FEBS 19675
`
`FEBS Letters 421 (1998) 27 31
`
`Topological isomers of human uroguanylin:
`interconversion between biologically active and inactive isomers
`
`Naoyoshi Chinoa;*, Shigeru Kuboa, Tetsuya Kitania, Takuya Yoshidab, Ryosuke Tanabeb,
`Yuji Kobayashib, Masamitsu Nakazatoc, Kenji Kangawad, Terutoshi Kimuraa
`
`aPeptide Institute, Inc., Protein Research Foundation, 4 1 2 Ina, Minoh, Osaka 562, Japan
`bFaculty of Pharmaceutical Sciences, Osaka University, 1 6 Yamadaoka, Suita, Osaka 565, Japan
`cThird Department of Internal Medicine, Miyazaki Medical College, Miyazaki 889 16, Japan
`dNational Cardiovascular Center Research Institute, 5 7 1 Fujishirodai, Suita, Osaka 565, Japan
`
`Received 21 October 1997; revised version received 1 December 1997
`
`Abstract The solution structures of the two compounds of
`human uroguanylin (I and II), which were generated during
`disulfide bond forming reaction, were found to be topological
`isomers by 1H-nuclear magnetic resonance spectroscopy. These
`isomers are interconvertible in aqueous media at rates which vary
`with the pH and temperature of the solution. Because compound
`I is active in the cGMP producing assay, but compound II is not,
`this interconversion may be useful for evaluating the activity of
`human uroguanylin both in vivo and in vitro.
`z 1998 Federation of European Biochemical Societies.
`
`Key words: Uroguanylin; Guanylin; Topological isomer;
`Interconversion; HPLC analysis; Biological activity
`
`1. Introduction
`
`Uroguanylin and guanylin were discovered as endogenous
`peptide hormones in mammals based upon their structure
`similarity to heat stable enterotoxins (STs) secreted by patho
`genic bacteria. The primary structures of uroguanylin and
`guanylin from human, rat (mouse) and opossum have been
`reported as being comprised of 15 or 16 amino acid residues
`[1 5]; the human and rat peptide sequences are shown in Fig.
`1. The sequence similarity among them is high and four Cys
`residues in all the peptides are conserved. These Cys residues
`participate in the formation of the two intramolecular disul
`¢de linkages, one between Cys4 and Cys12 and the other be
`tween Cys7 and Cys15. Uroguanylin and guanylin, as well as
`ST, are reported to be involved in the regulation of salt and
`water transport in the intestinal tract and kidney. In addition,
`these peptides are known to stimulate cGMP production by
`activating the guanylyl cyclase C in both enterocytes and T84
`colon cancer cells. Therefore, endogenous uroguanylin and
`guanylin are suggested to play important roles in intestinal
`and renal dysfunction and salt dependent hypertension [6].
`In our previous paper on the chemical synthesis of human
`uroguanylin using a two step selective disul¢de forming meth
`od, two compounds (I and II) were found to be generated
`upon analyzing the second disul¢de bond forming reaction
`
`*Corresponding author. Fax: +81 (727) 29 4124.
`E mail: chino@prf.or.jp
`
`Abbreviations: ST, heat stable enterotoxin; cGMP, cyclic 3P,5P gua
`nosine monophosphate; RP HPLC, reversed phase high performance
`liquid chromatography; CD, circular dichroism; NMR, nuclear
`magnetic resonance; NOE, nuclear Overhauser effect; NOESY,
`NOE spectroscopy; MD, molecular dynamics; RMSD, root mean
`square deviation; GdnHCl, guanidine hydrochloride; NEM, N
`ethylmaleimide; TFA, trifluoroacetic acid; DMSO, dimethyl sulfoxide
`
`by reversed phase high performance liquid chromatography
`(RP HPLC) at 40‡C [7]. A typical chromatogram for the sep
`aration of a 1:1 mixture of these compounds is shown in Fig.
