Patent Application: US-99775704-A

Abstract:
the use of a monocarboxylate transporter protein or a functional part , derivative and / or analogue thereof for altering transport of a thyroid hormone or a functional part , derivative and / or analogue thereof across a membrane . the monocarboxylate transporter protein preferably comprises mct - 8 . an isolated molecule capable of specifically binding at least part of an mct protein , at least part of a ligand of an mct protein , or a nucleic acid encoding the mct and / or ligand , is also herewith provided . regulation of the bioavailability of thyroid hormone in a tissue enables interfering with diseases . the invention also provides pharmaceutical compositions comprising a compound capable of binding the mct protein or capable of influencing the binding or transporting of a ligand of the mct protein . methods for diagnosis and / or treatment of a disease , such as a disorder of thyroid metabolism , non - thyroidal illness , obesity or cardiovascular illness , are also provided , as well as bioassays for identifying or detecting a candidate drug capable of binding to , or influencing at least part of , the mct protein .

Description:
the invention is further explained in the following examples . the examples do not limit the scope of the invention ; they merely serve to exemplify the invention . xenopus oocytes were isolated and injected with 4 . 6 ng rat mcts crna . after 3 days , groups of 10 injected or uninjected oocytes were incubated for 60 mm at 25 c with 10 nm radioactive iodothyronines in 0 . 1 ml medium . uptake of iodothyronines was determined as previously described ( 18 ). the results are shown in fig1 . transport of amino acids by mcts in oocytes xenopus oocytes were isolated and injected with crna coding for rat mct8 or with crnas coding for the heavy chain ( 4f2 ) and the light chain ( lat1 ) of the heterodimeric human l - type amino acid transporter . after 3 days , groups of 10 injected or uninjected oocytes were incubated for 60 mm at 25 c with 10 μm radioactive leu , tyr , trp or phe in 0 . 1 ml medium . amino acid uptake was determined as previously described ( 18 ). the results are shown in fig2 . xenopus oocytes were isolated and injected with rat mcts crna . after 3 days , groups of 10 injected or uninjected oocytes were incubated for 60 mm at 25 c with radioactive t4 and increasing concentrations of non - radioactive t4 in 0 . 1 ml medium . t4 uptake was determined as previously described ( 18 ). the results are shown in fig3 . time course of t3 uptake by mcts in oocytes xenopus oocytes were isolated and injected with rat mcts crna . after 3 days , groups of 10 injected or uninjected oocytes were incubated for 5 - 60 mm at 25 c with 10 nm radioactive t3 in 0 . 1 ml medium . t3 uptake was determined as previously described ( 18 ). the results are shown in fig4 . materials — nonradioactive l - iodothyronines , 3 , 3 ′, 5 - triiodothyroacetic acid ( triac ) and n - bromoacetyl - 3 , 3 ′, 5 - triiodothyronine ( bract5 ) were obtained from henning berlin . d - iodothyronines , phe and tyr were purchased from sigma , leu was obtained from merck , and trp and bromosulfophthalein ( bsp ) were purchased from fluka . [ 3 ′, 5 ′˜ 125i ] t4 , [ 3 ′. 125i ] ts and carrier - free na ′ 251 were purchased from amersham biosciences . all other 125i - labeled compounds were prepared as previously described ( 21 ). 3h - labeled leu , phe , tyr , and trp were purchased from amersham biosciences . all other chemicals were of reagent grade . cloning of rat mct8 - primers for rt - pcr were designed to regions of high homology in the 5 ′ and 3 ′ untranslated region ( utr ) sequences of human ( 05315 ) and mouse ( af045692 ) mcts . the sense primer was 5 ′ agct ctcgag cggcaagccacagtcag 3 ′ ( seq id no : __ ) corresponding to the mouse sequence from nucleotide ( nt ) 145 ( the coding sequence starts at nt 175 ) and contained a xhoi restriction site ( underlined ). the antisense primer was 5 ′ aaatg cggccg cttctccgttggggtct 3 ′ ( seq id no : __ ) corresponding to the mouse sequence ending at nt 2242 ( the coding sequence ends at nt 1872 ) and contained a