Patent Application: US-66150505-A

Abstract:
the present invention relates to thioxothiazolidinone compounds for use as pharmaceuticals , to pharmaceutical compositions comprising these compounds , and to the use of said small - molecule compounds for the manufacture of pharmaceutical compositions for the treatment of conditions dependent on leukocyte cell migration , such as leukaemia and inflammatory diseases . said compounds inhibit leukaemia cell migration by stabilizing the active conformation of the α m integrin i domain .

Description:
we have identified a novel class of small molecule ligands , which stabilize the active conformation of the α m i domain . these compounds are structurally distinct from the previously characterized α m and α l i domain antagonists ( bansal et al ., 2003 ; kelly et al ., 1999 ; liu et al ., 2001 ; shimaoka et al ., 2003 ; weitz - schmidt et al ., 2001 ). imb - 10 , the most potent of the identified compounds , increased the binding of recombinant α m i domain to its ligands prommp - 9 and fibrinogen . remarkably , imb - 10 also made α m β 2 integrin - expressing cells highly resistant to the effect of the cation chelator edta , consistent with the chemical &# 39 ; s role as a stabilizer of the active α m i domain . the imb - 10 compound was also a highly potent inhibitor of α m β 2 integrin - mediated leukemia cell migration . although the compounds of formula i were identified as inhibitors of ddgw - peptide bearing phage binding to the α m i domain , they failed to inhibit the prommp - 9 / α m i domain interaction . this difference may be traced to the fact that we initially identified the ddgw peptide and the prommp - 9 / α m β 2 integrin interaction in a calcium - containing buffer ( stefanidakis et al ., 2003 ). our data suggests that ddgw peptide preferentially binds to the closed conformation of the i domain , whereas prommp - 9 binds both open and closed conformation , although preferring the open conformation . the inability of imbs to inhibit prommp - 9 interaction with the i domain clearly indicates that the imbs and ddgw bind to different sites . most small - molecule compounds are uncharged and thus may not occupy the same binding site as a charged peptide or protein ligand . such charged sequence motifs are typical for integrin ligands ( arnaout et al ., 2002 ). in accordance with this , all small - molecule ligands of the α l i domain are allosteric antagonists ( kelly et al ., 1999 ; last - barney et al ., 2001 ; liu et al ., 2001 ; weitz - schmidt et al ., 2001 ). indeed , it may be difficult to identify small - molecular compounds that directly mimic the action of such charged peptide ligands . in this respect , phage display can provide novel ligands to sites that cannot be well occupied by small - molecule compounds used in high - throughput screenings . apparently , phage display can reveal biologically important sites , which would remain unnoticed in small - molecule screenings . the activation state of the recombinant α m and α l i domains can be regulated by a site distinct from the ligand - binding metal ion dependent adhesion site ( midas ) ( kallen et al ., 1999 ; xiong et al ., 2000 ). a single point mutation ile 316 → gly near the c terminus of the α m i domain locks the i domain in the constitutively active , open conformation ( xiong et al ., 2000 ). in the closed α m i domain structure this ile 316 residue lies in a hydrophobic socket that is in a nearly analogous location , where lovastatin binds in the α l i domain ( kallen et al ., 1999 ). in the absence of a ligand , a closely balanced equilibrium between the open and closed conformation of the α m i domain is evident , with 10 - 12 % of the protein present in the open conformation . instead , the α l i domain is exclusively in the closed conformation ( mccleverty and liddington , 2003 ). thus there is a high possibility to obtain ligands for the active conformation of the α m i domain . although imb - 10 did not inhibit prommp - 9 binding , it was far more potent inhibitor of leukemia cell migration than the ddgw peptide . the imb - 10 interfered only with β 2 integrin - dependent migration , as there was no effect on the migration of ht1080 fibrosarcoma cells , which express other integrins . the ability of imb - 10 to inhibit cell migration on fibrinogen appears to be independent on gelatinase activity , as the small - molecule gelatinase inhibitor ( inhi ) did not block cell migration . furthermore , imb - 10 did not inhibit pericellular gelatinase - dependent proteolysis of upar . collectively our data indicates that the inhibition of leukemia cell migration by imb - 10 is caused primarily due to enhanced adhesion and not by inhibition of integrin - regulated gelatinase activity . too strong adhesion inhibits cell motility ( palecek et al ., 1997 ) and this phenomenon may be utilized to develop antagonists of cell migration as exemplified by our studies . screening of the compound library . a combinatorial library of 10 000 small molecules was purchased from chembridge ( san diego , calif .). a competition assay with the ddgw peptide bearing phage was set up by immobilizing 20 ng / well recombinant α m i domain - gst fusion in 96 - well plates ( michishita et al ., 1993 ). the compounds were used in pools comprising eight compounds , each at a 5 μm concentration and dmso at a 1 . 