`2. We have so far clari¢ed the following characteristics for
`compounds I and II: (i) each compound can be isolated to
`a purity greater than 99% as determined by RP HPLC; (ii)
`both have identical primary structures, molecular weights and
`disul¢de connectivity patterns according to examination by
`suitable analytical methods; and (iii) signi¢cant di¡erences
`exist between them in the optical rotation value and their
`biological activity. During the course of our previous study,
`Skelton et al. reported that two clearly separable signal con
`nectivities were detected in the analysis of the amino termi
`nally extended or deleted human guanylin derivatives by nu
`clear magnetic resonance (NMR) spectroscopy. Based on
`these observations and structural re¢nements, they proposed
`that the heterogeneity of the NMR signals of human guanylin
`derivatives originated from the topological isomerism of the
`peptide, although such isomers were unseparable on RP
`HPLC under the various analytical conditions used [8]. In con
`trast, we found in a previous study that human des Leu16 uro
`guanylin and rat guanylin, both of which terminate the peptide
`chains at the fourth Cys residue like human guanylin, were
`detected as two base line separable peaks on RP HPLC when
`the analytical temperature was decreased to 8‡C, although these
`peptides were eluted in a broad but single peak at 40‡C [7]. From
`these observations, we assumed that the two well separable hu
`man uroguanylin compounds on RP HPLC were similar topo
`logical isomers with respect to the peptide backbone as reported
`for human guanylin derivatives. However, this assumption re
`quired de¢nite con¢rmation by experimental evidence.
`In the present study, we analyzed the solution structures of
`both compounds by NMR in an aqueous medium to gain
`further insight into the characteristics of the isolated com
`pounds I and II of human uroguanylin. We report here con
`
`Fig. 1. Primary structures of human and rat uroguanylin and gua
`nylin. Two intramolecular disul¢de linkages are shown at the top of
`the sequences.
`
`0014 5793/98/$19.00 (cid:223) 1998 Federation of European Biochemical Societies. All rights reserved.
`PII S 0 0 1 4 5 7 9 3 ( 9 7 ) 0 1 5 2 7 5
`
`Bausch Health Ireland Exhibit 2011, Page 1 of 5
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`28
`
`N. Chino et al./FEBS Letters 421 (1998) 27 31
`
`2.4. RP HPLC analysis
`RP HPLC was performed on a Shimadzu Model LC 6A with a
`YMC ODS column (4.6U150 mm). Standard conditions for analyses
`of the two compounds of human uroguanylin were isocratic elution at
`25.5% acetonitrile in 0.1% TFA at 40‡C. Analyses of 15 residue gua
`nylin and uroguanylin derivatives were carried out both at 40‡C and
`8‡C under linear gradient conditions; 1% to 60% acetonitrile (25 min)
`in 0.1% TFA. Absorbance was monitored at 220 nm.
`
`2.5. Stability of compounds I and II in solution
`Each isolated compound of human uroguanylin was dissolved in
`50% AcOH, 0.05% TFA or 50 mM NH4OAc at pH 7.7 containing
`0.25 M guanidine hydrochloride (GdnHCl) at a concentration of
`1 mg/200 Wl. Half of each prepared solution was incubated at 37‡C
`and the remaining half was kept at ambient temperature (15 20‡C). In
`the case of the analysis in the presence of N ethylmaleimide (NEM), each
`compound was dissolved in the same bu¡er as above at pH 7.7 which
`contains a slight molar excess amount of NEM. The change in purity
`of each peptide was analyzed at 24 h intervals by RP HPLC. The
`amount of each compound in the individual solution was calculated
`by integration of the corresponding peak areas on the chromatogram.
`
`2.6. Bioassay
`Accumulation of cGMP in T84 cells was measured following a
`reported procedure [9].
`
`3. Results
`
`3.1. Solution structure of compounds I and II of human
`uroguanylin
`In order to elucidate the secondary structure di¡erence be
`tween compounds I and II of human uroguanylin, CD spectra
`of each compound were recorded at pH 7.0 (Fig. 3). A neg
`ative band around 200 nm was detected for both compounds,
`but the ellipticity was much greater in compound I, suggesting
`a di¡erence in their structures.