noti restriction site ( underlined ). isolated rat liver mrna was in vitro reverse transcribed and amplified using the reverse transcription system from promega and subjected to pcr using the above primers . the 2038 bp product was ligated into pgemt - easy and sequenced . insertion of a flag - tagged mct8 construct into the oocyte expression vector pgem - hejuel - for expression in oocytes it was decided to append an 8 amino acid “ flag ” epitope to the c - terminus of mct8 in order to allow detection of expression by immunofluorescence microscopy and western blotting . for this purpose the stop codon of mct8 was removed by performing pcr using the same sense primer as above , but with the modified antisense primer , 5 ′ acag cggccg caaatgggctcttcaggtgttg 3 ′ ( seq id no : __ ) which lacks the stop codon . this pcr product was ligated into pgemt - easy before being excised with ecori and ligated into the flag vector , pcmv - tag4a ( stratagene ), which had previously been digested with the same restriction enzyme and dephosphorylated . the flag epitope - tagged mct8 was then ligated into the xenopus oocyte expression vector pgem - hejuel which contains the 5 ′- and 3 ′- utrs of the xenopus b - globin flanking a multiple cloning site . the stability of the transcribed mct8 mrna sandwiched between the untranslated globin sequence is likely to be enhanced in the oocyte as was found to be the case for other mcts ( 22 ). the mct8 - flag insert was prepared using pcr with primers flanking the mcts - flag insert in pcm ˜ v ˜ tag4a and containing suitable restriction sites for the insertion into the pgem - hejuel vector . the sense primer was 5 ′ gcgg ggatcc acacgtcagtcccctagcca 3 ′ ( seq id no : __ ) and contained a bamhi restriction site ( underlined ) whilst the antisense primer was 5 ′ cvi ′ a tctaga taaggtaccgggccctact 3 ′ ( seq id no : __ ) and contained an xbai restriction site ( underlined ). following successful pcr amplification , the product was digested with bamhi and xbai and ligated into the pgem - hejuel vector digested with the same restriction enzymes . the correct identity of the product was established using an ecori digest and confirmed with sequencing . x . laevis oocyte expression — mct8 crna was obtained by in vitro transcription using the ampliscribe high yield ™ t7 rna transcription kit ( epicentre ) after linearization of pgem - hejuel containing the mcts cdna with noti . oocytes were isolated and allowed to recover overnight at 18 c in modified barth &# 39 ; s solution containing 20 iu / ml penicillin and 20 gg / ml streptomycin as described before ( 23 ). the next day , oocytes were injected with crna coding for mct8 , and further incubated for 3 days at 18 c in modified barth &# 39 ; s solution until analysis . uninjected or water injected oocytes were used as controls . western blotting of oocyte membranes — crude oocyte membranes were prepared using 10 oocytes harvested 3 days after microinjection with mcts or mct1 crna or the equivalent volume of water . oocytes were suspended in 500 pl of buffer ( 10 mm hepes , 83 mm nacl , 1 mm mgcl2 , ph 7 . 9 , containing 4 mg / ml pepstatin a , leupeptin , antipain and 0 . 5 mm phenylmethylsulfonyl fluoride and benzamidine ) and homogenized by vigorous vortex mixing followed by several passes through an 18 gauge needle . the homogenate was centrifuged at 2000 rpm for 5 mm at 4 c and the supernatant was collected , whilst the pellet was re - homogenized in 500 pl buffer and centrifuged as before . both the supernatants were then mixed and centrifuged at 100 , 000 g for 60 mm at 4 c and the pellet resuspended in 30 pl buffer and 30 pl of sds - page sample buffer . following separation by sds - page western blotting was performed using mouse anti - flag monoclonal antibody ( sigma ) or the specific mct1 antibody with detection by enhanced chemiluminescence ( ecl ) as described previously ( 24 ). preparation and staining of oocytes for immunofluorescence confocal microscopy — oocytes were first embedded in pieces of chicken liver and then placed on pieces of cork , covered in o . c . t . embedding compound ( tissue - tek , sakura finetek europe bv , the netherlands ) and frozen in liquid - nitrogen cooled isopentane . frozen sections ( 5 μm ) were cut , placed on silanized slides and air - dried at room temperature for 1 h before fixing with ice - cold acetone for 10 mm . permeabilization and staining were then carried out as previously described ( 24 ) using a mouse monoclonal anti - flag antibody and tritc - conjugated anti - mouse igg secondary antibody . samples were mounted with mowiol ( calbiochem ) and examined with a leica tcs - nt confocal scanning microscope ( 63 × 1 . 