Patent Application: US-57130109-A

Abstract:
the invention relates in general to tyrosine dephosphorylation . more specifically , the invention relates to methods and compositions for monitoring intracellular tyrosine dephosphorylation at the single cell level . the invention further relates to techniques that can be used as aid in the development of novel therapeutics , and monitor regulation of intracellular tyrosine phosphatase activity at the single cell level .

Description:
the inventors developed methods based on flow cytometry and fluorescence microscopy to detect intracellular dephosphorylation of a fluorescent ptp substrate in cells incubated with said substrate . the inventors tested this method using pcap peptides , which have several characteristics that make them more suited than traditional fluorescent non - peptidic substrates ( for example difmup ) to single - cell detection of ptp activity . peptide substrates show some enzyme specificity and can be used to detect the intracellular activity of a single or a few ptps . ptyr peptides cannot be used for this scope since the detection approach used for ptyr peptides is incompatible with single - cell assays , thus preventing the use of ptyr peptides in a cell - based assay for ptp inhibitors . the inventors developed a highly sensitive continuous , peptide - based assay which is ideal for intracellular detection of ptp activity . such an assay also provides a high - throughput method to rapidly screen for ptp inhibitors , providing lead compounds that are already optimized for cell permeability , minimal cellular toxicity , and enzyme specificity . although optimized for pcap peptides , the flow cytometry and fluorescence microscopy - based methods developed by the inventors will work as well with non peptidic fluorescent substrates , for example difmup and similar compounds , after the appropriate optimization steps have been taken . the syntheses of enantiomerically pure coumaryl amino propionic acid ( cap ), methoxy - cap ( mcap ), phosphorylated cap ( pcap ) and fluorinated derivatives in high yield from inexpensive commercially available starting materials are shown in scheme 1 . 14 , 15 all of these probes are readily incorporated into peptides using standard fmoc - based solid phase peptide synthesis methodologies . 16 upon excitation ˜ 340 nm , the inventors found that cap containing peptides are over 10 4 times more fluorescent than pcap containing peptides ( emission wavelength ˜ 460 nm ) 15 , indicating that ptp - catalyzcd hydrolysis of pcap - containing peptides should result in a highly sensitive , fluorescent direct assay for ptp activity . the inventors recently reported that pcap - containing peptides provide sensitive substrates for ptp activity . 15 a linear increase in fluorescence ( excitation at 340 nm and emission at 460 nm ) is observed when dade - pcap - gpaa - nh 2 ( peptide 1 ) is dephosphorylated by yop , a ptp isolated from yersinia enterocolitica . the pcap - containing peptide 1 was also efficiently turned over by human ptps including t cell ptp ( tcptp ), 15 indicating that it could serve as a valuable probe for ptp activity in cells . the inventors have shown that pcap peptides are dephosphorylated by ptps , but are not recognized by serine / threonine phosphatases , which makes them particularly suitable as probes to detect intracellular ptp activity ( fig1 ). a pcap peptide of sequence edne - pcap - tare ( 9lck394pcap ), which is efficiently hydrolyzed by ptpn22 , was incubated with three classes of serine - threonine phosphatases in the optimal buffer for each enzyme , and fluorescence of the reaction was monitored over time using a plate reader with excitation at 340 nm and emission at 460 nm . the amount of enzyme used was normalized to be the same activity on difmup . the serine - threonine phosphatases were ineffective at hydrolysis of this peptide . to determine if these peptides are suitable substrates for cell - based screening of ptp inhibitors , the inventors further assessed dephosphorylation in microinjected cells . using fluorescence microscopy , intracellular dephosphorylation of the microinjected peptides was observed as recognized by an increase in fluorescence over time and was inhibited by a known ptp inhibitor ( fig2 ). in order to demonstrate that flow cytometry can be used to detect intracellular dephosphorylation of the peptides , the inventors measured cap fluorescence in flow cytometry using cap - conjugated silica beads . the inventors found that they can detect an intense fluorescence signal analyzing the cap beads in the facsaria system , using the violet laser ( exciting at 407 mn ), and a 450 / 40 nm bandpass filter for detection . in principle , any facs system equipped with the appropriate excitation and emission filters could be used . in order to demonstrate that pcap peptide