High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.     

Two- and three dimensional combinatorial chemistry from multicomponent Grignard reagents

The conjugate addition of five component Grignard reagents to methyl ecgonidine was used to create libraries of 3-substituted tropanes. By variation in the reagent combination in 10 such 5-membered sublibraries, a library of 25 compounds was made in a two-dimensional format. Screening of this library led to identification of two new potent monoamine transporter ligands that were subsequently synthesized. The most potent compound in this library was (1R,2S,3S,5S)-3-(3,4-dimethylphenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester, which inhibited dopamine transporter (hDAT) binding and reuptake with a K(i) of 26 and 20 nM, respectively. The conjugate addition to a 5-membered library of methyl ecgonidine analogues with variation of nitrogen substituent was also carried out and used to create 15 sublibraries of 25 compounds, which displayed 125 compounds in a three-dimensional format. From this 3D library, several potent dopamine transport inhibitors were likewise identified and synthesized. The most potent hDAT inhibitor discovered was (1R,2S,3S,5S)-3-(3,4-dimethylphenyl)-8-pentyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester. The study also showed that 3-alkyltropanes were poor inhibitors of monoamine transporters.     

Molecular design of N,N-disubstituted 2-aminothiazolines as selective carboxylesterase inhibitors

Selective carboxylesterase inhibitors are employed as modulators of hydrolytic metabolism of ester or amide-containing drugs. Using the Molecular Field Topology Analysis (MFTA), the models for the relationships between the structures and inhibitory activities of 5-halomethyl-2-aminothiazolines against acetylcholinesterase, butyrylcholinesterase, and carboxylesterase were built, the molecular design was performed, and a focused library of potentially active and selective carboxylesterase inhibitors was proposed.     

Identification of inhibitors against p90 ribosomal S6 kinase 2 (RSK2) through structure-based virtual screening with the inhibitor-constrained refined homology model

P90 ribosomal S6 kinase 2 (RSK2), which was shown to be overexpressed in human cancers, is a serine/threonine kinase and a potential target for cancer treatment. RSK2 comprises two terminal kinase domains (NTKD and CTKD) that can be inhibited by binding with different types of inhibitors at the ATP binding sites. In the absence of a crystal structure of RSK2, we constructed a model for the 3D structure of the RSK2 NTKD by homology modeling and stepwise constrained refinement with the reported inhibitors using a molecular docking method. Structure-based virtual screening was subsequently performed against a library containing commercially available compounds using the refined model. This resulted in the identification of seven novel RSK2 inhibitors with IC?? values ranging from 2.4 to 14.45 μM.     

Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor

Small-molecule kinase inhibitors are predominantly discovered in pure protein assays. We have discovered an inhibitor of Rho-kinase (ROCK) through an image-based, high-throughput screen of cell monolayer wound healing. Using automated microscopy, we screened a library of approximately 16,000 compounds finding many that affected cell migration or cell morphology as well as compounds that blocked mitotic progression. We tested approximately 200 compounds in a series of subassays and chose one, 3-(4-pyridyl)indole (Rockout), for more detailed characterization. Rockout inhibits blebbing and causes dissolution of actin stress fibers, phenocopying Rho-kinase inhibitors. Testing Rho-kinase activity in vitro, Rockout inhibits with an IC50 of 25 microM ( approximately 5-fold less potent than Y-27632) but has a similar specificity profile. We also profile the wound healing assay with a library of compounds with known bioactivities, revealing multiple pathways involved in the biology.     

A genome-wide overexpression screen in yeast for small-molecule target identification

We describe a multicopy gene suppression screen of drug sensitivity in Saccharomyces cerevisiae that facilitates the identification of cellular targets of small molecules. An array of yeast transformants harboring a multicopy yeast genomic library was screened for resistance to growth inhibitors. Comparison of array growth patterns for several such inhibitors allowed the differentiation of general and molecule-specific genetic suppressors. Specific resistance to phenylaminopyrimidine (1), an inhibitor identified from a kinase-directed library, was associated with the overexpression of Pkc1 and a subset of downstream kinases. Components of two other pathways (pheromone response/filamentous growth and Pho85 kinase) that genetically interact with the PKC1 MAPK signaling cascade were also identified. Consistent with the suppression screen, inhibitor 1 bound to Pkc1 in yeast cell lysate and inhibited its activity in vitro. These results demonstrate the utility of this approach for the rapid deconvolution of small-molecule targets.     