32 na oil immersion objective ). transport measurements in oocytes — oocytes were washed and preincubated at 18 c in standard uptake solution ( 100 mm nacl , 2 mm kcl , 1 mm cacl2 , 1 mm mgcl2 , 10 mm hepes , and 10 mm tris , ph 7 . 5 ). usually , transport was tested by incubation of groups of 5 - 10 oocytes for 2 - 60 mm at 25 c with 10 nm [ 1251 ] iodothyronines or 10 μm [ 3h ] amino acids in 0 . 1 ml standard uptake solution . the possible na + dependence of transport was tested by preincubation and incubation in na + - free uptake solution in which nacl was replaced by choline chloride . the influence of the temperature on the uptake rate was tested by the incubation of oocytes with uptake solutions at different temperatures between 4 and 37 c . the influence of albumin on uptake of t3 was tested by the addition of 0 . 1 % and 0 . 5 % bsa to the standard uptake solution . the incubation was stopped by aspiration of the medium , and oocytes were washed 4 times at 4 c with standard uptake solution containing 0 . 1 % bsa . the substrate specificity of mct8 was investigated by incubation of oocytes with different putative radioactive ligands , including t4 , t3 , 3 , 3 , 5 ′- triiodothyronine ( rt 3 ), 3 , 3 ′. diiodothyronine ( t2 ), na - sulfonated t4 ( t4 sulfamate , t4ns ) and 4 ′- oh - sulfonated t4 ( t4 sulfate , t4s ), and the amino acids leu , phe , tyr and trp . specificity of transport of 1251 - t4 and 12 ˜ i . t3 was measured in the presence of putative competitors , including 10 μm unlabeled iodothyronine derivatives such as d - and l - iodothyronines , triac and bract3 , and 100 pm tyr , trp or bsp . analysis of rt3 metabolism in oocytes — groups of 10 oocytes were incubated for 2 - 60 mm at 25 c with 10 nm [&# 39 ; 25iirta . after incubation , medium was collected , and 2 groups of 5 oocytes were counted separately and homogenized in 0 . 1 ml 0 . 1 m naoh as described before ( 25 ). lysates were cleared by centrifugation . lysates ( in duplicate ) and incubation media were acidified with 0 . 1 m hcl and analyzed by sephadex lh - 20 chromatography ( 21 ). the different products were successively eluted with 0 . 1 m hcl ( iodide ), water ( conjugates ), and 1 % nh4oh in ethanol ( iodothyronines ). transport kinetics — saturation of iodothyronine uptake in mct8 crna injected oocytes was analyzed in incubations containing labeled and unlabeled t4 , t3 , or rt3 at final concentrations of 1 nm - 30 μm . apparent km values were calculated by fitting the plot of uptake rate ( v ) versus ligand concentration ( 5 ) to the michaelis menten equation : v = vmax /( 1 + km / s ), wherein vmax is the maximum uptake rate , and km the michaelis constant . calculations were performed using the slide write plus program version 5 . 01 ( advanced graphics software ). statistics . results are expressed as means ± sem . statistical significance was determined using the student &# 39 ; s t test for unpaired observations . cloning of rat mct8 — the coding sequence of rat mct8 was cloned using rt - pcr from rat liver mrna as described in the experimental section and has been assigned the accession code nivij47216 ( gi : 22219453 ). the translated protein sequence is shown in fig5 where it is aligned with human ( 1105315 ) and mouse ( af045692 ) mct8 sequences . as would be predicted , the rat and mouse sequences show very few differences with only four amino acids changes and the insertion of a 20 amino acid repeat in the mouse pest sequence which is absent in the rat and human sequences . the predicted molecular mass of the protein is 60 . 