`Further structural analysis of the two compounds was car
`ried out by NMR in 10 mM sodium phosphate at pH 3.7 and
`10‡C. From the distance constraints elucidated from the NOE
`data, the ensembles of 10 solution structures of compounds I
`and II were deduced with reasonably good convergence
`(RMSDs for compounds I and II backbone atoms are
`0.67 A(cid:238) and 0.48 A(cid:238) , respectively) if the less well de¢ned ami
`no terminal 3 residues and the carboxyl terminal Leu residue
`were excluded. The energy minimized average structures of
`
`Fig. 3. CD spectra of compounds I and II of human uroguanylin in
`10 mM sodium phosphate at pH 7.0.
`
`Fig. 2. Separation of the isolated compounds I and II of human ur
`oguanylin (1:1 mixture) on RP HPLC under isocratic conditions.
`See Section 2 for details of the analytical conditions.
`¢rmation that the two compounds of human uroguanylin are
`indeed topological isomers. We then report on their stability
`in solution and in the solid state. These experiments demon
`strate that the topological isomers of human uroguanylin are
`not
`stable in solution and are readily interconvertible,
`although they are stable during storage in a freezer as pow
`ders. This has important implications for evaluating the bio
`logical activity of human uroguanylin because compound I
`elicits the activity in the cGMP production assay, whereas
`compound II primarily aborts it [9].
`
`2. Materials and methods
`
`2.1. Peptide synthesis
`Two compounds (I and II) of human uroguanylin were obtained
`following the reported procedure [7]. Brie£y, the protected peptide
`was elongated on resin using an ABI 430A peptide synthesizer by
`applying Boc/Bzl chemistry. Pairs of the Cys residues which form
`intramolecular disul¢de bonds were selectively protected by orthogo
`nally cleavable groups, 4 methylbenzyl and acetamidomethyl. After
`treatment with anhydrous hydrogen £uoride, two intramolecular di
`sul¢de bonds were formed successively with K3[Fe(CN)6] and then
`with iodine. Two compounds generated during the second disul¢de
`bond formation in MeOH/50% AcOH (1:1, v/v) were isolated by RP
`HPLC, lyophilized and stored in a freezer until use. Human des
`Leu16 uroguanylin, rat guanylin and rat uroguanylin 15 were synthe
`sized by applying the same strategy as for human uroguanylin. The
`resulting major peaks from RP HPLC were isolated at 40‡C and
`stored in a freezer after the usual workup including lyophilization.
`
`2.2. CD measurement
`The CD spectrum was recorded on a JASCO J720 spectropolarim
`eter with a cell path length of 0.1 cm at 25‡C. The sample was dis
`solved in 10 mM sodium phosphate bu¡er at pH 7.0 at a concentra
`tion of 100 WM.
`
`2.3. NMR measurement and structure calculation
`All the spectra were recorded on a Bruker DRX 500 spectrometer
`at 10‡C. The peptide was dissolved in 10 mM sodium phosphate at
`pH 3.7 containing 10% deuterated water (D2O) at a peptide concen
`tration of 5 mM. Assignment of proton resonances was achieved
`according to the standard method developed by Wuthrich [10]. The
`nuclear Overhauser e¡ect (NOE) distance constraints for compounds
`I and II were derived from two dimensional NOESY spectra acquired
`for 24 h with mixing times of 120 and 250 ms, respectively. All the
`structure calculations were performed with the program X PLOR.
`
`Bausch Health Ireland Exhibit 2011, Page 2 of 5
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`N. Chino et al./FEBS Letters 421 (1998) 27 31
`
`29
`
`Fig. 4. Average favored solution structures of compound I (a) and compound II (b) of human uroguanylin.
`
`compounds I and II are shown in Fig. 4. Both favored struc
`tures are depicted by ¢xing the loop structure composed of the
`residues 7 12 (central loop) to have the same spatial arrange
`ment at their bottoms. As is obvious from the deduced struc
`ture of compound I shown in Fig. 4a, one segment comprising
`the amino terminal residues 1 7 and the disul¢de bond be
`tween Cys4 and Cys12 is located at the left top side of the
`central loop and another segment encompassing the carboxyl
`terminal residues 12 16 and the disul¢de bond between Cys7
`and Cys15 is at the right top side. In contrast, the structure of
`compound II, shown in Fig. 4b, revealed that the above two
`segments extend outwards and directly opposite from the cen
`tral loop. Therefore, the solution structure di¡erence between
`the two compounds lies merely in the orientation of these two
`
`segments from the central loop, clearly demonstrating that the
`well separable two compounds of human uroguanylin on RP
`HPLC are topological isomers.
