Patent Application: US-63172505-A

Abstract:
the present invention provides for methods and systems for detecting steroids . examples of such steroids include estrogen , progesterone , androgen , testosterone , and derivatives and analogs thereof . systems useful for carrying out the method include tripartite constructs including a dna - binding domain , a ligand binding domain , and an activation domain . the present invention provides numerous improvements over previous diagnostic systems for detection of steroids , such advantages include that the method allows for detection of steroid analogs and derivatives , whose structures may not yet be known , the method is generally applicable to a wide variety of organisms , and numerous ligand binding domains may be used in conjunction with the present system .

Description:
the present invention relates to methods and systems for detecting steroids . in contrast to previously known methods for steroid detection , the present invention is able to detect derivatives and analogs of naturally occurring steroids . any molecule that binds with the ligand binding domain of the tripartite construct can be detected by the method of the present invention . thus the invention provides for detection of previously unknown molecules that affect the steroid hormone receptor . such molecules may include derivatives of anabolic steroids being manufactured as athletic performance aids . the systems and methods of the present invention provide for use of short nucleic acid sequences in each of three domains forming the tripartite system . advantageously , the use of short nucleic acid sequences encoding these protein domains makes it unnecessary to make and manipulate entire proteins for each portion of the construct . additionally , the tripartite systems of the present invention are extremely versatile because various tripartite constructs may be created by mixing and matching domains . in a particular embodiment , the expression plasmids of the invention may be used in the generation of cell lines or cellular systems that express the proteins described herein . such cell lines exhibiting ligand - dependent transactivation pathways may be used to identify molecules that impact the steroid hormone receptor transactivation pathway . this expression system has utility in methods for assaying materials for agonistic or antagonistic activity toward the steroid hormone receptor of interest in each system . for example , assays may be established whereby intact cells expressing the proteins of the invention are contacted with agents or materials suspected of affecting the intracellular activity of the steroid hormone receptor , and the affect of such agents on ligand - dependent transactivation activity measured . the effect of such agents on the ligand - dependent transactivation activity may be measured in any number of ways . for example , such cell systems may utilize a reporter system in which the production of the reporter signal is dependent on ligand - dependent transactivation . numerous reporters may serve equally well in this application including , but not limited to , β - galactosidase , alkaline phosphatase , fluorescent green protein , luciferase and the like . assays involving the cell based systems of the invention may be formatted in any number of configurations . particularly useful for evaluating large numbers of agents and materials are high throughput screening formats . traditionally , such assays were typically formatted in 96 well plates . however , 384 , 864 , 1536 and larger well plates may be used in such high throughput assay systems . these systems are often automated using robotic technologies to allow manipulation and processing of large numbers of samples . the agents or materials that may be evaluated in the various assay methods of the invention for potential agonistic or antagonistic affects include but are not limited to small molecules , polymers , peptides , polypeptides , proteins , immunoglobulins or fragments thereof , oligonucleotides , antisense molecules , peptide - nucleic acid conjugates , ribozymes , polynucleotides and the like . biochips , dna chips or dna microarrays can also be used with the present invention . a “ biochip ” comprises a suitable solid substrate . by “ substrate ” or “ solid support ” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the tripartite constructs , either as part of a plasmid or used alone , and amenable to at least one detection method . as will be appreciated by those in the art , the number of possible substrates are very large , and include , but are not limited to , glass and modified or functionalized glass , plastics ( including acrylics , polystyrene and copolymers of styrene and other materials , polypropylene , polyethylene , polybutylene , polyurethanes , teflonj , etc . ), polysaccharides , nylon or nitrocellulose , resins , silica or silica - based materials including