Development and validation of high-throughput screening assays for poly(ADP-ribose) polymerase-2 inhibitors

Poly(ADP-ribose) polymerase-1 and -2 (PARP1/2) are two key facilitators of DNA repair and are implicated in the pathogenesis of cancers and several chronic diseases. Inhibitors of PARP1/2 have shown powerful therapeutic effects in the treatment of cancer, cerebral ischemia, and inflammation. In addition, evidence from several studies suggests unique functions for PARP2 in genome surveillance, spermatogenesis, adipogenesis, and T cell development, and PARP2-specific inhibitors might have many other applications. To acquire PARP1/2 inhibitors, many high-throughput screening (HTS) assays for PARP1 inhibitors have been developed. However, detailed screening assays for PARP2 inhibitors have not been reported. Herein, three HTS assays for PARP2 inhibitors were developed and validated with reference inhibitors in each case. The results suggest that the HTS assays for PARP2 inhibitors using chemical quantification of NAD⁺, biotin-based quantification of PAR, and ELISA quantification of PAR are sensitive, robust, and cost effective.     

Discovery of potent and selective PARP-1 and PARP-2 inhibitors: SBDD analysis via a combination of X-ray structural study and homology modeling

We disclose herein our efforts aimed at discovery of selective PARP-1 and PARP-2 inhibitors. We have recently discovered several novel classes of quinazolinones, quinazolidinones, and quinoxalines as potent PARP-1 inhibitors, which may represent attractive therapeutic candidates. In PARP enzyme assays using recombinant PARP-1 and PARP-2, the quinazolinone derivatives displayed relatively high selectivity for PARP-1 and quinoxaline derivatives showed superior selectivity for PARP-2, and the quinazolidinone derivatives did not have selectivity for PARP-1/2. Structure-based drug design analysis via a combination of X-ray structural study utilizing the complexes of inhibitors and human PARP-1 catalytic domain, and homology modeling using murine PARP-2 suggested distinct interactions of inhibitors with PARP-1 and PARP-2. These findings provide a new structural framework for the design of selective inhibitors for PARP-1 and PARP-2.     

Purification and characterization of proteinase inhibitors from winged bean (Psophocarpus tetragonolobus (L.) DC.) seeds

Seven proteinase inhibitors were isolated from winged bean seeds by ion-exchange chromatographies. These inhibitors had molecular weights of around 20,000, included four half-cystine residues, and were Kunitz-type inhibitors. Two (WTI-2 and 3) inhibited bovine trypsin strongly and four (WCI-1, 2, 3, and 4) inhibited bovine alpha-chymotrypsin, but in different ways. One mole of WCI-2 or -3 could inhibit 2 mol of alpha-chymotrypsin. The remaining inhibitor (WTCI-1) could bind both bovine trypsin and alpha-chymotrypsin at the molar ratio of 1:1, but not simultaneously. All four chymotrypsin inhibitors cross-reacted with rabbit anti-WCI-3 serum, while the other inhibitors did not.     

Substrate analog inhibitors of HIV-1 protease containing phenylnorstatine as a transition state element

Substrates of HIV-1 protease are classified into three groups (A, B and C) based on the amino acid residues present at P1' and P2' sites. Replacement of the scissile amide bond by phenylnorstatine in representative substrate analog sequences from class A, B and C, yielded inhibitors of HIV-1 protease. Of the twelve inhibitors synthesized in this series, class C substrate analog inhibitors are more potent inhibitors (Ki's 3.3-24 microM) than either class A or class B inhibitors. In this series of inhibitors, the (2S,3S) isomer of phenylnorstatine is preferred over the other isomers as a "transition state element" for design of inhibitors of HIV-1 protease.     