can be used to monitor intracellular ptp activity by flow cytometry , the inventors internalized pcap peptides into jtag cells by nucleofection and cell fluorescence was analyzed by flow cytometry . the peptides were found to be dephosphorylated in the cells and inhibition of ptp activity by a known ptp inhibitor could be detected using this method ( fig3 - 5 ). the inventors believed that cell - based screening using pcap peptides or other fluorescent ptp substrates internalized in cells and detecting cell fluorescence by flow cytometry or microscopy could yield inhibitor leads that are optimized for cell permeability and enzyme specificity and minimal cellular toxicity , thus considerably speeding up the development of therapeutic ptp inhibitors . in order to demonstrate the utility of this invention , the inventors have synthesized several cell - permeable tagged peptides for use in cellular imaging experiments , and have optimized their uptake into cells . cap in n - terminal fusion with two types of cell - penetrating peptides ( cpp ), a penetratin derived from amino acids 43 - 58 of the antennapedia homeodomain ( ant = rqikiwfqnrrmkwk ) and a polyarginine peptide ( r 8 ) were synthesized . human jurkat tag ( jtag ) cells with cap - ant and cap - r8 peptides were incubated and analyzed by flow cytometry . intense cell fluorescence from internalized ant - and r8 - peptides can be easily detected by flow cytometry . in jtag cells r8 seemed to be a more efficient cap - peptide carrier than ant . fig6 shows cellular fluorescence after incubation of one hour with 12 . 5 um ant - cap or r8 - cap in the presence of 0 . 5 % serum . several additional experiments were performed in order to optimize cellular uptake by variation of concentration of peptides and incubation buffers . for jtag cells the inventors found that incubation of cells with 10 - 15 um cap - r8 in rpmi with o . 5 %, serum for 1 h leads to high cell fluorescence . in order to increase carrier efficiency and cargo distribution in the cell cytosol , cell - permeable peptides were conjugated with lipid chains , and the cellular uptake was tested through flow cytometry and confocal microscopy . the inventors synthesized r7 - cap conjugated with n - terminal 14c and 16c lipidic chains through a beta - alanine ( betaala ) spacer ( c14 - betaala - r7 - cap , c16 - betaala - r7 - cap ). treatment of jtag t cells with these lipid - r7 peptides showed that c14 and c16 were able to substantially increase efficacy of internalization as compared to the non - lipidic tags . through confocal microscopy , the inventors found that conjugation of r7 - cap with the longer hydrocarbon chains not only increases the cellular internalization of the peptides , but also increases the cytosolic / nuclear localization ratio of cell fluorescence . as used herein the term “ cell ” refers to or describes hela , cos , mefs , jurkat , jurkat tag ( jtag ), insect cells , primary cells , or any cell with ptp activity . the following examples are intended to illustrate , but not to limit , the scope of the invention . while such examples are typical of those that might be used , other procedures known to those skilled in the art may alternatively be utilized . indeed , those of ordinary skill in the art can readily envision and produce further embodiments , based on the teachings herein , without undue experimentation . pcap and the related probes cap , mcap , pcapf n , capf n , and mcapf n can be synthesized using a slight modification of the published procedure , as outlined in scheme 1 . 20 , 21 the pcap residue can be incorporated into peptides using standard solid phase amino acid coupling procedures . specifically , rink amide resin ( 100 mg , 7 . 4 × 1 − 5 mol ) can be used as the solid support , and each new amino acid ( 5 equiv ) is coupled to the growing chain using 2 -( 6 - chloro - 1 - h - benzotriazole - 1 - yl )- l , l3 , 3 ,- tetramethy1 uranium hexafluorophosphosphate ( hctu ) as the coupling agent . in the case of the cap - based amino acids , benzotriazol - 1 - oxytripyrrolidinophosphonium hexafluorophosphate ( pybop ) and n - hydroxybenzotriazole ( hobt ) are more effective as coupling agents . all amino acids are allowed to couple for 1 . 5 h . the phosphate protecting groups on the pcap moiety can be removed by treating the resin with 20 equiv trimethylsilyliodide ( tmsi ) ( 0 . 1 113 ml , 0 . 74 mmol ) in anhydrous dichloromethane ( 1 ml ). the beads are then washed with dichloromethane , followed by dimethylformamide ( dmf ). removal of the n - terminal fmoc group is accomplished by twice agitating the resin with 1 ml of 2 % 1 , 8 - diazabicyclo [ 5 . 4 . 0 ] undec - 7 - ene ( dbu ) in dmf . the peptides are then released from the resin under acidic conditions ( 1 ml of 95 % trifluoroacetic acid , 2 . 5 % h 2 o , 2 . 