KR-39038, a Novel GRK5 Inhibitor, Attenuates Cardiac Hypertrophy and Improves Cardiac Function in Heart Failure.

G protein-coupled receptor kinase 5 (GRK5) has been considered as a potential target for the treatment of heart failure as it has been reported to be an important regulator of pathological cardiac hypertrophy. To discover novel scaffolds that selectively inhibit GRK5, we have identified a novel small molecule inhibitor of GRK5, KR-39038 [7-((3-((4-((3-aminopropyl)amino)butyl)amino)propyl) amino)-2-(2-chlorophenyl)-6-fluoroquinazolin-4(3H)-one]. KR-39038 exhibited potent inhibitory activity (IC50 value=0.02 µM) against GRK5 and significantly inhibited angiotensin II-induced cellular hypertrophy and HDAC5 phosphorylation in neonatal cardiomyocytes. In the pressure overload-induced cardiac hypertrophy mouse model, the daily oral administration of KR-39038 (30 mg/kg) for 14 days showed a 43% reduction in the left ventricular weight. Besides, KR-39038 treatment (10 and 30 mg/kg/ day, p.o.) showed significant preservation of cardiac function and attenuation of myocardial remodeling in a rat model of chronic heart failure following coronary artery ligation. These results suggest that potent GRK5 inhibitor could effectively attenuate both cardiac hypertrophy and dysfunction in experimental heart failure, and KR-39038 may be useful as an effective GRK5 inhibitor for pharmaceutical applications.

Synthesis and SAR of 5-aryl-furan-2-carboxamide derivatives as potent urotensin-II receptor antagonists.

The synthesis and biological evaluation as potential urotensin-II receptor antagonists of a series of 5-arylfuran-2-carboxamide derivatives 1, bearing a 4-(3-chloro-4-(piperidin-4-yloxy)benzyl)piperazin-1-yl group, are described. The results of a systematic SAR investigation of furan-2-carboxamides with C-5 aryl groups possessing a variety of aryl ring substituents led to identification of the 3,4-difluorophenyl analog 1y as a highly potent UT antagonist with an IC50 value of 6 nM. In addition, this substance was found to display high metabolic stability, and low hERG inhibition and cytotoxicity, and to have an acceptable PK profile.

Identification of N-(5-(phenoxymethyl)-1,3,4-thiadiazol-2-yl)acetamide derivatives as novel protein tyrosine phosphatase epsilon inhibitors exhibiting anti-osteoclastic activity.

Cytosolic protein tyrosine phosphatase epsilon (cyt-PTPε) plays a central role in controlling differentiation and function of osteoclasts, whose overactivation causes osteoporosis. Based on our previous study reporting a number of cyt-PTPε inhibitory chemical compounds, we carried out a further and extended analysis of our compounds to examine their effects on cyt-PTPε-mediated dephosphorylation and on osteoclast organization and differentiation. Among five compounds showing target selectivity to cyt-PTPε over three other phosphatases in vitro, two compounds exhibited an inhibitory effect against the dephosphorylation of cellular Src protein, the cyt-PTPε substrate. Moreover, these two compounds caused destabilization of the podosome structure that is necessary for the bone-resorbing activity of osteoclasts, and also attenuated cellular differentiation of monocytes into osteoclasts, without affecting cell viability. Therefore, these findings not only verified anti-osteoclastic effects of our cyt-PTPε inhibitory compounds, but also showed that cyt-PTPε expressed in osteoclasts could be a putative therapeutic target worth considering.

Cardioprotective effect of KR-33889, a novel PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cells and isolated rat hearts.