Hierarchical virtual screening of the dual MMP-2/HDAC-6 inhibitors from natural products based on pharmacophore models and molecular docking

The dual-target inhibitors tend to improve the response rate in treating tumors, comparing with the single-target inhibitors. Matrix metalloproteinase-2 (MMP-2) and histone deacetylase-6 (HDAC-6) are attractive targets for cancer therapy. In this study, the hierarchical virtual screening of dual MMP-2/HDAC-6 inhibitors from natural products is investigated. The pharmacophore model of MMP-2 inhibitors is built based on ligands, but the pharmacophore model of HDAC-6 inhibitors is built based on the experimental crystal structures of multiple receptor–ligand complexes. The reliability of these two pharmacophore models is validated subsequently. The hierarchical virtual screening, combining these two different pharmacophore models of MMP-2 and HDAC-6 inhibitors with molecular docking, is carried out to identify the dual MMP-2/HDAC-6 inhibitors from a database of natural products. The four potential dual MMP-2/HDAC-6 inhibitors of natural products, STOCK1 N-46177, STOCK1 N-52245, STOCK1 N-55477, and STOCK1 N-69706, are found. The studies of binding modes show that the screened four natural products can simultaneously well bind with the MMP-2 and HDAC-6 active sites by different kinds of interactions, to inhibit the MMP-2 and HDAC-6 activities. In addition, the ADMET properties of screened four natural products are assessed. These found dual MMP-2/HDAC-6 inhibitors of natural products could serve as the lead compounds for designing the new dual MMP-2/HDAC-6 inhibitors having higher biological activities by carrying out structural modifications and optimizations in the future studies.     

Unexpected small molecules as novel SIRT2 suicide inhibitors

A series of sirtuin inhibitor candidates were assembled based on an intermediate ester (1a) our accidently discovered. After screening and evaluation, several SIRT2 selective inhibitors were identified, which can inhibit all the deacetylation, defatty-acylation and debenzoylation of SIRT2. Among these inhibitors, compound 1e was the best SIRT2 selective inhibitors. The primary study on the inhibitory mechanism indicated that compound 1e may be a suicide inhibitor acting as an irreversible way. Given almost all reported sirtuin inhibitors are non-covalent, sirtuin covalent inhibitors are still need to be developed. These findings will facilitate for further development of SIRT2 selective and suicide inhibitors.     

Isolation and characterization of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) from human rheumatoid synovial fluid.

The tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 were purified to apparent homogeneity from human rheumatoid synovial fluid (HRSF). The inhibitors were isolated by dissociation of non-covalent gelatinase/TIMP complexes. TIMP-1 migrated as a single polypeptide with Mr 28,500 on SDS-PAGE, while the Mr of TIMP-2 was 21,000. The inhibitory activity was stable under heat and acid pH. N-terminal sequence data were obtained for the first 15 residues of both inhibitors and showed identity to the human fibroblast inhibitors TIMP-1 and TIMP-2. This is the first demonstration that TIMP-1 and TIMP-2 can be directly purified from human rheumatoid synovial fluid. The complex formation between the metalloproteinase inhibitors and leucocyte metalloproteinases was shown by immunoblotting.     

Differential inhibitor sensitivity between human kinases VRK1 and VRK2

Human vaccinia-related kinases (VRK1 and VRK2) are atypical active Ser-Thr kinases implicated in control of cell cycle entry, apoptosis and autophagy, and affect signalling by mitogen activated protein kinases (MAPK). The specific structural differences in VRK catalytic sites make them suitable candidates for development of specific inhibitors. In this work we have determined the sensitivity of VRK1 and VRK2 to kinase inhibitors, currently used in biological assays or in preclinical studies, in order to discriminate between the two proteins as well as with respect to the vaccinia virus B1R kinase. Both VRK proteins and vaccinia B1R are poorly inhibited by inhibitors of different types targeting Src, MEK1, B-Raf, JNK, p38, CK1, ATM, CHK1/2 and DNA-PK, and most of them have no effect even at 100 μM. Despite their low sensitivity, some of these inhibitors in the low micromolar range are able to discriminate between VRK1, VRK2 and B1R. VRK1 is more sensitive to staurosporine, RO-31-8220 and TDZD8. VRK2 is more sensitive to roscovitine, RO 31-8220, Cdk1 inhibitor, AZD7762, and IC261. Vaccinia virus B1R is more sensitive to staurosporine, KU55933, and RO 31-8220, but not to IC261. Thus, the three kinases present a different pattern of sensitivity to kinase inhibitors. This differential response to known inhibitors can provide a structural framework for VRK1 or VRK2 specific inhibitors with low or no cross-inhibition. The development of highly specific VRK1 inhibitors might be of potential clinical use in those cancers where these kinases identify a clinical subtype with a poorer prognosis, as is the case of VRK1 in breast cancer.     

