10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-formyl-DDACTHF): a potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway

The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding gamma- and alpha-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC(50) for 3=60nM) that exceeded their enzyme inhibition potency [K(i) (3)=6 and 1 microM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS(-)) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar K(i)'s versus E. coli GAR Tfase and only modestly enhanced K(i)'s versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding gamma-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (K(i) for 3, 14nM against rhGAR Tfase versus 6 microM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.     

Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as anti-leukemia agents by inhibiting Btk and PI3Kδ

Btk inhibitors and PI3Kδ inhibitors play crucial roles in the treatment of leukemia, and studies confirmed that the synergetic inhibition against Btk and PI3Kδ could gain an optimal response. Herein, a series of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives were designed and synthesized as dual Btk/PI3Kδ kinases inhibitors for the treatment of leukemia. Studies indicated that most compounds could suppress the proliferation of multiple leukemia or lymphoma cells (Raji, HL60 and K562 cells) at low micromolar concentrations in vitro. Further kinase assays identified several compounds could simultaneously inhibit Btk kinase and PI3Kδ kinase. Thereinto, compound 16b exhibited the best inhibitory activity (Btk: IC₅₀?=?139?nM; PI3Kδ: IC₅₀?=?275?nM) and showed some selectivity against PI3Kδ compared to PI3Kβ/γ. Finally, the SAR of target compounds was preliminarily discussed combined with docking results. In brief, 16b possessed of the potency for the further optimization as anti-leukemia drugs by inhibiting simultaneously Btk kinase and PI3Kδ kinase.     

Identification of novel, selective and potent Chk2 inhibitors

A series of isothiazole carboxamidine compounds were synthesized and discovered as novel and selective inhibitors for Chk2. They are not active against the related Chk1 kinase. The structure-activity relationship studies were performed on the scaffold, and enzymatic kinetic analysis showed they are simple ATP competitive inhibitors with K(i) values as low as 11 nM for Chk2. Computer modeling studies were employed to comprehend the mechanism of action and SAR of these compounds.     

Carbonic anhydrase inhibitors. Inhibition of the membrane-bound human and bovine isozymes IV with sulfonamides

An inhibition study of the human and bovine membrane-associated isozymes of carbonic anhydrase (CA, EC 4.2.1.1), hCA IV and bCA IV, with a series of sulfonamides and sulfamates, some of which are widely clinically used, such as acetazolamide, methazolamide, ethoxzolamide, topiramate, dorzolamide, dichlorophenamide, celecoxib, and valdecoxib among others, is reported. In contrast to bCA IV, which is generally strongly inhibited by most of these derivatives, hCA IV has a rather different inhibition profile. Several of these compounds such as acetazolamide, ethoxzolamide, and bromosulfanilamide are potent hCA IV inhibitors (K(i)'s of 74-93 nM), others, such as celecoxib and some halogenated sulfanilamides are medium potency inhibitors (K(i)'s of 450-880 nM) whereas most of them are weak hCA IV inhibitors (methazolamide: 6.2 microM; dorzolamide 8.5 microM; topiramate 4.9 microM; dichlorophenamide: 15.3 microM). The hCA IV/bCA IV inhibition ratios for all the investigated compounds ranged between 1.05 (for acetazolamide) and 198.37 (for dorzolamide). Based on these results, we doubt that hCA IV is indeed one of the main contributors to the intraocular pressure (IOP) lowering effects of sulfonamide CA inhibitors, in addition to hCA II, as hypothesized earlier by Maren et al. (Mol. Pharmacol.1993, 44, 901-906). Indeed, both the very good hCA IV inhibitors (acetazolamide and ethoxzolamide) as well as the quite weak hCA IV inhibitors (methazolamide, dorzolamide, or dichlorophanamide) are effective in lowering IOP when administered either systemically or topically. The membrane-associated isozyme which probably is critical for aqueous humor secretion is hCA XII and not hCA IV.     