Structure Based Library Approach to Photosynthesis Inhibitors:The Solid Phase Synthesis of Hydro-Uracils and Thiouracils

Combinatorialchemistryhasemergedasanefficienttoolforchedriststosynthesize"comPoundslibraries"fortheraPididentificationandoPtforizationofnewleads-lSolidphaseorganicsynthesisprovidesaraPidmeansforpreparationofcomPoundslibraries,andhasbeenusedfortheconstrUctionofoligomericcomPounds,'andsmallorganicmoleculessuchasbenzodiazepine,'9-lactams,'andquinolones.'ThemajortargetfortheinhibitionofphotosystemII(PSII)istheDlprotein,apolyPePtidewithamolecularweightof32kDa.'Wehavedevelopedmodelsoftheher-b…     

A three-hybrid approach to scanning the proteome for targets of small molecule kinase inhibitors

In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets.     

Discovering potent inhibitors against c-Met kinase: molecular design, organic synthesis and bioassay

The receptor tyrosine kinase c-Met is an attractive target for therapeutic treatment of cancers nowadays. The discovery of small molecule inhibitors is of special interest in the blockade of the c-Met kinase pathway. Here, we initiated our study from compound 1a, a novel inhibitor against c-Met kinase. A substructure similarity search against the SPECS database and chemical synthesis methods were performed to obtain a series of pyrazolidine-3,5-dione derivatives. Through the enzyme-based assay against c-Met kinase, 4 compounds (1c, 1e, 1m and 1o) showed potential inhibitory activity, with IC(50) values mostly less than 10 μM. Based on the structure-activity relationship (SAR) and binding mode analysis, a focused combinatorial library was designed by the LD1.0 program. Taking into account ADMET properties and synthesis accessibility, seven candidate compounds (5a-g) were successfully synthesized. The activity of the most potent compounds 5b (IC(50) = 0.46 μM) was 20 fold higher than that of the lead 1a. Taken together, our findings identified the pyrazolidine-3,5-dione derivatives as potent inhibitors against c-Met kinase and demonstrated the efficiency of the strategy in the development of small molecules against c-Met kinase.     

新型吡咯并[2,1-f][1,2,4]三嗪酪氨酸激酶抑制剂的设计与合成

以对甲基苯磺酰甲基异腈和反-2-丁烯酸甲酯为原料,经过11步反应合成了7个4,5,6-三取代的新型吡咯并[2,1-f][1,2,4]三嗪酪氨酸激酶抑制剂,总收率3.2%～10.0%,其结构经1H NMR和MS 表征.     

Diversity-oriented synthesis of pochonins and biological evaluation against a panel of kinases

Pochonins A-F were recently characterized as six new members of the naturally occurring family of 14-membered resorcylic acid lactones. As there are a high number of ATPase and kinase inhibitors among natural resorcylic lactones, a library based on the pochonin scaffold, with five points of diversity, was prepared which includes diversity beyond that of the natural analogues. The library was synthesized by using solid-supported reagents amenable to automation. Testing the library for its inhibition against a panel of 24 kinases at 10 microM afforded a >14 % hit rate. These results demonstrate the potential of the resorcylides towards the inhibition of therapeutically relevant kinases.     

Solid-phase synthesis of a farnesylated CaaX peptide library: inhibitors of the Ras CaaX endoprotease

A solid-phase method, based on Kaiser's p-benzophenone oxime resin, was developed for the synthesis of a series of N-acetyl-S-(E, E-farnesylated) Ca(1)a(2)X tetrapeptides as potential inhibitors of recombinant Ras and a-factor converting enzyme (RCE). N-Acetyl-S-(E, E-farnesyl)-L-cysteine was coupled to resin-bound a(1)a(2) dipeptide using HOBt/DCC activation in conjunction with N-BOC chemistry. The protected farnesylated tripeptide was cleaved from the resin with simultaneous addition of the X residue by treating the resin-bound farnesylated Ca(1)a(2) tripeptide with L-amino acid benzyl ester tosylates under mildly acidic conditions. The benzyl ester was saponified, and the resulting carboxylate precipitated by ether to afford a library of tetrapeptides as a mixture of diastereomers at the cysteine center. The peptides were evaluated as inhibitors of recombinant yeast RCE endoprotease (yRCE) to obtain information about the affinity of the enzyme for the a(1)a(2)X portion of the Ca(1)a(2)X moiety.     