Oxidative stress plays a critical role in cardiac injury during ischemia/reperfusion (I/R). Despite a potent cardioprotective activity of KR-33889, a novel poly (ADP-ribose) polymerase inhibitor, its underlying mechanism remains unresolved. This study was designed to investigate the protective effects of KR-33889 against oxidative stress-induced apoptosis in rat cardiomyocytes H9c2 cells and isolated rat hearts. H2O2 caused severe injury to H9c2 cells, mainly due to apoptosis, as revealed by TUNEL assay. However, KR-33889 pretreatment significantly attenuated H2O2-induced apoptosis of H9c2 cells, which was accompanied by decrease in expression of both cleaved caspase-3 and Bax and increase in Bcl-2 expression and the ratio of Bcl-2/Bax. KR-33889 also significantly enhanced the expression of anti-oxidant enzymes including heme oxygenase-1, Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase, thereby inhibiting production of intracellular ROS. Furthermore, KR-33889 reversed H2O2-induced decrease in phosphorylation of Akt, GSK-3ß, ERK1/2, p38 MAPK, and SAPK/JNK during most H2O2 exposure time. In globally ischemic rat hearts, KR-33889 inhibited both I/R-induced decrease in cardiac contractility and apoptosis by increasing Bcl-2, decreasing both cleaved caspase-3 and Bax expression, and enhancing expression of anti-oxidant enzymes. Taken together, these results suggest that KR-33889 may have therapeutic potential to prevent I/R-induced heart injury in ischemic heart diseases mainly by reducing oxidative stress-mediated myocardial apoptosis.

A novel urotensin II receptor antagonist, KR-36676, prevents ABCA1 repression via ERK/IL-1ß pathway.

Urotensin II (U-II), the most potent vasoconstrictor peptide known to date, is expressed at a high level in vascular smooth muscle cells (VSMC) and endothelial cells, whereas its receptor, urotensin (UT) receptor, is abundant in monocytes and macrophages of atherosclerotic lesions. U-II is highly present in the coronary arteries of the atherosclerotic patients compared to normal subjects. Recently, U-II was shown to down-regulate ATP binding cassette transporter-A1 (ABCA1) expression, which is responsible for reverse cholesterol transport in macrophages of atherosclerotic lesions. However, the mechanism of this observation was not clearly elucidated. Previous studies also revealed that the proinflammatory cytokine interleukin-1ß (IL-1ß) repressed ABCA1 expression. To clarify the signaling pathway involved with respect to U-II-induced ABCA1 down-regulation, we investigated whether IL-1ß was involved. Our results provided that U-II repressed ABCA1 through an ERK/ IL-1ß pathway. We further demonstrated that U-II receptor antagonist KR-36676 decreased IL-1ß production and significantly led to a recovery of ABCA1 expression at both mRNA and protein levels. In previous investigations, U-II receptor antagonists have been shown to protect atherosclerosis in cell and animal models. Our results imply that U-II receptor antagonist KR-36676 might be a potent candidate for treating atherosclerosis, and leading to a recovery of ABCA1 expression, affected by the ERK/IL-1ß pathway.

A novel urotensin II receptor antagonist, KR-36996, improved cardiac function and attenuated cardiac hypertrophy in experimental heart failure.

Urotensin II and its receptor are thought to be involved in various cardiovascular diseases such as heart failure, pulmonary hypertension and atherosclerosis. Since the regulation of the urotensin II/urotensin II receptor offers a great potential for therapeutic strategies related to the treatment of cardiovascular diseases, the study of selective and potent antagonists for urotensin II receptor is more fascinating. This study was designed to determine the potential therapeutic effects of a newly developed novel urotensin II receptor antagonist, N-(1-(3-bromo-4-(piperidin-4-yloxy)benzyl)piperidin-4-yl)benzo[b]thiophene-3-carboxamide (KR-36996), in experimental models of heart failure. KR-36996 displayed a high binding affinity (Ki=4.44±0.67nM) and selectivity for urotensin II receptor. In cell-based study, KR-36996 significantly inhibited urotensin II-induced stress fiber formation and cellular hypertrophy in H9c2UT cells. In transverse aortic constriction-induced cardiac hypertrophy model in mice, the daily oral administration of KR-36996 (30mg/kg) for 14 days significantly decreased left ventricular weight by 40% (P<0.05). In myocardial infarction-induced chronic heart failure model in rats, repeated echocardiography and hemodynamic measurements demonstrated remarkable improvement of the cardiac performance by KR-36996 treatment (25 and 50mg/kg/day, p.o.) for 12 weeks. Moreover, KR-36996 decreased interstitial fibrosis and cardiomyocyte hypertrophy in the infarct border zone. These results suggest that potent and selective urotensin II receptor antagonist could efficiently attenuate both cardiac hypertrophy and dysfunction in experimental heart failure. KR-36996 may be useful as an effective urotensin II receptor antagonist for pharmaceutical or clinical applications.