Kinetic characterization of novel pyrazole TGF-beta receptor I kinase inhibitors and their blockade of the epithelial-mesenchymal transition

Transforming growth factor beta (TGF-beta) signaling pathways regulate a wide variety of cellular processes including cell proliferation, differentiation, extracellular matrix deposition, development, and apoptosis. TGF-beta type-I receptor (TbetaRI) is the major receptor that triggers several signaling events by activating downstream targets such as the Smad proteins. The intracellular kinase domain of TbetaRI is essential for its function. In this study, we have identified a short phospho-Smad peptide, pSmad3(-3), KVLTQMGSPSIRCSS(PO4)VS as a substrate of TbetaRI kinase for in vitro kinase assays. This peptide is uniquely phosphorylated by TbetaRI kinase at the C-terminal serine residue, the phosphorylation site of its parent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by only TbetaRI and not TGF-beta type-II receptor kinase, indicating that the peptide is a physiologically relevant substrate suitable for kinetic analysis and screening of TbetaRI kinase inhibitors. Utilizing pSmad3(-3) as a substrate, we have shown that novel pyrazole compounds are potent inhibitors of TbetaRI kinase with K(i) value as low as 15 nM. Kinetic analysis revealed that these pyrazoles act through the ATP-binding site and are typical ATP competitive inhibitors with tight binding kinetics. More importantly, these compounds were shown to inhibit TGF-beta-induced Smad2 phosphorylation in vivo in NMuMg mammary epithelial cells with potency equivalent to the inhibitory activity in the in vitro kinase assay. Cellular selectivity analysis demonstrated that these pyrazoles are capable of inhibiting activin signaling but not bone morphogenic protein or platelet-derived growth factor signal transduction pathways. Further functional analysis revealed that pyrazoles are capable of blocking the TGF-beta-induced epithelial-mesenchymal transition in NMuMg cells, a process involved in the progression of cancer, fibrosis, and other human diseases. These pyrazoles provide a foundation for future development of potent and selective TbetaRI kinase inhibitors to treat human disease.     

Non-competitive inhibitors of metabotropic glutamate receptor 5 (mGluR5)

Based on a pharmacophore alignment on known non-competitive mGluR5 inhibitors applying 4SCan technology, a new lead series was identified and further structurally investigated. K(i)'s as low as around 100 nM were achieved.     

Kinetics, in silico docking, molecular dynamics, and MM-GBSA binding studies on prototype indirubins, KT5720, and staurosporine as phosphorylase kinase ATP-binding site inhibitors: the role of water molecules examined

With an aim toward glycogenolysis control in Type 2 diabetes, we have investigated via kinetic experiments and computation the potential of indirubin (IC?? > 50 μM), indirubin-3'-oxime (IC?? = 144 nM), KT5720 (K(i) = 18.4 nM) and staurosporine (K(i) = 0.37 nM) as phosphorylase kinase (PhKγtrnc) ATP-binding site inhibitors, with the latter two revealed as potent inhibitors in the low nM range. Because of lack of structural information, we have exploited information from homologous kinase complexes to direct in silico calculations (docking, molecular dynamics, and MMGBSA) to predict the binding characteristics of the four ligands. All inhibitors are predicted to bind in the same active site area as the ATP adenine ring, with binding dominated by hinge region hydrogen bonds to Asp104:O and Met106:O (all four ligands) and also Met106:NH (for the indirubins). The PhKγtrnc-staurosporine complex has the greatest number of receptor-ligand hydrogen bonds, while for the indirubin-3'-oxime and KT5720 complexes there is an important network of interchanging water molecules bridging inhibitor-enzyme contacts. The MM-GBSA results revealed the source of staurosporine's low nM potency to be favorable electrostatic interactions, while KT5720 has strong van der Waals contributions. KT5720 interacts with the greatest number of protein residues either by direct or 1-water bridged hydrogen bond interactions, and the potential for more selective PhK inhibition based on a KT5720 analogue has been established. Including receptor flexibility in Schr?dinger induced-fit docking calculations in most cases correctly predicted the binding modes as compared with the molecular dynamics structures; the algorithm was less effective when there were key structural waters bridging receptor-ligand contacts.     