25 % concentration . after preincubation of the compounds in the wells , ddgw phage was added ( 3 × 10 8 transducing units / well ). as controls , soluble ddgw peptide and a control peptide , and an unrelevant phage were included in every plate . phage binding was detected with an anti - phage antibody as described ( bjorklund et al ., 2004 ). pools with inhibitory activity were re - tested as single compounds . the ddgw - phage inhibiting activity of these hits at a 10 μm concentration is shown in supplementary data . to calculate the ic 50 values for the compounds , dilution series from the ddgw peptide and compounds were made ( 10 μm to 150 nm ) and ddgw phage binding was measured . no inhibitor ( dmso as a vehicle ) was used as 100 % binding after subtracting the background value obtained with an irrelevant control phage . prommp - 9 and fibrinogen binding to the α m integrin i domain . the catalytically inactive prommp - 9 - δhc - e 402 q mutant was prepared via site - directed mutagenesis from the wild - type prommp - 9 - δhc and was purified using gelatin - sepharose ( bjorklund et al ., 2004 ). the resulting mmp - 9 with this mutation is structurally identical to the wild type protein ( rowsell et al ., 2002 ). prommp - 9 - δhc - e 402 q , intact prommp - 9 or fibrinogen ( 100 ng / well ) was coated on microtiter wells in tbs followed by saturation of the wells with 1 % bsa in pbs / 0 . 05 % tween20 . soluble α m integrin i domain - gst fusion ( 2 . 5 μg / ml ) was added in the presence or absence of peptides or compounds in 0 . 1 % bsa / tbs / 0 . 05 % tween20 / 1 mm cacl 2 / 1 mm mgcl 2 , and incubated for one hour . in some experiments 10 mm cacl 2 or 10 mm mgcl 2 were used instead of 1 mm cacl 2 / 1 mm mgcl 2 . bound gst fusion was detected with anti - gst antibody ( 1 : 2000 dilution ) and peroxidase - conjugated anti - goat antibody ( 1 : 2000 dilution ). inhibition of antibody binding to the integrins . the α m i domain or purified α m β 2 integrin ( 50 ng / well ) were coated on microtiter wells followed by saturation of the wells with 1 % bsa in pbs / 0 . 05 % tween20 . the ddgw peptide or the compounds at concentrations indicated were preincubated for 30 minutes with the integrin , followed by addition of the antibodies lm2 / 1 , mem170 , okm - 10 , ib4 or a control igg at a 1 μg / ml final concentration . the antibodies lm2 / 1 and mem170 recognize the α m integrin i domain , whereas the epitope for okm - 10 is located outside the α m i domain ( koivunen et al ., 2001 ; li et al ., 1995 ; stefanidakis et al ., 2003 ). the antibodies were incubated for 45 minutes followed by detection of the bound antibodies with a peroxidase conjugated anti - mouse antibody . cell culture . human monocytic leukemia thp - 1 , oci - aml - 3 acute myeloid leukemia and human ht1080 fibrosarcoma cells were cultured as described ( koivunen et al ., 1999 ; koivunen et al ., 2001 ; stefanidakis et al ., 2003 ). the cell viability was measured using an 3 -[ 4 , 5 - dimethylthiazol - 2 - yl ]- 2 , 5 - diphenyl tetrazolium bromide assay as described ( bjorklund et al ., 2004 ). cell adhesion and migration . cell adhesion was performed as described ( koivunen et al ., 2001 ). briefly , thp - 1 cells ( 50 000 / well ) were stimulated with 50 nm pdbu ( 4β - phorbol - 12 , 13 - dibutyrate , sigmaaldrich ) and allowed to bind to the fibrinogen coated microtiter wells ( 10 μg / ml ) in the presence or absence of compounds or dmso in serum - free rpmi medium containing 0 . 1 % bsa . non - adherent cells were removed by gentle washing with pbs or 2 . 5 mm edta in pbs , and adherent cells were quantitated with a phosphatase assay ( koivunen et al ., 2001 ). alternatively , thp - 1 cells were stimulated with 20 nm pdbu and allowed to adhere on uncoated plastic overnight . nonadherent cells were removed by washing with pbs followed by six washes with 2 . 