A Novel Urotensin II Receptor Antagonist, KR-36996 Inhibits Smooth Muscle Proliferation through ERK/ROS Pathway.

Urotensin II (UII) is a mitogenic and hypertrophic agent that can induce the proliferation of vascular cells. UII inhibition has been considered as beneficial strategy for atherosclerosis and restenosis. However, currently there is no therapeutics clinically available for atherosclerosis or restenosis. In this study, we evaluated the effects of a newly synthesized UII receptor (UT) antagonist, KR- 36996, on the proliferation of SMCs in vitro and neointima formation in vivo in comparison with GSK-1440115, a known potent UT antagonist. In primary human aortic SMCs (HASMCs), UII (50 nM) induced proliferation was significantly inhibited by KR-36996 at 1, 10, and 100 nM which showed greater potency (IC50: 3.5 nM) than GSK-1440115 (IC50: 82.3 nM). UII-induced proliferation of HASMC cells was inhibited by U0126, an ERK1/2 inhibitor, but not by SP600125 (inhibitor of JNK) or SB202190 (inhibitor of p38 MAPK). UII increased the phosphorylation level of ERK1/2. Such increase was significantly inhibited by KR-36996. UII-induced proliferation was also inhibited by trolox, a scavenger for reactive oxygen species (ROS). UII-induced ROS generation was also decreased by KR-36996 treatment. In a carotid artery ligation mouse model, intimal thickening was dramatically suppressed by oral treatment with KR-36996 (30 mg/kg) which showed better efficacy than GSK-1440115. These results suggest that KR-36996 is a better candidate than GSK-1440115 in preventing vascular proliferation in the pathogenesis of atherosclerosis and restenosis.

Benzo[b]thiophene-2-carboxamide derivatives as potent urotensin-II receptor antagonists.

Members of a series of benzo[b]thiophene-2-carboxamide derivatives, possessing an N-(1-(3-bromo-4-(piperidin-4-yloxy)benzyl)piperidin-4-yl) group, were synthesized and evaluated as urotensin-II receptor antagonists. The results show that these substances have potent UT binding affinities. Observations made in a systematic SAR investigation of the effects of a variety of substituents (R(1) and R(2)) at the 5- and 6-positions in the benzo[b]thiophene-2-carboxamide moiety on UT binding affinities led to identification of the 5-cyano analog 7f as a highly potent UT antagonist with an IC50 value of 25nM. Despite having a good metabolic stability, 7f is a potent inhibitor of CYP isozyme and displays an unsuitable PK profile.

A urotensin II receptor antagonist, KR36676, decreases vascular remodeling and inflammation in experimental pulmonary hypertension.

The pathophysiological implications of binding of urotensin II (U-II) to urotensin II receptor (UT) in pulmonary arterial hypertension (PAH) have been proposed recently. Besides high expression of U-II in experimental models and patients with PAH, U-II has been shown to increase proliferation of pulmonary vascular smooth muscle cells and inflammatory responses, which were critical for PAH pathophysiology. However, the direct role of the urotensinergic system in the pathogenesis of PAH is yet to be understood. The aim of the present study was to determine whether a novel UT antagonist, KR36676, attenuates the pathophysiological progression of PAH in an animal model of PAH. PAH was induced by a single subcutaneous injection of monocrotaline (MCT, 60mg/kg) in rats. All the animals received KR36676 (30mg/kg/day) or vehicle by oral gavage. Three weeks after MCT-injection, changes in hemodynamic parameters, extent of right ventricular hypertrophy, fibrosis and pulmonary vascular remodeling, and degree of protein expression were determined. Oral administration of KR36676 effectively decreased the MCT-induced increase in right ventricular systolic pressure, hypertrophy and fibrosis. Furthermore, wall thickness of pulmonary arterioles, proliferation of pulmonary vascular cells, and inflammatory response significantly decreased in the KR36676-treated group following MCT injection compared to that in the MCT-treated vehicle group. These preventive effects of KR36676 are mediated, at least in part, by suppression of ERK1/2 and NF-κB signaling pathways. The novel UT antagonist, KR36676, effectively prevented MCT-induced PAH progression and pulmonary vascular remodeling in rat model. Our findings support the therapeutic efficacy of UT antagonist in PAH prevention and elucidate the possible underlying mechanisms of action.