silicon and modified silicon , carbon , metals , inorganic glasses , plastics , etc . in general , the substrates allow optical detection and do not appreciably fluoresce . generally the substrate is planar , although as will be appreciated by those in the art , other configurations of substrates may be used as well . for example , the probes may be placed on the inside surface of a tube , for flow - through sample analysis to minimize sample volume . similarly , the substrate may be flexible , such as a flexible foam , including closed cell foams made of particular plastics . in a particular embodiment , the surface of the biochip and the probe ( i . e ., tripartite system or tripartite construct ) may be derivatized with chemical functional groups for subsequent attachment of the two . thus , for example , the biochip is derivatized with a chemical functional group including , but not limited to , amino groups , carboxy groups , oxo groups and thiol groups , with amino groups being particularly useful . using these functional groups , the probes can be attached using functional groups on the probes , for example using linkers as are known in the art . in addition , in some cases , additional linkers , such as alkyl groups ( including substituted and heteroalkyl groups ) may be used . in a particular embodiment , a biochip includes various tripartite systems of the present invention such that a single biochip may be used to screen for estrogen , progesterone , testosterone , androgen , etc . and each steroid may be monitored in duplicate , triplicate or higher frequency . the tripartite systems may be attached to the biochip by techniques know to those skilled in the art . another configuration of the invention is the use of reporter genes that provide viability under selective growth conditions . such reporter genes include , but are not limited to , the his3 and ura3 biosynthetic genes . such viability selection assays can be used in a variety of schemes , including , but are not limited to , the identification of tripartite constructs with altered specificity in ligand recognition or the identification of new agonists . expression of some reporter genes , including , but are not limited to , ura3 and can1 , results in lethality when cells are grown in the presence of nontoxic compounds that are converted to a toxic compound by the enzyme encoded by the reporter genes . such reporter genes and compounds that can be converted into toxic forms can be used in schemes to reduce expression of the reporter genes , allowing the identification of antagonists . another feature of the invention includes kits to facilitate the use of the compositions and methods disclosed herein . exemplary kits would include the expression plasmids of the invention , and / or variants thereof . also , included would be protocols for use of the compositions of the invention for the particular application and the necessary reagents to carry out the application . such reagents may include , but are not limited to , buffers , solvents , media and solutions , substrates and cofactors , vectors and host cells , and detection or reporter reagents . accessory items may include vials , vessels , reaction chambers and instructions . serum contains a myriad of components ( i . e ., sugars , amino acids , etc .) that may in some situations affect the assay . to at least partially circumvent potential inhibitory or non - specific effects , extracted samples may be tested . steroids may be separated from serum by any method known in the art . in a particular embodiment , strata - x solid phase extraction cartridges from phenomenex ( torrence , calif .) are used to partially purify steroids from serum . the tripartite system may be used to monitor estrogen levels of per - or post - menopausal women , for example . whole blood collected from the individual is separated and purified as described above . at least 3 aliquots of 50 - 200 μl extracted samples are added to a well plate . 50 - 200 μl of estrogen methanol is added to three unused wells , which will serve as controls running in triplicate . 100 μl of host cells , expressing the tripartite protein containing estrogen receptors and possessing dna capable of expressing β - galactosidase , is added to each of the wells . the cells are grown at 30 ° c . in a shaking water bath or in a temperature controlled air incubator for 18 hr . 