Comparison of the Effect of Calpain Inhibitors on Two Extralysosomal Proteinases: The Multicatalytic Proteinase Complex and m-Calpain

Abstract: The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of β-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC₅₀ values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.     

Novel BACE1 inhibitors possessing a 5-nitroisophthalic scaffold at the P2 position

Recently, we reported substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell assays. We also designed and synthesized non-peptidic and small-sized inhibitors possessing a heterocyclic scaffold at the P(2) position. By studying the structure-activity relationship of these inhibitors, we found that the σ-π interaction of an inhibitor with the BACE1-Arg235 side chain played a key role in the inhibition mechanism. Hence, we optimized the inhibitors with a focus on their P(2) regions. In this Letter, a series of novel BACE1 inhibitors possessing a 5-nitroisophthalic scaffold at the P(2) position are described along with the results of the related structure-activity relationship study. These small-sized inhibitors are expected improved membrane permeability and bioavailability.     

A specific pharmacophore model of sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors

Sodium-dependent glucose co-transporter 2 (SGLT2) plays a pivotal role in maintaining glucose equilibrium in the human body, emerging as one of the most promising targets for the treatment of diabetes mellitus type 2. Pharmacophore models of SGLT2 inhibitors have been generated with a training set of 25 SGLT2 inhibitors using Discovery Studio V2.1. The best hypothesis (Hypo1(SGLT2)) contains one hydrogen bond donor, five excluded volumes, one ring aromatic and three hydrophobic features, and has a correlation coefficient of 0.955, cost difference of 68.76, RMSD of 0.85. This model was validated by test set, Fischer randomization test and decoy set methods. The specificity of Hypo1(SGLT2) was evaluated. The pharmacophore features of Hypo1(SGLT2) were different from the best pharmacophore model (Hypo1(SGLT1)) of SGLT1 inhibitors we developed. Moreover, Hypo1(SGLT2) could effectively distinguish selective inhibitors of SGLT2 from those of SGLT1. These results indicate that a highly predictive and specific pharmacophore model of SGLT2 inhibitors has been successfully obtained. Then Hypo1(SGLT2) was used as a 3D query to screen databases including NCI and Maybridge for identifying new inhibitors of SGLT2. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. And several compounds selected from the top ranked hits have been suggested for further experimental assay studies.     

Purification and properties of proteinaceous trypsin inhibitors in the skin mucus of pufferfish Takifugu pardalis

A screening assay for inhibitory activity against trypsin in skin mucus from 29 species of fishes reveals a wide distribution of trypsin inhibitors in skin mucus and relatively high antitryptic activity in pufferfish of the family Tetraodontidae. Two trypsin inhibitors termed TPTI 1 and 2 were purified to homogeneity from the skin mucus of Takifugu pardalis by salting out, lectin affinity, anion exchange FPLC and gel filtration HPLC. Both inhibitors are acidic glycoproteins, with an apparent molecular mass of 57 kDa in SDS-PAGE, pI below 4 and 1.9% reducing sugar for TPTI 1 and with an apparent molecular mass of 47 kDa in SDS-PAGE, pI 5.2 and 0.8% reducing sugar for TPTI 2. The inhibitors effectively repress the catalytic activity of trypsin and alpha-chymotrypsin, and therefore can be classified as serine protease inhibitors. The inhibitory constants against trypsin were 4.9x10(-8) M for TPTI 1 and 3.9x10(-8) M for TPTI 2. Both inhibitors react with trypsin at a molar ratio of 1:1, although TPTI 1 reversibly inactivates the proteolytic activity of trypsin non-competitively and TPTI 2, competitively. The trypsin inhibitors in the skin mucus of T. pardalis may function as defense substances to neutralize serine proteases released by invasive pathogens.     