Biochemical,cellular and structural characterization of novel and selective ERK3 inhibitors

Triazolo[4,5-d]pyrimidin-5-amines were identified from kinase selectivity screening as novel ERK3 inhibitors with sub-100 nanomolar potencies in a biochemical assay using MK5 as substrate and with an attractive kinase selectivity profile. ERK3 crystal structures clarified the inhibitor binding mode in the ATP pocket with impact on A-loop, GC-loop and αC-helix conformations suggesting a potential structural link towards MK5 interaction via the FHIEDE motif. The inhibitors also showed sub-100 nM potencies in a cellular ERK3 NanoBRET assay and with excellent correlation to the biochemical IC₅₀s. This novel series provides valuable tool compounds to further investigate the biological function and activation mechanism of ERK3.     

Isoindol-1,3-dione and isoindol-1-one derivatives with high binding affinity to beta-amyloid fibrils

Based on the structural features of Indoprofen and PIB, a series of isoindol-1,3-diones 1a-k and isoindol-1-ones 2a-l were designed and synthesized. These 23 compounds were evaluated by competitive binding assay against aggregated Abeta42 fibrils using [(125)I]TZDM. All the isoindolone derivatives showed very good binding affinities with K(i) values in the subnanomolar range (0.42-0.94 nM). Among them, isoindol-1,3-diones 1i and 1k and isoindol-1-ones 2c and 2i exhibited excellent binding affinities (K(i)=0.42-0.44 and 0.46-0.49 nM) than those of Indoprofen (K(i)=0.52 nM) and PIB (K(i)=0.70 nM). These results suggest that isoindolones could be served as a scaffold for potential AD diagnostic probes to monitor Abeta fibrils.     

Discovery of novel selective Janus kinase 2 (JAK2) inhibitors bearing a 1H-pyrazolo[3,4-d]pyrimidin-4-amino scaffold

Janus kinases (JAKs) regulate various cancers and immune responses and are targets for the treatment of cancers and immune diseases. A new series of 1H-pyrazolo[3,4-d]pyrimidin-4-amino derivatives were synthesized and optimized by introducing a functional 3,5-disubstituted-1H-pyrazole moiety into the C-3 moiety of pyrazole template, and then were biologically evaluated as potent Janus kinase 2 (JAK2) inhibitors. Among these molecules, inhibitors 11f, 11g, 11h and 11k displayed strong activity and selectivity against the JAK2 kinase, with IC₅₀ values of 7.2?nM, 6.5?nM, 8.0?nM and 9.7?nM, respectively. In particular, the cellular inhibitory assay and western blot analysis further support the JAK2 selectivity of compound 11g also in cells. Furthermore, compound 11g also exhibited potent inhibitory activity in lymphocytes proliferation assay and delayed hypersensitivity assay. Taken together, the novel JAK2 selective inhibitors discovered in this study may be potential lead compounds for new drug discovery via further development of more potent and selective JAK2 inhibitors.     

2-((1H-Azol-1-yl)methyl)-N-arylbenzamides: novel dual inhibitors of VEGFR-1/2 kinases

Novel potent derivatives of (azol-1-yl)methyl-N-arylbenzamides with improved solubility (>3mM) are described as ATP-competitive inhibitors of vascular endothelial growth factor receptor 2 (VEGFR-2). Many compounds display VEGFR-2 inhibitory activity reaching IC(50)<100 nM in the enzymatic assay. The compounds also inhibit the related tyrosine kinase, VEGFR-1, with similar potencies. Several compounds containing bulky lipophilic substituents at the benzamide pharmacophore yielded 10- to 17-fold selectivity for the VEGFR-2 versus VEGFR-1 kinase.     

Aminopyrimidinone cdc7 kinase inhibitors

We have investigated the SAR of a series of pyrimidinone-containing Cdc7 kinase inhibitors. A wide range of amine substitutions give potent compounds with activities (K(i)) less than 1nM. Kinase selectivity is reasonable and cytotoxicity corresponds to inhibition of MCM2 phosphorylation.     