25 μl of cells from each well are transferred to a secondary well plate and 25 μl of beta - glo ( promega , madison wis .) are added to each well of the secondary plate . after 30 min incubation at room temperature , luminescence is detected using a bio - rad lumimark luminometer . the detection level of the present invention is 10 - 1000 pg / ml . for comparison , the mean level of estradiol in healthy women is 50 - 150 pg / ml and in menopausal women is 10 - 20 pg / ml . the tripartite system of the present invention may be used to detect and measure progesterone levels . low progesterone levels have been linked to increased risk of miscarriage for pregnant women . whole blood is drawn from an individual and processed as described above . in this embodiment , however , progesterone is used as the control and the host cells contain tripartite constructs with progesterone receptors rather than estrogen receptors . progesterone levels detectable by this method range from 1000 - 100 , 000 pg / ml . during the first trimester of pregnancy , progesterone levels range from 10 to 90 ng / ml ( 10 , 000 - 90 , 000 pg / ml ). for pregnant women with lower levels , physicians may prescribe progesterone therapy . the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . yeast strain w303 yarup16 mata / matα ade2 - 1 / ade2 - 1 , can1 - 100 / can1 - 100 , his3 - 11 , 151his3 - 11 , 15 , trpl - 1 / trpl - 1 , ura3 - 1 / ura3 :: lex8op - lacz , leu2 - 3 , 112 / leu2 :: lex8op - luciferase was transformed with plasmids encoding the various tripartite receptor fusion proteins ( see table 1 ) generating the mutants listed in table 2 . lex8op contains 4 full operators or 8 half - lexa binding sites . danazole , 5a - dihydrot fluoxymesterone , methyl t , oxymethelone were purchased from sigma ( st . louis , mo .). methenolone , testosterone , dihydrotestosterone , norethandrolone , stanozolol , oxandrolone , mesterolone , methenolone acetate , nandrolone , methandrostenolone , trans - dihydrotestosterone , methyldihydrotestosterone , androsterone , trans - dihydroandrosterone , epitestosterone , epiandrosterone , androstenediol , ethiocholanolone were purchased from steraloids ( newport , r . i .). androgen receptor inhibitors 1 -( 2 - chlorophenyl )- 1 -( 4 - chlorophenyl )- 2 , 2 - dichloroethane and flutamide were purchased from sigma , and cyproterone acetate was purchased from steraloids ( newport , r . i .). total testosterone was extracted from serum using either ether or meth - tert butyl ether ( mtbe ). for ether extractions , 200 μl of serum was added to a 13 - mm glass tube . 400 μl of ether was added to the serum and the tube was vortexed briefly . after a 5 min incubation at room temperature , the samples were centrifuged for 5 min at 2000 g and then placed at − 80 ° c . for 30 min to freeze the aqueous layer . the liquid organic layer , containing the extracted testosterone , was transferred by pouring into a new glass tube . the ether was evaporated by incubating the samples at 60 ° c . to resuspend the testosterone , 350 μl of pbs containing 0 . 5 % bsa was added to the tube . 50 μl of sample was added to 50 μl of cells for the bioassay . extraction with mtbe was similar except 1 . 25 ml of mtbe with 3 % phosphoric acid was added to 200 μl of serum in a 2 ml eppendorf tube . the mixture was vortexed gently ( at low setting ). after a 5 min incubation , the tube was centrifuged at 17 , 000 g . after freezing the aqueous layer at − 80 ° c ., 750 μl of supernatant was transferred with a pippettman to a 13 mm glass tube . the solvent was the evaporated and the sample resuspended according to the ether extraction . to prepare serum samples for extraction , 500 ul , nine - hundred microliters of serum was added to an equal volume of water . the tube was vortexed and heated for 5 min at 70 ° c . solid phase extraction columns , strata - x , 60 mg ( phenomenex , torrance , calif .) were conditions with 1 ml of methanol followed by 1 ml of water . sample was then applied to the column . the column was washed with 1 ml of water then vacuum was applied for 30 sec to remove residual water from the column . steroids were eluted with 1 ml methanol ; a vacuum was then applied for 30 sec to elute residual methanol . fifty to one hundred microliters of eluent was added to each well in a 96 - well plate and dried at 60 ° c . fig1 illustrates a schematic tripartite system according to an embodiment of the present invention . a yeast cell comprises an inactive lexa - dbd : ar - lbd : vp16 - ad protein in the cytoplasm , which may be bound to a heat shock protein ( hsp ). upon binding with an androgen that has crossed the cell membrane , the protein becomes activated and enters the nucleus . in the nucleus , the lexa end of the active tripartite system binds to a lexa binding site or operator ( op ) of a lacz reporter gene . this binding interaction activates transcription of β - galactosidase , which may be monitored via known luminescent detection techniques . plasmid ( lexa - dbd : er : vp16 ) encoding the lexa dna binding domain fused to estrogen response element and vp16 activation domain ( lexa - dbd : er - lbd : vp16 ) was obtained from balasubramanian and morse 1999 . parup21 was generated by ligating the spei - xhoi fragment encoding the lexa - dbd : er - lbd : vp16 fusion from plasmid lexa : er : vp16 into spei - xhoi digested gel purified prs413 - gpd ( mumberg et al . 1994 ; mumberg et al . 1995 ; ronicke et al . 