1 kda . expression of the flag - tagged mct8 in xenopus oocytes — we initially sought to express the mct8 in oocytes but the antibody we raised to the c - terminus of the protein ( which we have used successfully for production of antibodies against other mcts ) failed to detect native and recombinant rat mct8 . thus , we expressed mct8 with a flag epitope attached to the c - terminus as described in the experimental section . the cloning strategy used means that the c - terminus is extended by the following sequence ( flag - tag underlined ) caavitsefdiklidtvdle dykdijddk ( seq id no : __ ) giving a predicted molecular mass 63 . 3 kda . in fig6 , we present western blots using an anti - flag antibody of membranes derived from oocytes injected with water ( control ), mct1 ( 26 ) or mct8 - flag crna . a band of about 63 kda is present only in the membranes from the mct8 crna - injected oocytes ( lanes 3 and 4 ) confirming the presence of the expressed flag - tagged mct8 . the band at 38 kda is a flag - sensitive band in x . laevis oocytes . plasma membranes from oocytes injected with mct1 crna showed no response to the flag antibody but did show a 43 kda protein corresponding to mct1 with the specific mct1 antibody ( lane 2 , lower part ). the latter failed to detect the mct8 as would be expected in view of the lack of similarity between the c - termini of mct8 and mct1 . in order to confirm that mct8 was expressed at the plasma membrane we performed immunofluorescence microscopy on sections of oocytes , again using the anti - flag antibody . the data are shown in fig7 and reveal that mct8 is strongly expressed at the plasma membrane . water - injected oocytes showed no such expression ; nor did the secondary antibody alone detect any protein at the plasma membrane . functional characterization — transport studies were performed to investigate the function of mct8 expressed in x . laevis oocytes . fig8 shows the time course of uptake of t3 and t . 1 in uninjected oocytes and in oocytes injected with 4 . 6 ng mct8 crna . expression of mct8 induced a ˜ 10 - fold increase in initial uptake of t3 and t4 compared with uninjected oocytes . this graph also shows that the uptake of t3 and t4 into mct8 crna - injected oocytes was only linear for the first 4 mm . therefore , all further transport experiments were performed at 2 mm incubations . the difference in uptake of t3 and t4 in oocytes injected with mct8 crna is not statistically significant . transport of t3 was independent of na + as the same results were obtained using medium with choline chloride instead of nacl ( fig8 a ). however , t4 transport by mct8 showed a modest but consistent inhibition in the absence of na + ( fig8 b ). fig9 shows the influence of the amount of crna injected on the uptake of 10 nm t3 and 1 ′ 4 . the results indicate that the lowest amount of mct8 crna ( 0 . 23 ng ) injected already induced a 5 - fold increase in t3 transport and a 7 . 3 - fold increase in t4 transport . maximum induction of t3 and t4 transport was found after injection of 1 . 15 - 2 . 3 ng of mct8 crna . therefore , oocytes were further injected with 4 . 6 ng mct8 crna for maximum induction of iodothyronine transport . to test the temperature dependence of mct8 - mediated iodothyronine transport , oocytes were incubated with 10 nm [ 1251 ] t 3 or [ 1251 ] t4 for 2 mm at 4 - 37 c . fig1 shows significant uptake for t3 and t4 into the oocytes at 4 c , with marked increases if the temperature was increased to 15 , 25 and 37 c , which is above the usual ambient temperature for frog oocytes . exposure to 37 c was tolerated by the oocytes during this short incubation time ; prolonged incubation at 37 c resulted in disintegration of the cells . the results show identical temperature dependence of transport of t4 and t3 by mct8 . fig1 shows the effects of addition of 0 . 1 % and 0 . 5 % bsa on the uptake of t 3 in oocytes . both in uninjected oocytes and in mct8 crna - injected oocytes , t 3 uptake was decreased concentration - dependently by bsa . however , the fold stimulation of t 3 uptake by mct8 increased from 75 in the absence of bsa to 14 in the presence of 0 . 1 % bsa , and further to 25 in the presence of 0 . 