MYLAN EXHIBIT - 1014
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland - IPR2022-00722
`
`

`

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`Copyright © 1980 International Union of Pure and
`Applied Chemistry
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`First published 1980
`
`British Library Cataloguing in Publication Data
`International Symposium on Frontiers of Bioorganic
`Chemistry and Molecular Biology,Moscow and
`Tashkent, 1978
`Frontiers of bioorganic chemistry and molecular
`biology. - (International Union of Pure and
`Applied Chemistry. IUPAC symposium series).
`1. Biological chemistry - Congresses
`2. Chemical reactions - Congresses
`I. Title
`II. Ananchenko, S N
`574.1'9283
`QP514.2
`ISBN 0-08-023967-6
`7
`
`Il. Series
`79-41671
`
`In order to makethis volume available as economical-
`ly and as rapidly as possible the author's typescript
`has been reproducedinits original form. This method
`has its typographical limitations but it is hoped that
`they in no way.distract the reader.
`
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`Printed in Great Britain by A. Wheaton & Co., Ltd., Exeter
`
`

`

`RECOGNITION OF PEPTIDE HORMONES
`AND KININS: MOLECULAR ASPECTS
`OF THE PROBLEM
`
`G. Chipens, F. Mutulis and S. Galaktionov
`Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR, Riga,
`USSR
`
`Abstract - Some aspects of space structure complementarity of oligopeptide
`hormones and receptor sites and the possible contribution of intramolecular
`ionic-type interactions to the stability of the "biological" conformation
`of peptide bioregulator molecules have been discussed. The theoretical con-
`formational analysis of the bradykinin molecule performed earlier revealed
`close proximity of the C-terminal carboxyl group and the guanino group in
`the arginine residue; a cyclic analogue of bradykinin has been synthesized,
`in which the close location of the two groups was stabilized by covalent
`bonding. The CD spectra of the synthesized compound were identical with
`those of bradykinin, and the new compound was active in eliciting a strong
`and prolonged depressor effect in rats.
`
`Interaction of molecules or complex systems thereof underlies most regulatory processes ope-
`rative in the living cells and organisms. According to our present knowledge, interaction of
`a low-molecular components - "effector" (hormones, antigens, various modulators, substrates
`or inhibitors of enzymatic reactions, etc.) with high-molecular components - "receptor sys-
`tem" (cell membrane receptors, antibodies, enzymes, etc.) results in a mutually-induced alte-
`ration of the stereo-electronic structure of the components and, consequently,
`in altered
`functional properties of the resultant complex. Regulatory processes are known to be charac-
`terized by high specificity, owing to the correspondence of geometrical
`forms and the appro-
`priate spacing of functional groups in the effector-receptor pair. As evidenced by X-ray
`analysis, specific recognition and binding occuring in the course of protein-protein inter-
`action is provided by a small area on their surface characterized by rigid structure with
`loops or ledges on the effector molecule and hollows - "pockets" or "slots" on the receptor
`(Refs.1-4). The active centres of protein effectors (enzyme inhibitors, antibodies, etc.)
`comprise, on the average, 6-8 amino acid residues (Refs. 1-5). Geometry of these "recognition
`and binding" centres is spatially stabilized by means of loops or f-bends and is "cemented"
`by the overall space structure of the protein globule. The ends of the "active" fragments
`(especially in the case of mini-proteins) are frequently immobilized by means of disulphide
`bonds (Refs.6 & 7).
`that the process of
`The concept of biochemical universality makes it conceivable to expect
`"recognition" and binding of peptide hormones and kinins to receptors located on the cell
`membrane would be equally determined by similar "active" regions (containing 6-8 amino acid
`residues) on the peptide molecules. However,
`in contrast with the proteins,
`the space struc-
`ture of peptide effectors in solution is not so well-defined: it is most likely that there
`exists an equilibrium of several equally stable conformers in solution,
`the best suited of
`them being "selected" by the receptor. The existence of such a limited set of conformers,
`characterized by relatively rigid space structures seems to be a prerequisite for the purpo-
`seful transfer of information to occur at the molecular level and for the effector-receptor
`interaction involving peptide effectors to be specific (Ref.8).
`Extremely interesting,
`in this respect, are the peptide space structure data obtained using
`semi-empirical conformational analysis, which provides evaluation of intramolecular confor-
`mational energy for each possible molecular conformation as a measure of its stability
`(Refs.9-10). Having acquired similar data for a number of peptide molecules, it may be pos-
`sible to establish the common principles shared by the space organization of the peptides.
`In fact, our studies along this line have led us to the establishment of a number of charac-
`teristic features inherent in the structural organization of low-molecular peptides which
`are well-correlated with our earlier findings on their functional organization (Ref.8). Thus,
`the conformational calculations performed in our laboratory for the molecules of biologically
`active peptides - bradykinin, angiotensin, Met-enkephalin, tuftsin, reveal the presence in
`all cases of a more or less limited set of stable conformers characterized by compact struc-
`tures with N- and C-terminal parts located in close proximity, hence, quasicyclic structure
`being a prominent feature of their space organization (Refs.11-14). In each case,
`the obser-
`ved structure of the molecule is determined by the overall system of intramolecular interac-
`tions as a whole; one can discern, however, several structural elements, each of them contri-
`
`99
`
`