5 % triisopropylsilane ) and purified twice by rp - hplc , using either a c18 column , a diphenyl column , or both ( varian , inc .). the resulting peptides will have acylated n - termini and amides on the c - termini . after characterization using maldi - tof spectrometry , stock solutions of each peptide will be made by dissolving the peptides in dmso . adherent cells ( hela cells , cos cells , or mefs or any cells adherent or made to adhere to slides by any means ) can be plated directly on microscope chamber slides the night before the experiment at a subconfluent density . suspension cells ( jtag , or any other cells which spontaneously grows in suspension or is made to grow in suspension , e . g . by continuous agitation ) are added to wells of a plate at a concentration of up to 10 million / ml . for incubation with the cell permeable cap peptides , cells are washed once with pbs and resuspended in culture medium containing 0 . 5 % serum . equal volumes of appropriate dilutions of peptide stock solutions are added to the wells / slides in order to achieve the final concentration of peptides in the medium . after incubation in a 5 % co 2 incubator at 37 ° c . for the appropriate time , cells are washed in order to remove any non - internalized peptide . total amount of cells , peptide concentration , peptide to cell ratio and time of incubation should be optimized for each cell type and peptide used . cells can be imaged with a fluorescence microscope equipped with appropriate excitation and emission filters . cells are added to wells of a plate at a concentration of up to 10 million / ml . cells are washed once and resuspended in culture medium containing 0 . 5 % serum . equal volumes of appropriate dilutions of peptide stock solutions are added to the wells / slides in order to achieve the final concentration of peptides in the medium . after incubation in a 5 % co 2 incubator at 37 ° c . for the appropriate time , cells are washed and resuspended in hank &# 39 ; s balanced salt solution ( hbss ) containing 2 . 5 mg / ml bsa and 5 mm edta and subjected to flow cytometry analysis . total amount of cells , peptide concentration , peptide cell ratio and time of incubation should be optimized for each cell type and peptide used . cells can be analyzed with any flow cytometry system equipped with appropriate excitation and emission filters . cell - based high throughput screening of small molecule ptp inhibitors by flow cytometry cells are treated with pcap peptides or other fluorescent ptp substrate and varying concentrations of candidate ptp inhibitors in a 96 - well format , and flow cytometry analysis is performed using a uv - cytometer equipped with a multi - plate , loader ( mpl ) system , or with a high - content fluorescence microscope . the ptp substrate can be spontaneously cell - permeable or made cell - permeable by conjugation with tags or other chemical modification . cells are prepared for facs analysis and assessed for inhibition of ptp activity . amount of inhibitor and length of incubation should be analyzed for each inhibitor , substrate , and cell type used . cells and compounds are distributed in an initial 96 - well or higher number of wells plate , and a liquid handler can be used to pipette cells , compounds , and substrate in wells . the desired inhibitor will show activity in secondary cell - based assays , for example inhibitors of ptps which negatively regulate signal transduction through a certain pathway , will show activity in western blotting assays monitoring the activation of said signal transduction pathway . if using peptide substrates , the inhibitors will also show some degree of specificity for the target phosphatase , depending on the specificity of the peptide substrate used . also the cell - based assay in principle should detect uncompetitive and allosteric inhibitors in addition to competitive inhibitors . pcap peptides as specific substrates for such assays have been optimized . the novel assays can be carried out using non peptidic fluorescent ptp substrates , however pcap - peptides offer the obvious advantages of 1 ) increased similarity to physiological substrates and 2 ) increased specificity of for ptps versus other enzymes , and 3 ) the possibility of further optimizing the sequence of the peptide if needed . thus pcap peptides are particularly suitable as probes for the shown novel applications including cell - based hts for ptp inhibitors . many modifications and variations of the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof and therefore only such limitations should be imposed as are indicated by the appended claims . all patent and literature references cited in the present specification are hereby incorporated by reference in their entirety . 2 . mustelin , t . ; vang , t . ; bottini , n . nature rev . immunology 2005 , 5 , 43 - 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