NSAID-induced gut inflammation and vasoconstriction: Causes and potential reversal with beta-CGRP - A hypothesis

Traditional non-steroidal anti-inflammatory drugs, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitors control inflammation. While these drugs are formulated to reduce one of the cardinal signs of inflammation by reducing prostaglandin levels at the site of inflammation, COX-1 inhibitors induce inflammation in the stomach as well as the small bowel. The COX-2 inhibitors, a large portion of the non-steroidal anti-inflammatory drug market, provide a gastro-intestinally safer class of drugs. However, COX-2 inhibitors induce vasoconstriction via actions in renal and cardiovascular tissues. Since COX-2 inhibitors also have anticancer potential, it is worthwhile to design drug formulations that will not cause hypertension or cardiovascular damage. An attempt has thus been made in this article to formulate a hypothesis to circumvent the COX inhibitors induced inflammation and vasoconstriction through COX independent activation of calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide found throughout the vascular and sensory nervous system.     

Novel binding mode of a potent and selective tankyrase inhibitor

Tankyrases (TNKS1 and TNKS2) are key regulators of cellular processes such as telomere pathway and Wnt signaling. IWRs (inhibitors of Wnt response) have recently been identified as potent and selective inhibitors of tankyrases. However, it is not clear how these IWRs interact with tankyrases. Here we report the crystal structure of the catalytic domain of human TNKS1 in complex with IWR2, which reveals a novel binding site for tankyrase inhibitors. The TNKS1/IWR2 complex provides a molecular basis for their strong and specific interactions and suggests clues for further development of tankyrase inhibitors.     

Targeting transcription of MCL-1 sensitizes HER2-amplified breast cancers to HER2 inhibitors

Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.Subject terms: Breast cancer, Target identification     

Thalidomide and its analogues as cyclooxygenase inhibitors

Thalidomide showed cyclooxygenase (COX)-1/2 inhibitory activity with a potency comparable to that of aspirin. Structural development studies of thalidomide resulted in potent COX-1/2 inhibitors, and COX-1-selective and COX-2-selective inhibitors.     

Novel non-peptidic and small-sized BACE1 inhibitors

Recently, we reported substrate-based beta-secretase (BACE1) inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. These inhibitors showed potent BACE1 inhibitory activities (approximately 1.2 nM IC(50)). In order to improve in vivo enzymatic stability and permeability across the blood-brain barrier, these penta-peptidic inhibitors would need to be further optimized. On the other hand, non-peptidic inhibitors possessing isophthalic residue at the P(2) position were reported from other research groups. We selected isophthalic-type aromatic residues at the P(2) position and an HMC isostere at the P(1) position as lead compounds. On the basis of the design approach focused on the conformer of docked inhibitor in BACE1, we found novel non-peptidic and small-sized BACE1 inhibitors possessing a 2,6-pyridinedicarboxylic, chelidamic or chelidonic residue at the P(2) position.     

Evaluation of 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-diones as inhibitors of human protein kinase CK2

Protein kinase CK2 (Casein Kinase 2) is an extremely pleiotropic Ser/Thr kinase with high constitutive activity. The observation of CK2 deregulations in various pathological processes suggests that CK2 inhibitors may have a therapeutic value, particularly as anti-neoplastic and antiviral drugs. Here, we present the 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-diones as a novel potent class of CK2 inhibitors. We identified this class of inhibitors by high-throughput docking of a compound collection in the ATP-binding site of human CK2. The most active compounds are 2-(4,5,6,7-tetraiodo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanoic acid and 2-(4,5,6,7-tetraiodo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)acetic acid with IC(50) values of 0.15 microM and 0.3 microM, respectively. These inhibitors are ATP-competitive and they only minimally inhibit the activities of protein kinases DYRK1a, MSK1, GSK3 and CDK5. Binding modes for the most active inhibitors are proposed.