5 mm edta in pbs . cell migration was done using transwells ( 5 μm pore size ) coated with 40 μg / ml llg - c4 - gst or fibrinogen ( koivunen et al ., 2001 ). thp - 1 and oci - aml - 3 cells ( 50 000 cells / 100 μl ) were allowed to migrate overnight in 10 % fbs / rpmi medium in the presence or absence of peptides or compounds and stimulating cell migration with 40 nm pdbu . gelatinase - selective inhibitor i ( inhi ) at a 50 μm concentration ( calbiochem ) was used to evaluate the level of gelatinase - dependent migration on fibrinogen . migrated cells were stained with crystal violet and counted ( koivunen et al ., 2001 ). migration of ht1080 cells on serum coated transwells was conducted as described ( koivunen et al ., 1999 ). cellular cleavage of the urokinase - plasminogen activator receptor . pericellular gelatinase - dependent upar cleavage was performed as described ( bjorklund et al ., 2004 ). briefly , oci - aml - 3 cells were stimulated with pdbu or left untreated and cultured for 48 hours in the presence of 20 μm imb - 10 , the gelatinase - selective inhibitor ( inhi ) or vehicle ( dmso ). upar and the cleaved form of upar d2 + 3 was detected with western blotting using a polyclonal antibody to upar ( 399r , american diagnostica ). neutrophil emigration in vivo . the mouse experiments were approved by the ethical committee for the animal experiments at the university of helsinki . inflammation was induced in balb / colahsd female maintained in the viikki laboratory animal centre by injecting 1 ml 4 % thioglycollate broth in pbs i . p . followed by intravenous injections of chemicals ( 200 μl at a 20 , 5 or 1 μg / ml concentration ) or pbs after five minutes . cells emigrated in the peritoneum after 3 or 24 h were counted using a hemocytometer . statistical significance between groups in the peritonitis model was calculated with one - way anova and found differences were analyzed by bonferroni pairwise multiple comparison tests . results with p & lt ; 0 . 01 were deemed significant . we screened a combinatorial library in order to identify small - molecules , which would block leukemia cell migration by inhibiting the previously characterized interaction between prommp - 9 and the leukocyte integrins . several compounds were identified in this screen , most of which had a common thioxothiazolidinone substructure ( fig1 and fig7 ). the structures of ic3b / α m β 2 interaction blocking compounds ( bansal et al ., 2003 ) are shown for comparison ( fig1 a ). the newly identified compounds specifically inhibited ddgw - phage binding to the α m i domain , but had no effect on binding of crv - peptide to the c terminal domain of mmp - 9 ( bjorklund et al ., 2004 ) ( data not shown ). representative compounds imb - 2 , - 6 , - 8 and - 10 ( fig1 b ) were tested to measure their ic 50 values for the inhibition of ddgw - phage binding to the α m i domain ( fig2 a ). the best compound , imb - 10 had an ic 50 value of 0 . 4 ± 0 . 2 μm , six - fold better than the ic 50 value 2 . 6 ± 0 . 5 μm for the soluble ddgw peptide . these compounds also inhibited ddgw phage binding to the α l i domain ( not shown ), but this interaction is significantly weaker than the binding to the α m i domain ( stefanidakis et al ., 2003 ). we next evaluated the effect of the compounds on prommp - 9 binding to α m i domain . the α m i domain binding site in mmp - 9 is mapped to the ddgw - like negatively charged sequence present in the catalytic domain of mmp - 9 . however , it is unknown if the i domain binding activity is dependent on the catalytic activity of the mmp - 9 as gelatinase - selective inhibitors block this interaction ( stefanidakis et al ., 2003 ). to resolve this question , we prepared a catalytically inactive prommp - 9 mutant lacking the collagen v - like hinge region and the c - terminal domain ( prommp - 9 - e 402 q - δhc ). strong binding of soluble α m i domain - gst to immobilized prommp - 9 - e 402 q - δhc was observed and the level of binding was comparable to intact prommp - 9 ( fig2 b ). gst alone did not bind prommp - 9 . the interaction of α m i domain to prommp - 9 - e 402 q - δhc was inhibited by the ddgw peptide ( fig2 c ). surprisingly , although identified by the ddgw phage assay , none of the chemicals inhibited binding of α m i domain to prommp - 9 - e 402 q - δhc ( fig2 c ) or prommp - 9 ( not shown ) at a 50 μm concentration . instead , the α m i domain binding activity was markedly enhanced by the imbs . the level of activation correlated with their ic 50 values in the phage assay . the α m i domain binding to fibrinogen was enhanced by even more in the presence of the chemicals ( fig2 d ). this unexpected behaviour suggested that ddgw and the identified chemicals do not compete for the same binding site and that the imbs might actually stabilize the active conformation of the α m i domain . the compounds of formula i thus show a novel activity causing enhanced ligand - binding activity in contrast to the previously identified small - molecule ligands to the α l i domain , which inhibit ligand binding . to investigate the possible stabilization of the α m i domain , binding assays were conducted in the presence of 10 mm ca 2 + or 10 mm mg 2 + to maintain the i domain in the inactive or active conformation , respectively . a strong binding of α m i domain to prommp - 9 - e 402 q - δhc was observed in the presence of magnesium and imb - 10 at a 10 