in the β3 subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of αIIbβ3 (RUC-1) that binds exclusively to the αIIb subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β3 as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2–IIbβ3 headpiece complex in 1 mM calcium ion (Ca2+)/5 mM Mg2+ at 2.6 Å revealed that RUC-2 binds to αIIb the way RUC-1 does, but in addition, it binds to the β3 MIDAS residue glutamic acid 220, thus displacing Mg2+ from the MIDAS. When the Mg2+ concentration was increased to 20 mM, however, Mg2+ was identified in the MIDAS and RUC-2 was absent. RUC-2′s ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg2+ concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other αIIbβ3 antagonists and may offer advantages as a therapeutic agent.
Kinetic solubility measurements using prototypical assay buffer conditions are presented for a ~58,000 member library of small molecules. Analyses of the data based upon physical and calculated properties of each individual molecule were performed and resulting trends were considered in the context of commonly held opinions of how physicochemical properties influence aqueous solubility. We further analyze the data using a decision tree model for solubility prediction and via a multi-dimensional assessment of physicochemical relationships to solubility in the context of specific ‘rule-breakers’ relative to common dogma. The role of solubility as a determinant of assay outcome is also considered based upon each compound’s cross-assay activity score for a collection of publicly available screening results. Further, the role of solubility as a governing factor for colloidal aggregation formation within a specified assay setting is examined and considered as a possible cause of a high cross-assay activity score. The results of this solubility profile should aid chemists during library design and optimization efforts and represents a useful training set for computational solubility prediction.
Continued examination of substituted 6-arylquinazolin-4-amines as Clk4 inhibitors resulted in selective inhibitors of Clk1, Clk4, Dyrk1A and Dyrk1B. Several of the most potent inhibitors were validated as being highly selective within a comprehensive kinome scan.
Clk1; Clk2; Clk3; Clk4; Dyrk1A; Dyrk1B; Pre-mRNA splicing; kinase inhibition; quinazoline
Pleasure-seeking deficits, including lack of libido, are a core feature of depression. Animal and preliminary clinical studies both suggest that phosphodiesterase 4 (PDE4) is a target for developing novel antidepressants. This study examined the potential involvement of PDE4 in the pathology of depression in both animal models and human postmortem brains. In humans, PDE4B and PDE4D levels were elevated in cingulate cortical tissue from individuals with major depressive disorder (MDD) compared to controls. Using the female urine smelling test (FUST), a recently refined method for monitoring sexual pleasure-seeking activity in mice, we found that icv infusion of novel, selective, and potent PDE4 inhibitors enhanced sexual pleasure-seeking activity in male mice that underwent the learned helplessness or serotonin depletion paradigms. The infusion also increased sexual pleasure-seeking activity in naïve male mice. The results suggest that PDE4 may be a plausible contributor to the sexual pleasure-seeking deficits seen in depressed patients; inhibiting PDE4 may restore these deficits.
PDE4; postmortem human brain; depression; pleasure-seeking activity
Chromosome band 9p24 is frequently amplified in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (HL). To identify oncogenes in this amplicon, we screened an RNA interference library targeting amplicon genes and thereby identified JAK2 and the histone demethylase JMJD2C as essential genes in these lymphomas. Inhibition of JAK2 and JMJD2C cooperated in killing these lymphomas by decreasing tyrosine 41 phosphorylation and increasing lysine 9 trimethylation of histone H3, promoting heterochromatin formation. MYC, a major target of JAK2-mediated histone phosphorylation, was silenced following JAK2 and JMJD2C inhibition, with a corresponding increase in repressive chromatin. Hence, JAK2 and JMJD2C cooperatively remodel the PMBL and HL epigenome, offering a mechanistic rationale for the development of JAK2 and JMJD2C inhibitors in these diseases.
Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described.
PKM2; pyruvate kinase; cellular metabolism; anti-cancer strategies; small molecule activators
Transforming growth factor–β (TGFβ) signaling drives aneurysm progression in multiple disorders, including Marfan syndrome (MFS), and therapies that inhibit this signaling cascade are in clinical trials. TGFβ can stimulate multiple intracellular signaling pathways, but it is unclear which of these pathways drives aortic disease and, when inhibited, which result in disease amelioration. Here we show that extracellular signal–regulated kinase (ERK) 1 and 2 and Smad2 are activated in a mouse model of MFS, and both are inhibited by therapies directed against TGFβ. Whereas selective inhibition of ERK1/2 activation ameliorated aortic growth, Smad4 deficiency exacerbated aortic disease and caused premature death in MFS mice. Smad4-deficient MFS mice uniquely showed activation of Jun N-terminal kinase–1 (JNK1), and a JNK antagonist ameliorated aortic growth in MFS mice that lacked or retained full Smad4 expression. Thus, noncanonical (Smad-independent) TGFβ signaling is a prominent driver of aortic disease in MFS mice, and inhibition of the ERK1/2 or JNK1 pathways is a potential therapeutic strategy for the disease.
Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase (PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells. Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone scaffold. The synthesis of these agents, structure activity relationships, analysis of activity at related targets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent the second reported chemotype for activation of PKM2.
Warburg effect; pyruvate kinase; cellular metabolism; anti-cancer strategies; small molecule activators
The metabolism of cancer cells is altered to support rapid proliferation. Pharmacological activators of a tumor cell specific pyruvate kinase isozyme (PKM2) may be an approach for altering the classic Warburg effect characteristic of aberrant metabolism in cancer cells yielding a novel anti-proliferation strategy. In this manuscript we detail the discovery of a series of substituted N,N′-diarylsulfonamides as activators of PKM2. The synthesis of numerous analogues and the evaluation of structure activity relationships are presented as well as assessments of mechanism and selectivity. Several agents are found that have good potencies and appropriate solubility for use as chemical probes of PKM2 including 55 (AC50 = 43 nM, maximum response = 84%; solubility = 7.3 μg/mL), 56 (AC50 = 99 nM, maximum response = 84%; solubility = 5.7 μg/mL) and 58 (AC50 = 38 nM, maximum response = 82%; solubility = 51.2 μg/mL). The small molecules described here represent first-in-class activators of PKM2
Warburg effect; pyruvate kinase; cellular metabolism; high-throughput screening; small molecule activators
Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas’ disease and African sleeping sickness, respectively. Both parasites rely on essential cysteine proteases for survival, cruzain for T. cruzi and TbCatB/rhodesain for T. brucei. A recent quantitative high-throughput screen of cruzain identified triazine nitriles, which are known inhibitors of other cysteine proteases, as reversible inhibitors of the enzyme. Structural modifications detailed herein, including core scaffold modification from triazine to purine, improved the in vitro potency against both cruzain and rhodesain by 350-fold, while also gaining activity against T. brucei parasites. Selected compounds were screened against a panel of human cysteine and serine proteases to determine selectivity, and a co-crystal was obtained of our most potent analog bound to Cruzain.
A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Synthesis, structure activity-relationships and the selectivity of a potent analogue against a panel of 402 kinases are presented. Inhibition of Clk4 by these agents at varied concentrations of assay substrates (ATP and receptor peptide) highly suggests that this chemotype is an ATP competitive inhibitor. Molecular docking provides further evidence that inhibition is the result of binding at the kinase hinge region. Selected compounds represent novel tools capable of potent and selective inhibition of Clk1, Clk4 and Dyrk1A.
kinase inhibition; pre-mRNA splicing; Clk; Dyrk1A