`
`3.2. Stability of compounds I and II of human uroguanylin
`Compounds I and II have distinctly di¡erent retention
`times on RP HPLC at 40‡C, and thus could be separately
`isolated at purities greater than 99%. The purities of the iso
`lated compounds were con¢rmed to be maintained for more
`than one year when each compound was stored in a freezer as
`an amorphous powder. However, mutual contamination,
`comprising 0.8% of the total peak areas, was seen when the
`purities of the isolated compounds were evaluated by RP
`HPLC after the compounds were lyophilized from 0.1%
`
`Fig. 5. Time course analyses of conversion of compound I (a) and compound II (b) of human uroguanylin in solution. The purity change of
`each compound was monitored by RP HPLC under isocratic conditions. The peak area of each converted compound was shown as a % of the
`sum of those for compounds I and II on the individual chromatogram.
`
`Bausch Health Ireland Exhibit 2011, Page 3 of 5
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`30
`
`N. Chino et al./FEBS Letters 421 (1998) 27 31
`
`TFA solution. These results suggest that while the two com
`pounds are stable once they are isolated and stored as pow
`ders at below 20‡C, their stability may be less in solution,
`that is, compounds I and II are interconvertible in solution.
`To examine whether such interconversion occurred during the
`synthesis and analysis, the degree of purity of each compound
`in 50% AcOH, 0.05% TFA and 50 mM NH4OAc (pH 7.7)
`containing 0.25 M GdnHCl was analyzed by RP HPLC.
`These solvents were chosen as mimicking the media employed
`for disul¢de bond formation, puri¢cation of the oxidized pep
`tides by RP HPLC and measurement of the biological activ
`ity, respectively. Each solution was kept at both ambient tem
`perature (15 20‡C) and 37‡C.
`RP HPLC analyses of the constructed solutions of com
`pounds I and II showed that the peak area of the starting
`compound decayed gradually and that of the converted ma
`terial (compound I in the case of compound II and vice versa)
`increased comprehensively in all the media we examined. This
`clearly demonstrates that the interconversion of the two com
`pounds indeed takes place in solution. The results of the anal
`yses of the interconversion for each compound are summar
`ized in Fig. 5 as a function of time. In the acidic milieu (0.05%
`TFA and 50% AcOH), the conversions proceeded in an al
`most comparable manner for both compounds. The slopes of
`the conversion rate in 0.05% TFA, however, were steeper than
`those in 50% AcOH, that is, the conversion from compound I
`to compound II and vice versa at 37‡C in 0.05% TFA was
`complete and resulted in a steady state in 2 days, whereas
`more than 4 days were required for the same processes in
`50% AcOH. The temperature dependence of the conversion
`was observed in both acidic media, in which the rate of the
`interconversion was always faster at 37‡C than that at room
`temperature (15 20‡C).
`In 50 mM NH4OAc bu¡er at pH 7.7, both compounds
`were generally more stable (slower conversion) than in acidic
`milieu. However, in contrast to the results in the two acidic
`solvents, their conversion rates were not identical in this neu
`tral bu¡er, that is, compound I was converted more slowly
`than compound II. In addition, the slope of the conversion
`rate of compound I at pH 7.7 and 37‡C was found to be lower
`than that in 0.05% TFA at ambient temperature. This rank
`order alteration in neutral bu¡er is distinct from all other
`experiments because regardless of the starting compounds,
`preferential conversion rates were always observed at 37‡C
`except for this particular case.