Blockade of Urotensin II Receptor Prevents Vascular Dysfunction.

Urotensin II (UII) is a potent vasoactive peptide and mitogenic agent to induce proliferation of various cells including vascular smooth muscle cells (VSMCs). In this study, we examined the effects of a novel UII receptor (UT) antagonist, KR-36676, on vasoconstriction of aorta and proliferation of aortic SMCs. In rat aorta, UII-induced vasoconstriction was significantly inhibited by KR-36676 in a concentration-dependent manner. In primary human aortic SMCs (hAoSMCs), UII-induced cell proliferation was significantly inhibited by KR-36676 in a concentration-dependent manner. In addition, KR-36676 decreased UII-induced phosphorylation of ERK, and UII-induced cell proliferation was also significantly inhibited by a known ERK inhibitor U0126. In mouse carotid ligation model, intimal thickening of carotid artery was dramatically suppressed by oral treatment with KR-36676 (30 mg/ kg/day) for 4 weeks compared to vehicle-treated group. From these results, it is indicated that KR-36676 suppress UII-induced proliferation of VSMCs at least partially through inhibition of ERK activation, and that it also attenuates UII-induced vasoconstriction and vascular neointima formation. Our study suggest that KR-36676 may be an attractive candidate for the pharmacological management of vascular dysfunction.

Evaluation of anti-coccidial effects of 1-[4-(4-nitrophenoxy)phenyl]propane-1-one and identification of its potential target proteins in Toxoplasma gondii.

Coccidiosis affects many vertebrates worldwide, but treatment with known anti-coccidial drugs causes several adverse side effects. There is a critical need for the development and evaluation of new drugs. The anti-coccidial effect of 1-[4-(4-nitrophenoxy)phenyl]propane-1-one (NPPP), a synthetic compound, was studied in vitro and in vivo. Treatment with NPPP showed anti-Toxoplasma activity in vitro with a lower EC50 value than pyrimethamine. In ICR mice infected with Toxoplasma gondii, oral administration of NPPP for 4 days showed statistically significant anti-Toxoplasma activity with lower numbers of tachyzoite than those of the negative control (p < 0.01). NPPP also exhibited strong anti-Eimeria activity in Eimeria tenella-infected chickens when treated for 4 days with orally administered NPPP at a dose of 100 mg/kg. Potential target proteins of NPPP were analyzed by proteomic profiles of T. gondii tachyzoites. Two hypothetical proteins were identified as possible targets of NPPP, a putative ortholog of vacuolar ATP synthase subunit C and a class I S-adenosylmethionine-dependent methyltransferase. Our data show that the NPPP might be an anti-coccidial drug candidate for clinical application against coccidial infections. Future investigations will focus on identifying the function of proteins regulated by NPPP.

Synthesis and SAR of thieno[3,2-b]pyridinyl urea derivatives as urotensin-II receptor antagonists.

The preparation and SAR profile of thieno[3,2-b]pyridinyl urea derivatives as novel and potent urotensin-II receptor antagonists are described. An activity optimization study, probing the effects of substituents on thieno[3,2-b]pyridinyl core and benzyl group of the piperidinyl moiety, led to the identification of p-fluorobenzyl substituted thieno[3,2-b]pyridinyl urea 6n as a highly potent UT antagonist with an IC50 value of 13nM. Although 6n displays good metabolic stability and low hERG binding activity, it has an unacceptable oral bioavailability.

Predicted ligands for the human urotensin-II G†protein-coupled receptor with some experimental validation.