`

`100
`
`Recognition of Peptide Hormones and Kinins
`
`buting to the stability of the quasicyclic molecular structures. The first to be mentioned
`in this connection are the glycin and proline residues, distinquishable from the rest of the
`natural amino acid residues present in the amino acid sequence of the above peptides, owing
`to the characteristic steric conditions of their backbone (Ref.15). The presence of proline
`in the peptide chain is known to substantially limit conformational lability of the peptide
`backbone,
`the effect not being confined to the position occupied by proline itself, but being
`also present in the preceding position (ref.16); consequently, proline plays the role of a
`conformationally rigid element in the peptide chain. Conversly,
`the glycine residue exhibits
`enhanced conformational lability and is a kind of “conformational
`joint~-hinge" providing
`close spacing of N- and C-terminal parts of the peptide chain. This fact is in good agreement
`with the results of calculations performed on the glycine-containing peptides bradykinin and
`Met-enkephalin, as well as the recent results with luliberin (Ref.17):
`the most stable back-
`bone conformations in all these peptides are characterized by the glycine residue conformat-
`ion which is sterically inconsistent for any other type of amino acid residues. The specific
`role of glycine and proline residues in peptide molecules is also indicated by their increas-
`ed relative content
`(~12%), as compared with proteins,
`in the total amino acid composition of
`short
`(up to 30 amino acid residues) peptides (Ref.18).
`The compact quasicyclic structures of peptides mentioned are stabilized predominantly by non-
`bonded interactions; an additional factor contributory to their stability is present
`in all
`the cases described above (Table 1), viz.strong electrostatic interaction between the functi-
`onal groups carrying alternative charges - guanidyl group of arginine residue,
`€-amino
`group
`of lysine residue or d-amino group and C-terminal carboxyl group. Such interaction occurs
`between ionogenic groups in the molecule on their close spacing,
`the process being accompan-
`ied by the appropriate hydrogen bonding. It is notable that in aqueous solution the above
`interactions are weakened by hydration, whereas in non-polar medium, i.e.
`in the course of
`effector-receptor complex formation they are significantly enhanced leading to increased re-
`lative content of quasicyclic conformers of the peptide effector molecule. The aforesaid
`gains indirect substantiation from the protein-protein interaction kinetics studies on enzy-
`mes binding to their specific inhibitors. The results of these studies demonstrated the oc-
`curance of desolvation of the interacting surfaces and charged side groups in amino acids
`during the early stages of interaction (Ref.] ). Furthermore, according to the results of our
`quantum chemical calculations carried out in vacuo,
`the energy of interaction between guani-
`dyl and carboxyl
`ions was estimated to be about -55 kcal/mole,
`the value comparable with the
`dissociation energy of covalent disulphide bond.
`
`TABLE 1. Quasi-cyclic structures of oligopeptides, as characterized by the
`
`data of semi-empirical conformational analysis
`
`Peptide
`
`Type of interaction
`
`Ref.
`
`
`
`Size of
`quasi-cycle
`aa 9>Pat1 oof a=
`
`
`
`Bradykinin
`Arg 6-G
`©... OOC(Arg’)
`1-9
`(11)
`ArgProProGlyPheSerProPheArg
`Angiotensin
`AsnArgValTyrValHisProPhe
`Met-Enkephaline
`TyrGlyGlyPheMet
`Tuftsin
`ThrLysProArg
`BPP
`pGlyTryProArgProGlulleProPro
`+ G* - delta guanidyno group:
`
`Arg?26-c* ...~00c(Phe®)
`
`. ss 00C (Met?)
`o-NHS
`Lyse-NH} ...00C(Arg*)
`Arg'é-c"
`... 00C(Pro”)
`
`2-8
`
`1-5
`2-4
`4-9
`
`(12)
`
`(13)
`(14)
`(23)
`
`_ wHewee NH,
`“SH2
`
`im-
`important
`the results obtained using semi-empirical conformational analysis suggest
`Thus,
`plication of ionogenic groups in the formation and maintenance of the space structure in the
`peptide molecules, especially during the process of their interaction with receptors. It is
`appropriate to recall,
`in this connection,
`that we had postulated previously the presence of
`typical elements in amino acid sequences in the molecules of biologically active low-molecu-
`lar peptides. These sequences contained an ionogenic basic amino acid incorporated between
`proline and valine residues, on the one side, and acidic amino acid and glycine, on the other
`side (Refs.8,20,21). These fragments were also shown to exhibit a rather wide range of non-
`specific biological action, but their conjugation with the "shortest" peptide fragments in-
`duced a significant rise in the specific activity of these fragments (Ref.22). However,
`there
`is no clear-cut evidence, at present, demonstrating direct implication of these fragments in
`the activation of receptor during effector-receptor interaction. At
`the same time,
`the fin-
`ding concerning the space organization of the peptide effectors described above serve,
`in our
`
`