`The above results de¢nitely demonstrate that the two com
`pounds are interconvertible in solution, therefore, it is tempt
`ing to conjecture that disul¢de linkage scission and reclosure
`is involved in the mechanism of the conversion. In order to
`address this, a stability test was done using the same bu¡er as
`above at pH 7.7, except NEM, a well known SH trapping
`reagent, was added. Analysis by RP HPLC showed that the
`slope of the conversion in the presence of NEM could be
`completely superimposed on that in its absence (data not
`shown), indicating that disul¢de bond opening does not occur
`during the conversion.
`
`4. Discussion
`
`Structure analyses of two compounds (I and II) of human
`uroguanylin by NMR were carried out in both dimethyl sulf
`oxide (DMSO) and aqueous solutions. Distinct chemical shifts
`
`were observed for both compounds in either medium, but
`more HK signal overlapping was detected in DMSO. There
`fore, the aqueous solution structures of the two compounds of
`human uroguanylin were re¢ned with a simulated annealing
`protocol, by which they were de¢nitely con¢rmed to be topo
`logical isomers. The elucidated backbone structure of the iso
`lated compound I (a biologically active component) is similar
`to that of the reported human guanylin isomer ‘A form’,
`which was determined in the mixture of the two topological
`isomers [8], as well as that of heat stable enterotoxin (ST) with
`three disul¢de bonds [11]. Although it has not been clari¢ed as
`to whether the human guanylin ‘A form’ is a biologically ac
`tive component or not, the similar backbone topology among
`these family peptides may be a prerequisite for expressing the
`cGMP producing activity. Analyses of the side chain orienta
`tion of the two compounds of human uroguanylin is now
`underway in our laboratory, however, in a preliminary result,
`side chain location in the putative active site region around
`Ala11, which is estimated from the active site of ST reported
`by Shimonishi et al. [12], seems to be di¡erent from each other
`(data not shown). Results of these analyses together with the
`experimental data for three dimensional backbone structure
`resolution will be reported in the near future (T. Yoshida et
`al., manuscript in preparation).
`In the RP HPLC analyses of the two human uroguanylin
`isomers, we had already established that they are separable at
`40‡C. However, separations of the human des Leu16 urogua
`nylin isomers, as well as the rat guanylin isomers, were pos
`sible only at lower temperatures such as 8‡C [7]. This separa
`tion characteristic has also been observed for a recently
`disclosed member of the uroguanylin and guanylin peptide
`family, rat uroguanylin 15 (unpublished result). Considering
`that the latter three peptides are composed of 15 residues with
`the sequence ending at the fourth Cys residue, we con¢rmed
`that the Leu residue at position 16 of human uroguanylin
`endows the topological isomers with a separation e⁄ciency
`signi¢cantly higher than the other shorter uroguanylin and
`guanylin family peptides on RP HPLC. In other words, the
`topological isomers of human uroguanylin may be stabilized
`signi¢cantly by the Leu residue lying outside the disul¢de
`linked loop structure.
`As far as we know, including the case of human guanylin
`[8], few features have been characterized to date for the iso
`lated individual topological isomers. In the present study, we
`have shown that these isomers of human uroguanylin are
`interconvertible without disul¢de bond opening when left
`standing in solution over a period of time (days). Analyses
`of the stability of the two compounds at acidic and neutral
`pH values suggest that the conversion rates are a¡ected by the
`ionization state of functional group(s) in the molecule. At
`acidic pH, both compounds are, in one sense, freely conver
`tible (same conversion rates) and eventually come to a 1:1
`equilibrium ratio. In contrast, conversions of both compounds
`at pH 7.7 seem to be hampered and thereby their rates are
`signi¢cantly decreased, especially for compound I. A more
`extensive analysis of the pH dependent conversion rates of
`the two isomers is now underway in our laboratory.