Discovery of 2-arylthieno[3,2-d]pyrimidines containing 8-oxa-3-azabi-cyclo[3.2.1]octane in the 4-position as potent inhibitors of mTOR with selectivity over PI3K

2-Aryl-4-morpholinothieno[3,2-d]pyrimidines are known PI3K inhibitors. This class of compounds also potently inhibited the homologous enzyme mTOR. Replacement of the morpholine group in these compounds with an 8-oxa-3-azabicyclo[3.2.1]octane group led to mTOR inhibitors with selectivity over PI3K. Optimization of the 2-aryl substituent led to the discovery of 2-(4-ureidophenyl)-thienopyrimidines as highly potent (IC₅₀ <1 nM) mTOR inhibitors with excellent selectivity (up to >1000-fold) over PI3K and good potency in a cellular proliferation assay (IC₅₀ <50 nM).     

A versatile high-throughput screen for inhibitors of lipid kinase activity: development of an immobilized phospholipid plate assay for phosphoinositide 3-kinase gamma

The family of phosphoinositide 3-kinases (PI3K) regulates fundamental cellular responses such as proliferation, apoptosis, motility, and adhesion. In particular, the PI3K gamma isoform plays a critical role in the control of cell migration. Despite the attractiveness of PI3-kinases as drug targets, drug discovery efforts have been hampered by the lack of appropriate lipid kinase assay formats suitable for high-throughput screening. The authors report the development of a simple and robust 384-well plate assay that is based on(33) P-phosphate transfer from radiolabeled [gamma(33) P]ATP to phosphatidylinositol immobilized on Maxisorp plates. The established assay format for PI3K gamma was easily adapted to the automated screening platform and was successfully employed for high-throughput screening. Enzymatic and inhibition characteristics of recombinant human PI3K gamma determined with the plate assay are in very good agreement with previously reported values determined in other assay formats. Maximal catalytic activity of PI3K gamma was observed at pH 7.0. The apparent K(m) value for ATP using a 1:1 mixture of phosphatidylinositol and phosphatidylserine was determined to be 7.3 microM (6.0-8.6 microM, 95% confidence interval [CI]). IC(50) values for known PI3-kinase inhibitors were determined to be 1.45 nM (1.17-1.80 nM, 95% CI) for wortmannin and estimated from partial inhibition data to be 1400, 2830, and 21,400 nM for quercetin, LY294002, and staurosporine, respectively. This novel assay approach allows for screening of inhibitors of lipid kinases in high-throughput mode and thereby may facilitate the identification of novel inhibitory structures for drug development.     

Inhibition of estrone sulfatase by aromatase inhibitor-based estrogen 3-sulfamates

Our rationale is based on the finding that estrone 3-sulfamate (EMATE, 2d), a typical estrone sulfatase (ES) inhibitor, can be hydrolyzed and the pharmacological effect of the free estrogen contributes to the bioactivity of the sulfamate. A number of 3-sulfamoylated derivatives of the good aromatase inhibitors, 2- and 4-halogeno (F, Cl, and Br) estrones and their estradiol analogs as well as 6beta-methyl and phenyl estrones, were synthesized and evaluated as inhibitors of ES in human placental microsomes in comparison with the lead compound EMATE. Among them, 2-chloro- and 2-bromoestrone 3-sulfamates (2b and 2c), along with their estradiol analogs 3b and 3c, were powerful competitive inhibitors with K(i)'s ranging between 4.0 and 11.3 nM (K(i) for EMATE, 73 nM). These four sulfamates as well as the 2-fluoro analogs 2a and 3a inactivated ES in a time-dependent manner more efficiently than EMATE, and 2-halogeno estrone sulfamates 2 also caused a concentration-dependent loss of ES activity. The results may be useful for developing a new class of drugs having a dual function, ES inhibition and aromatase inhibition, for the treatment of breast cancer.     

Novel halogenated nitrobenzylthioinosine analogs as es nucleoside transporter inhibitors

Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel halogenated nitrobenzylthioinosine analogs at the human es nucleoside transporter. Structure-activity relationships indicate the importance of hydrophobicity and electron withdrawing capacity of substituents at the para-position of the 6-position benzyl substituent. All of the compounds showed high binding affinity as shown by their ability to displace the fluorescent es transporter ligand, SAENTA-X8-fluorescein. Compound 16 (6-S-(para-iodobenzyl)-6-thioinosine) was the most tightly bound within the series with a K(i) of 3.88 nM (NBMPR exhibited a K(i) of 0.70 nM). This compound has higher affinity than the widely used nonnucleoside, nucleoside transport inhibitor, dipyridamole (K(i) = 8.79 nM), and may serve as a new lead compound.     

Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease,and 1 phosphatase

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhibition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentration of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demonstrating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential "hit" or false positives, depending on the assay format. Cy dyes (e.g., Cy3B and Cy5 ) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC compounds. This work demonstrates that fluorescent compound and light scattering interferences can be overcome by increasing the fluorophore concentration in an assay or by using longer wavelength dyes.     

Carbonic anhydrase inhibitors: inhibition of the human transmembrane isozyme XIV with a library of aromatic/heterocyclic sulfonamides

The first inhibition study of the transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes hCA XIV with a library of aromatic and heteroaromatic sulfonamides synthesized earlier is reported. Most of the inhibitors were sulfanilamide, homosulfanilamide and 4-aminoethyl-benzenesulfonamide derivatives, to which tails that would induce diverse physicochemical properties have been attached at the amino moiety. Several of these compounds were metanilamide, benzene-1,3-disulfonamide or the 1,3,4-thiadiazole/thiadiazoline-2-sulfonamide derivatives. The tails incorporated in these molecules were of the alkyl/aryl-carboxamido/ sulfonamido-, ureido- or thioureido-types. The sulfanilamides acylated at the 4-amino group with short aliphatic/aromatic moieties incorporating 2-6 carbon atoms showed modest hCA XIV inhibitory activity (K(I)-s in the range of 1.25-4.2 microM) which were anyhow better than that of sulfanilamide (K(I) of 5.4 microM). Better activity showed the homosulfanilamide and 4-aminoethyl-benzenesulfonamide derivatives bearing arylsulfonamido/ureido and thioureido moieties, with K(I)'s in the range of 203-935 nM. The best activity was observed for the heteroaromatic compounds incorporating 1,3,4-thiadiazole/thiadiazoline-2-sulfonamide and 5-arylcarboxamido/sulfonamido moieties, with K(I)'s in the range of 10-85 nM. All these compounds were generally also much better inhibitors of the other two transmembrane CA isozyme, hCA IX and XII. Thus, highly potent hCA XIV inhibitors were detected, but isozyme-specific inhibitors were not discovered for the moment.     

Cellular BRET assay suggests a conformational rearrangement of preformed TrkB/Shc complexes following BDNF-dependent activation

We developed a cellular Bioluminescent Resonance Energy Transfer (BRET) assay based on the interaction of TrkB fused to Renilla luciferase with the intracellular adaptor protein Shc fused to Enhanced Yellow Fluorescent Protein (EYFP). The TrkB agonist Brain Derived Neurotrophic Factor (BDNF) induced a maximum BRET signal as of 10 min with an EC₅₀ value of 1.4 nM, similar to the other endogenous agonists NT-3 and NT-4/5, 1.5 nM and 0.34 nM, respectively. Interestingly, measure of the BRET signal with increasing expression of Shc-EYFP, in the presence or absence of BDNF, suggested a conformational change of preformed TrkB/Shc complexes rather than Shc recruitment. Furthermore, the Y516F TrkB mutant deficient to bind Shc as well as the kinase-dead K572R TrkB mutant was unable to respond to BDNF and exhibited a lower basal BRET signal than that of the wild-type TrkB receptor, again suggesting a preformed complex with constitutive activity. The double YY706/707FF TrkB mutant in the kinase activation loop also showed reduced basal activity but surprisingly kept its capacity to enhance BDNF-induced interaction with Shc, though with less efficacy. The Trk selective kinase inhibitors K252a and BMS-9 blocked BDNF-induced BRET signal with similar potency (100–150 nM), the preferential c-Met inhibitor PF-2341006 being one order of magnitude less potent. Remarkably, in the absence of BDNF, K252a and BMS-9 also reduced basal activity to the level of the Y516F TrkB mutant, suggesting that these compounds were able to reduce the TrkB constitutive activity. BRET responses of mutants and to kinase inhibitors thus reveal a complex level of interaction between TrkB and Shc and suggest that this BRET assay could be of great utility to test blockers of TrkB signalling in a physiologically relevant context.