`

`Recognition of Peptide Hormones and Kinins
`
`101
`
`to emphasize the structural role of the detected fragments in the oligopeptide mo-
`
`opinion,
`lecules.
`The localization of ionogenic side chains, which participate in the interactions responsible
`for the closure of quasicyclic structures in non-cyclic peptides is somewhat similar to the
`localization of disulphide bonds, responsible for the closure of ring structures in cyclic
`peptides (see hypothesis on the equifunctionality of ionic and disulphide bonds (Refs.8,27).
`This can be viewed as another indication of the important role reserved for quasicyclic (or
`cyclic) structures in the molecules of biologically active peptides comparable size values
`(approximately, 6-8 amino acid residues) of functionally active disulphide cycles and tenta-
`tive quasicycles in the molecules of some peptides (Refs.8,27).
`The above consideration lead to assume that high selectivity and specificity observed during
`the processes of mutual "recognition" and peptide effector binding to the receptor are due to
`the interaction of conformationally rigid quasi-cyclic sturctures of the effectors with the
`receptor "pockets", providing large interacting areas and multiple contact sites. It can be
`expected, therefore,
`that covalent fixation of the stable quasi-cyclic structures of the ef-
`fector molecule "selected" by the receptor will result in increased interaction efficiency.
`The hypothesis was put to trial using bradykinin, a peptide endowed with high specific acti-
`vity, exhibiting a variety of biological effects. The stable conformations calculated for
`this compound demonstrated,
`in agreement with spectroscopy data (ref.24), considerable pre-
`valence of 4 types of quasi-cyclic structure for the peptide backbone (2 of them are depic-
`ted in Fig.l) in which guanidyl group of Arg
`is located close to C-terminal carboxyl group.
`
`
`
`Fig. 1. Two of the four types of the most stable quasi-cyclic bradykinin
`structures (Ref.24).
`
`these structures show highest stability also in the absence of
`According to calculations,
`electrostatic interaction of the above mentioned ionogenic groups.Bearing these results in
`mind, we undertook an attempt to synthesize bradykinin analogue in which mutual location of
`N- and C-terminal groups, as predicted by the calculations, was stabilized by covalent bon-
`
`