Selection of v-abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display

Methodologies for rapidly identifying cellular protein interactions resulting in posttranslational modification of one of the partners are lacking. Here, we select for substrates of the v-abl tyrosine kinase from two protein display libraries in which the protein is covalently linked to its encoding mRNA. Successive selection cycles from a randomized peptide library identified a consensus sequence closely matching that previously reported for the v-abl tyrosine kinase. Selections from a proteomic library derived from cellular mRNA identified several novel targets of v-abl, including a new member of a class of SH2 domain-containing adaptor proteins. Upon modification, several of the substrates obtained in these selections were found to be effective inhibitors of v-abl kinase activity in vitro. These experiments establish a novel method for identifying the substrates of tyrosine kinases from synthetic and cellular protein libraries.     

Inhibition of Wnt/β-catenin signaling by p38 MAP kinase inhibitors is explained by cross-reactivity with casein kinase Iδ/ɛ

Wnt/β-catenin signaling plays essential roles in embryonic development, adult stem cell maintenance, and disease. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed TAK-715 and AMG-548 as inhibitors of Wnt-3a-stimulated β-catenin signaling. TAK-715 and AMG-548 are inhibitors of p38 mitogen-activated protein kinase, which has been suggested to regulate activation of Wnt/β-catenin signaling. However, two highly selective and equally potent p38 inhibitors, VX-745 and Scio-469, did not inhibit Wnt-3a-stimulated β-catenin signaling. Profiling of TAK-715 and AMG-548 against a panel of over 200 kinases revealed cross-reactivity with casein kinase Iδ and ?, which are known activators of Wnt/β-catenin signaling. Our data demonstrate that this cross-reactivity accounts for the inhibition of β-catenin signaling by TAK-715 and AMG-548 and argue against a role of p38 in Wnt/β-catenin signaling.     

Solid phase combinatorial synthesis of a xanthone library using click chemistry and its application to an embryonic stem cell probe

We report the first solid phase synthesis of a xanthone library CX and its application to embryonic stem cell probe development. The CX library was further derivatised with an activated ester resin to provide an acetylated CX (CXAC) library. Screening of these libraries led to the discovery of a novel fluorescent mESC probe, CDb8.     

Engineered Fully Human Single-Chain Monoclonal Antibodies to PIM2 Kinase

Proviral integration site of Moloney virus-2 (PIM2) is overexpressed in multiple human cancer cells and high level is related to poor prognosis; thus, PIM2 kinase is a rational target of anti-cancer therapeutics. Several chemical inhibitors targeting PIMs/PIM2 or their downstream signaling molecules have been developed for treatment of different cancers. However, their off-target toxicity is common in clinical trials, so they could not be advanced to official approval for clinical application. Here, we produced human single-chain antibody fragments (HuscFvs) to PIM2 by using phage display library, which was constructed in a way that a portion of phages in the library carried HuscFvs against human own proteins on their surface with the respective antibody genes in the phage genome. Bacterial derived-recombinant PIM2 (rPIM2) was used as an antigenic bait to fish out the rPIM2-bound phages from the library. Three E. coli clones transfected with the HuscFv genes derived from the rPIM2-bound phages expressed HuscFvs that bound also to native PIM2 from cancer cells. The HuscFvs presumptively interact with the PIM2 at the ATP binding pocket and kinase active loop. They were as effective as small chemical drug inhibitor (AZD1208, which is an ATP competitive inhibitor of all PIM isoforms for ex vivo use) in inhibiting PIM kinase activity. The HuscFvs should be engineered into a cell-penetrating format and tested further towards clinical application as a novel and safe pan-anti-cancer therapeutics.     