Human Urotensin-II (U-II) is the most potent mammalian vasoconstrictor known.1 Thus, a U-II antagonist would be of therapeutic value in a number of cardiovascular disorders.2 Here, we describe our work on the prediction of the structure of the human U-II receptor (hUT2 R) using GEnSeMBLE (GPCR Ensemble of Structures in Membrane BiLayer Environment) complete sampling Monte Carlo method. With the validation of our predicted structures, we designed a series of new potential antagonists predicted to bind more strongly than known ligands. Next, we carried out R-group screening to suggest a new ligand predicted to bind with 7 kcal mol(-1) better energy than 1-{2-[4-(2-bromobenzyl)-4-hydroxypiperidin-1-yl]ethyl}-3-(thieno[3,2-b]pyridin-7-yl)urea, the designed antagonist predicted to have the highest affinity for the receptor. Some of these predictions were tested experimentally, validating the computational results. Using the pharmacophore generated from the predicted structure for hUT2 R bound to ACT-058362, we carried out virtual screening based on this binding site. The most potent hit compounds identified contained 2-(phenoxymethyl)-1,3,4-thiadiazole core, with the best derivative exhibiting an IC50 value of 0.581 µM against hUT2 R when tested in vitro. Our efforts identified a new scaffold as a potential new lead structure for the development of novel hUT2 R antagonists, and the computational methods used could find more general applicability to other GPCRs.

Synthesis and structure-activity relationship of naphtho[1,2-b]furan-2-carboxamide derivatives as melanin concentrating hormone receptor 1 antagonists.

The discovery that novel naphtho[1,2-b]furan-2-carboxamides containing linked piperidinylphenylacetamide groups serve as melanin concentrating hormone receptor 1 (MCH-R1) antagonists is described. An extensive structure-activity relationship (SAR) study, probing members of this family that contain a variety of aryl and heteroaryl groups at C-5 of the naphtho[1,2-b]furan-2-carboxamide skeleton and having different chain linker lengths, led to the identification of the 5-(4-pyridinyl) substituted analog 10b as a highly potent MCH-R1 antagonist with an IC50 value of 3 nM. This substance also displays good metabolic stability and it does not significantly inhibit cytochrome P450 (CYP450) enzymes. However, 10b has unacceptable oral bioavailability.

Synthesis and SAR study of pyrrolo[3,4-b]pyridin-7(6H)-one derivatives as melanin concentrating hormone receptor 1 (MCH-R1) antagonists.

The discovery and optimization of novel pyrrolo[3,4-b]pyridin-7(6H)-one MCH-R1 antagonists are described. A systematic SAR study probing the effects of aryl-, benzyl- and arylthio-substituents at the 2-position of the pyrrolo[3,4-b]pyridin-7(6H)-ones led to identification of the 2-[(4-fluorophenyl)thio] derivative 7b as a highly potent MCH-R1 antagonist. This compound also has favorable pharmacokinetic properties along with a high metabolic stability and a minimal impact on CYP isoforms and hERG.

5-AIQ inhibits H2O2-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3ß signaling pathway in H9c2 cardiomyocytes.

Poly(adenosine 5'-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H2O2-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H2O2-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H2O2-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3ß (GSK-3ß), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3ß activation. In addition, inhibiting the Akt/GSK-3ß pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H2O2-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3ß pathway.

Discovery of novel scaffolds for Rho kinase 2 inhibitor through TRFRET-based high throughput screening assay.

Recent advances in basic and clinical studies have identified Rho kinase (ROCK) as an important target potentially implicated in a variety of cardiovascular diseases and ROCK inhibitors were considered as a pharmacological strategy to prevent and treat cardiovascular diseases. To screen the small molecule compound library against ROCK, a high throughput screening (HTS) campaign was carried out using immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Z' value and signal to background (S/B) ratio were achieved at 0.76 and 5.27 for the pilot library screening of the most diverse set consisting of 15,040 compounds with a reasonable reconfirmation rate. From this screening campaign, four novel scaffolds, such as 3- nitropyridine, 4-methoxy-1,3,5,-triazine, naphthalene-1,4-dione, and 2,3-dihydro-1H-pyrrolo[2,3-b]quinoxaline, were yielded. Particularly, we found that 3-nitropyridine derivatives possess potent inhibitory activity and selectivity for ROCK. Our findings provide important information for the design of novel ROCK inhibitor.

4-arylphthalazin-1(2H)-one derivatives as potent antagonists of the melanin concentrating hormone receptor 1 (MCH-R1).