`

`102
`
`Recognition of Peptide Hormones and Kinins
`
`ding. The model compound, cyclo-(N-e'-lysine, 6-glycine)-bradykinin:
`
`Nil,~CH-CO-Pro-Pro-Gly-Phe~Gly-Pro-Phe-NH-CH-(CH).NH*Y-NH
`CHsoeCHpreong CHpee NH-CO
`NH,
`
`was synthesized using routine methods of peptide synthesis and was purified to produce a ho-
`mogeneous substance, as evidenced by chromatographic assays, with predicted molecular weight
`and amino acid and elementary composition. Comparison of
`CD spectra
`of bradykinin
`with those of its cyclic analogue in aqueous solution revealed their complete identity,
`thus
`confirming the hypothesis claiming likeness of their stable structures. Further evidence in
`favour of this hypothesis was presented by the results of biological testing which appeared
`to be even more telling (see Note a).
`The mode of bradykinin action on the smooth muscles is known to be subject to considerable
`variation depending on the tissue and species of origin and other factors (ref.25). It is
`noteworthy that competition studies performed on a large number of bradykinin analogues
`failed to provide so far any conclusive evidence of antagonistic relationship (refs.25 & 26).
`Peculiarity of bradykinin, as compared with other peptide compounds exhibiting myotropic ac-
`tivity,
`is further substantiated by the results of biological experiments carried out on its
`cyclic analogue. It must be noted, first and foremost,
`that
`the chemical modification caused
`a considerable change in the scope of its action. For example, bradykinin and cyclobradyki-
`nin were found to produce in rats depressor effect characterized by equal magnitude (Fig.2)>
`
`mm Hg
`
`BK
`
`CBK
`
`100
`
`80
`
`60
`40
`
`25 mkg/kg
`
`— 3 0
`sec.
`
`Time
`
`100
`
`50 mkg/kg
`
`Time
`
`Fig. 2. The effect of bradykinin (BK) and cyclobradykinin (CBK) on arterial
`pressure of urethane-anaesthetized rats.
`
`the effect being, however, more prolonged in the case of the latter compound. Depressor ac-
`tion of "linear" bradykinin, applied at 50 mg/kg was abolished 1-5 min following the admi-
`nistration, whereas cyclobradykinin at the same dose induced a sustained reduction in blood
`pressure by 30-40 mm Hg lasting for 2 hr; full recovery was not attained for several conse-
`cutive hours thereafter. On the other hand, cyclobradykinin appeared to be completely inac-
`tive when administered to guinea-pigs under the same conditions. Increase in vascular perme-
`ability in response to cyclobradykinin administration to rats was 25 times weaker than in
`the case of bradykinin. In vitro experiments with colon ascendens of the rat demonstrated
`lack of myotropic activity in cyclobradykinin; no antagonism towards bradykinin action was
`also noted in these experiments.
`It is obvious that the results presented above are far from being complete; nonetheless,
`characteristic mode of biological action found for the cyclic analogue of the non-cyclic
`peptide demonstrates feasibility of approach aimed at purposeful searching of "conformatio-
`
`Note a. PrelimaeypaOheeisabesePSKPiohxcbradykinin was performed in our laboratory by
`
`

`

`Recognition of Peptide Hormones and Kinins
`
`103
`
`nally limited" analogues of peptide molecules.
`It is known that
`the early attempts to carry out covalent fixation of particular fragments
`of the molecule which are brought
`in close proximity, met with little success (Ref.28). How-
`ever, recently there has been a report by Veber et al. who synthesized a highly active cyclic
`analogue of somatostatin (Ref.29). It is hoped that new analogues of other peptide hormones
`and kinins will be obtained in due course on the basis of our knowledge of the three-dimen-
`sional structure of their molecules.
`
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