Solid-phase combinatorial library of norstatine-type isosters by the nitroaldol reaction

A combinatorial library of norstatine-type peptide isosters as putative inhibitors of aspartic proteases is presented. The library was synthesized using a split-and-mix strategy designed to afford a one-bead-two-compounds library with the isosteric elements positioned centrally in peptide chains. Application of ladder synthesis during library generation enabled structure identification by MALDI-TOF mass spectroscopy. The library was screened against aspartic protease renin, and two types of inhibitors were identified, that is, XXX-psi[CHRCHOH)-XXX and an aldehyde arising from unreacted starting material. Selected hits were resynthesized and assayed in solution, revealing inhibitors of nanomolar potency.     

Synthesis of spirohydantoins and spiro-2,5-diketopiperazines via resin-bound cyclic alpha,alpha-disubstituted alpha-amino esters

A seven-step solid-phase synthesis of spirohydantoins and an eight-step solid-phase synthesis of spiro-2,5-diketopiperazines is reported. Key intermediate in the synthesis of both compound libraries is the resin-bound cyclic alpha,alpha-disubstituted alpha-amino ester, which can be obtained after selective homogeneous reduction of the aliphatic nitro ester using tin(II) chloride dihydrate. Nitro ester, in turn, is synthesized by a high-pressure-assisted [4 + 2] cycloaddition of resin-bound nitro alkene and butadiene, whereas nitro alkene is obtained by a Knoevenagel condensation of resin-bound nitro acetate with an imine. Novel spirohydantoins are obtained by isocyanate coupling with the resin-bound amino ester 5, followed by cyclization cleavage using a base. Novel spiro-2,5-diketopiperazines are obtained by PyBOP coupling of a Fmoc-protected amino acid with resin-bound amino ester, followed by Fmoc deprotection and an acid-assisted cyclization cleavage. After preparation of seven different resin-bound alpha,alpha-disubstituted alpha-amino esters, a 7 x 8 compound library of spirohydantoins was synthesized using eight different isocyanates, and a 7 x 8 compound library of spiro-2,5-diketopiperazines was synthesized using eight different Fmoc amino acids.     

Inhibition by protease inhibitors of binding of adrenal and sex steroid hormones.

Binding of steroid hormones is inhibited by protease inhibitors and substrates. The protease inhibitors phenylmethyl sulphonylfluoride, tosyl-lysine chloromethyl ketone, and tosylamide-phenylethyl-chloromethyl ketone and the protease substrates tosyl arginine methyl ester and tryptophan methyl ester eliminate specific binding of aldosterone, dexamethasone, dihydrotestosterone, estrogen, and progesterone to their respective receptors. These protease inhibitors and substrates also inhibit binding of progesterone to the 20,000 molecular weight mero-receptor formed from the progesterone receptor in chick oviduct. The binding of estradiol to rat alpha-fetoprotein is inhibited by the protease inhibitors and substrates but not by tryptophan or tryptophan amide, indicating the importance of an ester structure in the inhibition of steroid binding. Our results suggest that all steroid hormone receptors have a site with both common structural features and a role in the regulation of steroid hormone binding.     

Solid phase combinatorial library of phosphinic peptides for discovery of matrix metalloproteinase inhibitors

A solid phase combinatorial library of 165,000 phosphinic peptide inhibitors was prepared and screened for activity against MMP-12. The inhibitors of the library had the structure XXX-Gpsi(PO2H-CH2)L-XXX, in which X is an arbitrary amino acid and Gpsi(PO2H-CH2)L is a Gly-Leu phosphinic dipeptide analogue. The library was constructed as a one-bead-two-compounds library so that every bead contained a common quenched fluorogenic substrate and a different putative inhibitor. In addition, the inhibitor part was prepared by ladder synthesis. After incubation with MMP-12, beads containing active inhibitors were selected, and the inhibitor sequences were recorded using MALDI-TOF MS. Statistical analysis of the sequences obtained from 86 beads gave rise to a consensus sequence which was resynthesized along with 20 related sequences. Three truncated sequences and 16 sequences originally present on beads were also resynthesized. The inhibitors were investigated in an enzyme kinetic assay with MMP-12 showing that the compounds derived from the consensus sequence were strong inhibitors with Ki values down to 6 nM, whereas the sequences originally present on beads varied in potency with Ki values from micromolar to nanomolar. Truncated sequences derived from the consensus sequence were poor inhibitors of MMP-12.