`In the NMR study of human guanylin derivatives reported
`by Skelton et al. [8], they commented on the interconversion
`of the topological isomers: (i) based on line broadening ex
`periments at high temperature, the exchange must be very
`slow with a half life of seconds or longer, and (ii) in restrained
`
`Bausch Health Ireland Exhibit 2011, Page 4 of 5
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`

`

`N. Chino et al./FEBS Letters 421 (1998) 27 31
`
`31
`
`molecular dynamics (MD) calculations, transition from one
`state to the other could not be induced with a realistic force
`¢eld. In the present study, we observed by RP HPLC analyses
`that the half life of interconversion of the two human urogua
`nylin isomers in 0.05% TFA at 37‡C was about 1 day; these
`are the conditions under which the interconversion proceeds
`fastest. In the case of the human des Leu16 uroguanylin topo
`logical isomers, they were more unstable than those of the
`parental peptide because they were mutually contaminated
`by approximately 20 30% after isolation by RP HPLC at
`8‡C and subsequent concentration of the solvent at room
`temperature for 2 h (preliminary data). This phenomenon is
`also observed for rat uroguanylin 15, demonstrating that the
`topological isomers of the peptides with the fourth Cys resi
`due at their carboxyl termini survive as a main fraction after 2
`h even though the interconversion rate is faster than for hu
`man uroguanylin. These observations con¢rm that the inter
`conversion rates of the topological
`isomers of uroguanylin
`and guanylin family peptides are much longer than that of
`NMR and MD time scales.
`We have already reported that (i) in the chemical synthesis
`of human uroguanylin, the ratio of the topological isomers
`(compounds I and II) di¡ers signi¢cantly with the order of
`the two intramolecular disul¢de bond forming reactions, and
`that (ii) compound I shows signi¢cant activity in the cGMP
`producing assay, whereas compound II is practically inactive
`[7,9]. We have further clari¢ed recently that compound I iso
`lated after conversion from the inactive component, com
`pound II,
`is fully active. Furthermore, compound II is a
`weak agonist without antagonistic activity because the
`cGMP production in T84 cells by the simultaneous stimula
`tion with both compounds was equivalent to the sum of the
`individual stimulations (data not shown). In the former chem
`ical synthesis, the second disul¢de bond forming reaction pro
`ceeded very quickly (less than 20 min) and the reaction mix
`ture was analyzed immediately after quenching of the reaction
`with ascorbic acid. In the latter case, measurement of the
`biological activity was performed using freshly prepared sol
`ution of each compound and the response in T84 cells was not
`retarded. Therefore, we are certain that all these ¢ndings are
`correct even though the two compounds are interconvertible
`in solution. We have also elucidated the endogenous molec
`ular form of uroguanylin in humans by the combined analyses
`of RP HPLC and radioimmunoassay using antibodies speci¢c
`for each topological isomer [9]. As relatively long times and
`procedures were required for the isolation and quanti¢cation
`of the two isomers in the body, it is conceivable that the
`interconversion may occur during the course of the analysis.
`Actually, a low level of mutual cross reactivity contamina
`tions (3 3.5%) in each of the speci¢c antibodies were observed
`during characterization of the speci¢city of the respective anti
`bodies using two 125I Tyr0 human uroguanylin isomers as
`tracers. This implies the possible conversion of the two iso
`mers of the standard and/or radio labeled peptides, although
`such cross reactivity might be an intrinsic feature of the anti
`bodies. Nonetheless, this does not invalidate the reported re
`sults concerning the endogenous form and amount of human
`uroguanylin because care was always taken during each ex
`perimental step to detect the occurrence of the conversion and
`to minimize experimental errors by keeping the peptide solu
`tions at as a low temperature as possible. In the above NMR
`experiments, the structures of both compounds were deter
`
`mined without serious mutual contamination of signals prob
`ably because the measurements were carried out at a low
`temperature of 10‡C.
`In the present study, we found that human uroguanylin
`tends to isomerize topologically in solution, resulting in a
`mixture of the biologically active and inactive isomers. Inter
`estingly, the 24 amino acid peptide with an 8 residue exten
`sion at the amino terminus of human uroguanylin could be
`isolated from hemo¢ltrate pools and was found to accumulate
`cGMP in T84 cells, that is, amino terminally extended human
`uroguanylin is a biologically active form [13]. In the case of
`rat guanylin, its precursor with 94 amino acid residues as well
`as the amino terminally Asp extended peptide with 16 amino
`acid residues are both biologically inactive [14]. Most of the
`biologically active 15 residue peptide of rat guanylin is re
`ported to be generated by the arti¢cial cleavage at the Asp
`Pro bond in the precursor during the isolation process with
`hot AcOH [15]. Taken together, we speculate that exertion of
`the biological activity of the uroguanylin and guanylin family
`peptides is primarily determined by two factors: (1) peptide
`chain length and (2) topological isomerization.