1997 ). to make tripartite constructs with other lbd &# 39 ; s , the estrogen receptor lbd was removed and other nuclear hormone receptors were cloned in frame between the lexa - dbd and vp16 . the androgen receptor was amplified using primers oarup61 ( ggctgacatcggtcgacgcggtgtggaaatagatgggcttg ) ( seq . id . no . 11 ) and oarup62 ( ggggaattcccggggatcccatgtcagcccatctttctgaatg ) ( seq . id . no . 12 ) and p18 , ( origene , rockville md .) as template ( encoding the androgen receptor ). the progesterone receptor was amplified using primers oarup69 ( ctgacatcggtcgacgctttatgaaagagaaggggtttcaccatccc ) ( seq . id . no . 13 ) and oarup70 ( cccggggatcccacagttgattccaccactgatcaacc ) ( seq . id . no . 14 ) using image clone 5167591 as template encoding the progesterone receptor ( invitrogen , carlsbad , calif .). the pcr fragments encoding the androgen and progesterone receptor ligand binding domains were digested with bamhi - sali and ligated into the bamhi - sali site of parup21 replacing the estrogen receptor with the androgen receptor ( parup27 ) and progesterone receptor ( parup32 ), respectively . yeast were inoculated from a plate into synthetic media lacking leucine , uracil and histidine supplemented with 2 % dextrose . after 18 hr incubation at 30 ° c ., cells were washed with synthetic media lacking leucine , uracil and histidine supplemented with glycerol and resuspended in glycerol media to an absorbance at 600 nm of 0 . 03 . cells were grown for 4 hr then 50 μl of cells were added to each well of a 96 - well plate . 50 μl of steroid in phosphate buffered saline 0 . 5 % bsa . cells were grown at 30 ° c . in a shaking water bath for 18 hr . to detect β - galactosidase activity , 25 μl of cells and 25 μl of beta - glo ( promega , madison wis .) were transferred to a luminescent 96 - well plate ( fisher , pittsburgh , pa .). after 30 min incubation at room temperature , the reaction mixture was read using lumimark ( bio - rad , hercules , calif .). alternatively , 100 μl of cells grown as described above were added to dry spe extracted samples . the plate was covered with aeroseal sealing film ( research products international , palatine ill .). the cells were grown overnight and processed as described above . in order to generate an androgen inducible system , a fragment encoding the ligand - binding domain ( amino acid residues 670 - 919 ) of the androgen receptor was inserted between the lexa dna binding domain ( lexa - dbd ) and vp16 activation domain ( a strong transcriptional activator encoded by the herpes simplex virus ) creating parup27 ( see fig2 ). the ( lexa - dbd : ar - lbd : vp16 ) fusion is driven by the strong constitutive glycerol - 3 - phosphate dehydrogenase ( gpd ) promoter . parup27 was transformed into yarup16 , a diploid yeast strain containing an integrated copy of each of two reporters , ( lexa - 8op - lacz and lexa - 8op - luciferase ). both of these reporters contain eight copies of the binding site for the lexa dna binding protein inserted into the basal gall promoter . the bioassay is diagrammed in fig1 . the tripartite fusion is able to activate transcription of the reporters in the presence of androgen hormone . the sensitivity of the bioassay to testosterone was tested ( fig3 and 4a ). various concentrations of testosterone were added to pbs / bsa and incubated with yeast harboring the tripartite receptor system . as shown in fig3 and 4a , a concentration of 100 pg / ml was capable of detection . the dynamic range extended over several logs of luciferase units . the range between 100 pg / ml and 100 , 000 pg / ml for testosterone yielded the most linear response . for comparison , testosterone in men is typically about 4 - 8 ng / ml ( 4 , 000 - 8 , 000 pg / ml ), while in women it is about 0 . 1 - 0 . 54 ng / ml (˜ 100 pg / ml - 500 pg / ml ) or less . hormone receptor structures are conserved . to determine if other steroids could cross - react with a specific receptor ligand - binding domain , three tripartite systems were studied in the presence of androgen , estrogen , testosterone and progesterone , wherein each of the systems contained a different hormone receptor ligand binding domain ( e . g ., ar - lbd , er - lbd , and pr - lbd ). as shown in fig4 a , only non - physiological concentrations of estrogen were able to activate the reporter . estrogen and progesterone tripartite systems were also tested . as shown in fig4 b and 4c , each bioassay shows specificity , responding only to its specific ligand . to determine if the androgen tripartite system could detect androgen derivatives , dilutions of several different anabolic steroids were prepared and their induction potentials determined . all of the androgens tested induced expression of the reporter construct ( see fig5 ). consistent with the literature , dht was the strongest of those tested . as shown in fig6 , the bioassay failed to detect flutamide and 1 -( 2 - chlorophenyl )- 1 -( 4 - chlorophenyl )- 2 , 2 ,- dichloroethane , which are both anti - androgens , indicating the particular androgen binding domain utilized does not bind androgen inhibitors or , if it does , binding does not result in activation or expression of the reporter construct . in body fluids samples with high bioactivity using the androgen bioassay and a lower value for testosterone by ria or mass spectrometry ; this would suggest a high concentration of a compound with high androgen action that is not testosterone . the same concept for progesterone and estrogen receptors apply , respectively . to determine if there was a correlation between the yeast bioassay of the present invention and testosterone eci values , serum samples from thirty - six individuals were processed that had eci values ranging from 18 - 1910 ng / dl . as shown in fig7 , there was a strong correlation between bioactivity and eci values ( r = 0 . 94 , n = 36 ). in addition , the result suggests that some of the bioactivity from the sample set tested may be attributed to testosterone . elevated levels of androgen activity were found in sera from females using some formulations of birth control . the ability of medroxyprogesterone , ethynodial diacetate , 19 - norethindrone , 19 - norethindrone acetate , and levonorgestrel to induce transcription of the reporter through the androgen receptor tripartite were tested using the methods described above . several progestins activated the androgen bioassay . furthermore , the levonorgestrel (−/−) form had about a two - fold greater androgenic activity than the levonorgestrel (−/+) form ( data not shown ). serum samples were pre - treated by diluting with an equal volume of 0 . 9 mg / ml k 2 edta , ( 150 μl of serum was added to 150 μl of 0 . 9 mg / ml k 2 edta ), vortexed briefly and heated for 5 min at 72 c . pre - treated serum was added to yeast expressing the androgen or progesterone tripartite , prepared as follows . yeast were inoculated from a frozen stock into synthetic media lacking leucine , uracil , and histidine supplemented with 2 % dextrose . after overnight incubation at 30 ° c ., mid - log ( od600 nm 0 . 4 - 0 . 8 ) cells were washed with synthetic media lacking leucine , uracil , and histidine supplemented with glycerol and resuspended in 2 % glycerol media at an absorbance at 600 nm of 0 . 1 . twenty - five microliters of mid - log yeast were added to each well of a 384 - well plate using a , biomek 2000 ( beckman - coulter ). serum samples were diluted with an equal volume of 0 . 9 mg / ml k 2 edta , ( 150 μl of serum was added to 150 μl of 0 . 9 mg / ml k 2 edta ), vortexed briefly and heated for 5 min at 72 ° c . ten microliters of pre - treated sample was added to the plate . ten microliters of serially diluted compound in h 2 o was incubated with 25 μl of yeast ( thermolabsystems ). the plate was covered with aeroseal sealing film ( research products international , palatine ill . ), and placed on a plate shaker for 5 min and the plate was incubated for 17 - 18 hr at 28 ° c . in a humidified incubator . after overnight incubation , the plate was shaken for 5 min . to detect β - galactosidase activity , 25 μl of beta - glo ( promega ) was added to the cells incubated overnight using luminskan dispenser ( fisher scientific ). after about 2 - hr incubation at room temperature , the β - galactosidase in the reaction mixture was determined using luminskan luminometer ( thermolabsystems ). intra - assay variation is ˜ 6 . 0 %. the results indicate that there is a strong correlation , r 2 = 0 . 8576 between serum testosterone concentrations measured by both eci and bioactivity using the androgen assay with the exception of some women on birth control , see fig8 . the progesterone assay also showed a good correlation , r 2 = 0 . 9197 , between progesterone values measured by the centaur assay ( bayer healthcare ag , diagnostics division , tarrytown , n . y .) and progesterone tripartite system for normal individuals not taking birth control . females on some formulations of birth control pills , i . e . levonorgestrel , showed elevated androgen levels compared to untreated women . an increase was also observed with extracted samples . however , women on fourth generation progestin , i . e . drospireone , in general had lower androgen and progesterone levels . drospireone is structurally related to spironolactone an androgen receptor inhibitor . all of the compositions , methods and apparatus disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the compositions , methods and apparatus and in the steps or in the sequence of steps of the methods described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . all strains are isogenic , differing from each other in only the markers and / or plasmid indicated . they are all derived from w303 , thomas , b . j . and r . rothstein . 1989 elevated recombination rates in transcriptionally active dna . cell . 56 : 619 - 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