A novel series of 4-arylphthalazin-1(2H)-one linked to arylpiperidines were synthesized and evaluated as MCH-R1 antagonists. The results of an extensive SAR study probing the effects of substituents on the 4-arylphthalazin-1(2H)-one C-4 aryl group led to the identification of the 4-(3,4-difluorophenyl) derivative as a highly potent MCH-R1 inhibitor with an IC(50)=1nM. However, further investigations showed that this substance has unacceptable pharmacokinetic properties including a high clearance and volume of distribution.

KR33426, [2-(2,5-dichlorophenyl)-5-methyloxazol-4yl]carbonylguanidine, is a novel compound to be effective on mouse systemic lupus erythematosus.

B cell-activating factor (BAFF) is a key regulator of B lymphocyte development. Signals from BAFF are transmitted through binding to a specific BAFF receptor (BAFF-R). Here, we established screening method to find a specific inhibitor for the interference of BAFF-BAFF-R interactions. We screened oxazole-4-carbonylguanidine derivatives and selected KR33426, [2-(2,5-dichlorophenyl)-5-methyloxazol-4yl]carbonylguanidine, as a candidate to interfere BAFF-BAFF-R interactions. KR33426 inhibited BAFF-mediated anti-apoptotic effect on splenocytes as judged by hypodiploid cell formation. KR33426 also increased the degradation of procaspase-3 that was inhibited by BAFF protein. In addition, we examined whether KR33426 was effective on the treatment of systemic lupus erythematosus-like symptom in MRL(lpr/lpr) mouse. When 5 or 10mg/kg KR33426 was intraperitoneally administered to MRL(lpr/lpr) mice for 4 weeks, histopathological changes were ameliorated in the narrowed space between renal glomerulus and glomerulus capsule. KR33426 reduced B220(+) B cell population and B cell mitogen, lipopolysaccharide-stimulated lymphocyte proliferation in splenocytes. KR33426 attenuated an increase in CD43(-)IgM(+) immature pro-B and a decrease in CD21(+) IgM(+) T2-B and IgD(+) IgM(-)recirculating-B cells on B cell development. Data show that KR33426 inhibits BAFF-BAFF-R interactions and it is effective on the treatment of systemic lupus erythematosus-like symptom in MRL(lpr/lpr) mice. Thus, it suggests that KR33426 is a novel candidate to develop anti-autoimmune therapeutics by the interference of BAFF-BAFF-R interactions, specifically.

An increase in B cell apoptosis by interfering BAFF-BAFF-R interaction with small synthetic molecules.

B cell-activating factor (BAFF) transmitted signals through binding to specific BAFF receptors (BAFF-R) to regulate B cell survival and development. We used MTT assay to examine the cytotoxicity of chemicals, flow cytometry analysis to measure BAFF-BAFF-R interactions, and western blotting to detect BAFF protein. Here, we established screening method to find specific compounds to interfere with BAFF-BAFF-R interactions in WIL2-NS B lymphoblast cells. According to screening (imidazol-4-ylcarbonyl)guanidine or (oxazol-4-ylcarbonyl)guanidine derivatives, we selected KR32592, KR32673, KR33232, KR33341 and KR33426 as candidates to interfere with BAFF-BAFF-R interaction. No cytotoxicity was detected by KR32592, KR33232, and KR33426 at the concentration of 5 µM, and by KR32673, and KR33341 at the concentration of 0.5 µM. Cell population with BAFF-BAFF-R interactions was reduced by the pre-incubation of chemicals with human BAFF-murine CD8 (BAFF-muCD8). Cell population with BAFF-BAFF-R interactions was also decreased by pre-exposure of WIL2-NS cells to chemicals prior to the incubation with BAFF-muCD8. Chemicals also inhibited LPS-stimulated BAFF production from splenocytes. All these effects of chemicals may contribute to the inhibition of BAFF-mediated anti-apoptosis. These data demonstrate that chemicals interfering with BAFF-BAFF-R interaction may be screened with our experimental condition. It suggests that BAFF-BAFF-R interaction could be a chemical target to develop therapeutics for BAFF-mediated autoimmune diseases.