`Finally, we emphasize that the biological activity data of
`human uroguanylin and its derivatives, regardless of whether
`they are synthetic or natural products, may lead to confusing,
`variable potency results if the data are obtained after the
`peptide solutions have been left at room temperature for a
`few days, especially under acidic conditions.
`
`References
`
`[1] Currie, M.G., Fok, K.F., Kato, J., Moore, R.J., Hamra, F.K.,
`Du⁄n, K.L. and Smith, C.E. (1992) Proc. Natl. Acad. Sci. USA
`89, 947 951.
`[2] Wiegand, R.C., Kato, J., Huang, M.D., Fok, K.F., Kachur, J.F.
`and Currie, M.G. (1992) FEBS Lett. 311, 150 154.
`[3] Hamra, F.K., Forte, L.R., Eber, S.L., Pidhorodeckyj, N.V.,
`Krause, W.J., Freeman, R.H., Chin, D.T., Tompkins, J.A.,
`Fok, K.F., Smith, C.E., Du⁄n, K.L., Siegel, N.R. and Currie,
`M.G. (1993) Proc. Natl. Acad. Sci. USA 90, 10464 10468.
`[4] Kita, T., Smith, C.E., Fok, K.F., Du⁄n, K.L., Moore, W.M.,
`Karabatsos, P.J., Kachur, J.F., Hamra, F.K., Pidhorodeckyj,
`N.V., Forte, L.R. and Currie, M.G. (1994) Am. J. Physiol.
`266, F342 F348.
`[5] Miyazato, M., Nakazato, M., Matsukura, S., Kangawa, K. and
`Matsuo, H. (1996) FEBS Lett. 398, 170 174.
`[6] Forte, L.R. and Hamra, F.K. (1996) News Physiol. Sci. 11, 17
`24.
`[7] Chino, N., Kubo, S., Miyazato, M., Nakazato, M., Kangawa, K.
`and Sakakibara, S. (1996) Lett. Pept. Sci. 3, 45 52.
`[8] Skelton, N.J., Garcia, K.C., Goeddel, D.V., Quan, C. and Bur
`nier, J.P. (1994) Biochemistry 33, 13581 13592.
`[9] Nakazato, M., Yamaguchi, H., Kinoshita, H., Kangawa, K.,
`Matsuo, H., Chino, N. and Matsukura, S. (1996) Biochem. Bio
`phys. Res. Commun. 220, 586 593.
`[10] Wuthrich, K., Wider, G., Wagner, G. and Braun, W. (1982)
`J. Mol. Biol. 155, 311 319.
`[11] Ozaki, H., Sato, T., Kubota, H., Hata, Y., Katsube, Y. and
`Shimonishi, Y. (1991) J. Biol. Chem. 266, 5934 5941.
`[12] Yamasaki, S., Sato, T., Hidaka, Y., Ozaki, H., Ito, H., Hiraya
`ma, T., Takeda, Y., Sugimura, T., Tai, A. and Shimonishi, Y.
`(1990) Bull. Chem. Soc. Jpn. 63, 2063 2070.
`[13] Hess, R., Kuhn, M., Shulz Knappe, P., Raida, M., Fuchs, M.,
`Klodt, J., Adermann, K., Kaever, V., Cetin, Y. and Forssmann,
`W. G. (1995) FEBS Lett. 374, 34 38.
`[14] Yamaguchi, H., Nakazato, M., Miyazato, M., Kangawa, K.,
`Matsuo, H. and Matsukura, S. (1995) Biochem. Biophys. Res.
`Commun. 214, 1204 1210.
`[15] Wiegand, R.C., Kato, J. and Currie, M.G. (1992) Biochem. Bio
`phys. Res. Commun. 185, 812 817.
`
`Bausch Health Ireland Exhibit 2011, Page 5 of 5
`Mylan v. Bausch Health Ireland - IPR2022-00722
`
`