5 % bsa . substrate specificity — the substrate specificity of mct8 was investigated by incubation of oocytes with different putative radioactive ligands , including t4 , t3 , rt 3 , t2 , tins and t4s , and the amino acids leu , phe , tyr and trp . fig1 shows that in contrast to the rapid uptake of the different iodothyronines , t4ns and t4s and the amino acids are not transported by mct8 . mct8 failed to transport the different amino acids at concentrations ( 1 - 100 pm ) showing facile transport by the lat1 ( 18 ) and tat1 ( 19 , 20 ) amino acid transporters . we have also been unable to demonstrate any transport of [&# 39 ; 4c ] lactate , whereas in oocytes injected with rat mct1 crna , transport of [ 14c ] lactate was greatly stimulated compared to control oocytes , but no additional uptake of [ 125i1t4 was observed ( data not shown ). the specificity of iodothyronine transport by mcts was further addressed by investigating the uptake of [ 125i ] t 3 and [&# 39 ; 25i ] t4 in the absence or presence of structurally related compounds ( table 1 ). uptake of labeled t4 and t3 by mct8 was potently inhibited by 10 pm unlabeled l - t4 , d - t4 , l - t3 and dt3 , indicating that the interaction of t4 and t3 with mct8 is not stereospecific . lodothyronine uptake by mct8 is also potently inhibited by t3 analogs where the anh2 group is blocked ( bract3 ) or deleted ( triac ), indicating that this anh2 group is not important for interaction of iodothyronines with mct8 . the t - type amino acids tyr and trp hardly affected iodothyronine uptake by mct8 . the organic anion bsp proved to be a potent inhibitor . this , in contrast , to the weak effects of the bile acid taurocholate and the organic anion transport inhibitor probenecid ( data not shown ). in general , t3 transport by mct8 shows less inhibition by these compounds than mct8 - mediated t4 transport . it is remarkable that ligands and potent inhibitors of mct8 all carry halogen atoms . metabolism of rt 3 — previous studies have shown that x . laevis oocytes actively metabolize rt 3 by sulfation . since sulfotransferases are located intracellularly , we have used this property to investigate rt 3 internalization by oocytes . fig1 shows the time course of rt 3 uptake and subsequent metabolism in native and mct8 - expressing oocytes . at several time points , uninjected and mct8 crna - injected oocytes are homogenized , and the homogenates and medium samples are analyzed for rt 3 and rt 3 sulfate ( rt 3 s ) content . it is shown that already after 2 mm of incubation , rt 3 s is formed intracellularly in the mct8 crna - injected oocytes . after 10 mm of incubation , release of rt 3 s into the medium is observed . the production rate of rt 3 s is much higher in mct8 crna - injected oocytes than in uninjected cells . these findings demonstrate that mct8 - mediated uptake of iodothyronines indeed represents the internalization of these compounds . transport kinetics — the kinetics of mct8 - mediated iodothyronine transport were analyzed by incubation of oocytes with increasing t4 , t3 or rt3 concentrations ( 1 nm - 30 pm ). net transport by mct8 was calculated by subtracting uptake in uninjected oocytes from that in mct8 crna - injected oocytes . the results showed that transport of t4 , t3 and rt 3 by mct8 was saturable . michaelis - menten analysis of the data provided apparent km values of 4 . 7 pjv1 for t4 , 4 . 0 pm for t3 and 2 . 2 μm for rt3 ( fig1 ). 1 . larsen , p . r . and m . j . berry . 1995 . nutritional and hormonal regulation of thyroid hormone deiodinases . annu . rev . nutr . 15 : 323 - 352 . 2 . leonard , j . l . and j . kohrle . 1996 . intracellular pathways of iodothyronine metabolism . in the thyroid , l . e . braverman and r . utiger , editors . lippincott - raven , philadelphia , 125 - 161 . 3 . st . germain , d . l . and v . a . galton . 1997 . the deiodinase family of selenoproteins . thyroid 7 : 655 - 688 . 4 . hennemann , g . and t . j . visser . 1997 . thyroid hormone synthesis , plasma membrane transport and metabolism . in handbook of experimental pharmacology , vol 128 ; 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