μm concentration , whereas calcium nearly completely antagonized the effect of imb - 10 ( fig3 a ). binding in the presence of equimolar concentrations of ca 2 + and mg 2 + was intermediate to that of ca 2 + and mg 2 + alone . similar effect was obtained with α m i domain binding to fibrinogen ( fig3 b ). we next evaluated the ability of ddgw peptide and the chemicals to compete with antibody binding to immobilized α m i domain - gst fusion . the imbs inhibited mab lm2 / 1 binding in accordance their ddgw - peptide inhibitory potency , with 75 % inhibition by imb - 6 and - 10 at a 50 μm concentration ( fig4 a ). the compounds also inhibited the binding of another α m i domain specific antibody mem170 . the ddgw peptide or lovastatin had only a marginal effect on antibody binding . the imbs also inhibited lm 2 / 1 antibody binding to purified α m β 2 . binding of mab ib4 , was also inhibited by imb - 10 . the epitope of this antibody is located in the β 2 i - like domain ( fig4 b ). binding of ts1 / 22 and mem83 antibodies to α l i domain were similarly affected by the imb - 10 chemical , but not by imb - 8 . before conducting cell - based experiments , we tested possible toxicity of the chemicals . the compounds were incubated with thp - 1 monocytic leukemia and oci - aml - 3 acute myeloid leukemia cells for 48 h in serum - containing cell culture medium . the imb - 6 was toxic to both cell lines apparently due to low solubility , whereas imb - 2 , imb - 8 and imb - 10 had no significant effect on cell proliferation at a 50 μm concentration ( data not shown ). adhesion to uncoated cell - culture plastic is a hallmark of α m β 2 integrin activity ( yakubenko et al ., 2002 ). when thp - 1 cells were cultured on plastic in the presence of 20 nm pbdu , they became strongly adherent and were resistant to washing with pbs ( fig5 a ). the chemicals had no effect on this activity . interestingly , when the cells were washed with 2 . 5 mm edta , a significant portion of the imb - 10 treated cells resisted detachment and remained adherent ( fig5 a and b ). the imb - 8 compound or a chemical gelatinase inhibitor ( inhi ) did not have such an effect . increased resistance to detachment was also observed on fibrinogen substratum . again , the chemicals had no measurable effect on pbs washing , but imb - 10 treated cells were more resistant to washings with edta ( fig5 c ). similar results were obtained with oci - aml - 3 cells ( not shown ). in the absence of phorbol ester activation , imb - 10 did not increase cell adhesion indicating that it stabilizes the active i domain rather than activates it ( data not shown ). the effect of the compounds on cell migration was evaluated in a transwell assay . thp - 1 cells migrate on a synthetic llg - c4 - gst peptide coating in a β 2 integrin dependent manner ( koivunen et al ., 2001 ). here , imb - 10 potently inhibited cell migration as did the llg - c4 peptide ( fig6 a ). the less active compounds imb - 2 and - 8 did not siginificantly inhibit cell migration at a 25 μm concentration . at a 100 μm concentration , imb - 2 also became inhibitory ( not shown ). similar results were obtained by studying α m β 2 integrin - dependent migration on fibrinogen - coated transwells . again , imb - 10 completely inhibited migration of thp - 1 and oci - aml - 3 cells at a 25 μm concentration and imb - 2 and - 8 were less active ( fig6 b ). an inhibitory effect was also obtained by ddgw , but not by the gelatinase inhibitor inhi . the α m i domain - binding chemicals did not have any effect on β 2 integrin - independent cell motility . results for ht1080 fibrosarcoma cells are shown in fig6 c . the migration of these cells was partially inhibited with the gelatinase selective inhibitor inhi . as prommp - 9 interacts with α m β 2 integrin on the cell surface , we evaluated the effect of mbs on pericellular gelatinase activity . we have previously shown that pericellular proteolysis of urokinase - plasminogen activator ( upar ) is dependent on gelatinase activity . oci - aml - 3 cells stimulated with pdbu showed a high level of cleaved upar d2 + 3 form , which does not bind to the urokinase - plasminogen activator or α m β 2 integrin . the cleavage of upar could not be inhibited with imb - 10 at a 20 μm concentration , whereas 20 μm gelatinase - selective inhibitor ( inhi ) reduced upar proteolysis indicating that the activated integrin does not prevent gelatinase - mediated proteolysis ( fig6 d ). suppression of inflammation in vivo . as leukocyte integrins are needed for proper inflammatory response , we tested the functionality of imb - 10 in thioglycollate - induced peritonitis in mice . in this model , the ddgw peptide potently inhibits the emigration of activated neutrophils into the peritoneal cavity ( stefanidakis et al ., 2004 ). intravenously injected imb - 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