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Description  |
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FIELD OF THE INVENTION
The present invention is directed to anti-fibrotic agents useful in
controlling pathological fibrotic states in a variety of different
clinical disorders, diseases, and abnormalities; and is particularly
directed to methods for effectively inhibiting the enzymatic activity of
lysyl oxidase in-situ using adjacently positioned diamine analogue
substrates.
BACKGROUND OF THE INVENTION
Pathological fibrotic states are clinically apparent abnormalities caused
by the proliferation of fibroblasts, smooth muscle and other fibrogenic
cells; and by the laying down of collagen and other extracellular elements
typical of wound healing within specific tissues and organs of the body.
The pathology is not only the abnormally excess formation of collagen
polypeptide alpha chains, but also the critical modification using the
enzyme lysyl oxidase to create cross-linkages between adjacent collagen
chains and collagen molecules which is the basis of the structural
stability, maturation, and strength of collagen and scar tissue in
general. The cross-linking of the individual collagen alpha chains is the
major contributor to the tensile strength of the cross-linked fibrils.
Depending upon the location of the collagen chain formation and its
cross-linking via the enzyme lysyl oxidase, the abnormalities may take
form in a variety of clinically identifiable and diagnosed conditions
including: lung fibrosis, atherosclerosis, keloid, liver fibrosis, scar
tissue formation, diabetes, tumor development, and even post-operatively
in procedures such as radial keratotomy where scar formation is counter
productive to the purposes of the surgery. For more detailed information
and review of the relationship between the enzyme lysyl oxidase and the
creation of the pathological fibrotic state, the following publications
are recommended. [Kagan et al., Arteriosclerosis 1:287-291 (1981);
Chichester et al., Am. Rev. Respir. Dis. 124:709-713 (1981); Lerman et
al., Circ. Res. 53:378-388 (1983); Levene et al., Brit. J. Exp. Path.
49:152-159 (1968); Kogan, L. L. and L. Katzen, Ann. Ophthal. 15:842-845
(1983); Chvapil, M., Life Sciences 16:1345-1362 (1975) Arem, A. J. and R.
Misiorowski, J. Med. 7:239-248 (1976); and Knapp et al., Am. J. Pathol.
86:47-70 (1977)].
For these reasons, there has been considerable interest and research
investigations into the enzymatic activity and properties of lysyl oxidase
and its potential inhibition to prevent collagen chain cross-linking. It
has been proposed that by preventing the oxidative deamination of lysine
and hydroxylysine amino groups within the collagen alpha chains, which is
the enzymatic function and specific activity of lysyl oxidase, the
physical properties of the collagen scar tissue and the resulting fibrotic
pathological state could be substantially reduced. Much of the original
interest and research centered around the pathological defect found in
Marfan's Syndrome, in which a decreased formation of cross-linkages within
collagen and elastin fibers occurs. In prepared animal models, live rats
fed sweet pea meal derived from the seeds of Lathyrus odoratus developed
remarkably similar collagen cross-linking defects and skeletal
abnormalities. The active ingredient responsible for the production of
this defective cross-linking collagen fiber condition or "Lathyrism" was
found to be beta-aminopropionitrile, which blocks cross-linkages in
collagen and elastin fibers by inhibiting the enzyme lysyl oxidase. [Page
R. C. and E. P. Benditt, Biochemistry 6:1142-1147 (1976) and Proc. Soc.
Exp. Biol. Med. 124:454-459 (1967); Narayanan et al., Biochem. Biophys.
Res Commun. 46:745-751 (1971)]. Until very recently, it was believed that
the enzymatic activity of lysyl oxidase required the presence of both
metallic copper; and a specific cofactor, pyridoxal phosphate [Murray, J.
C. and C. R. Levene, Biochem. J. 167:463-467 (1977); Murray et al., Exp.
Mol. Pathol. 28:301-308 (1978)]. Using this enzyme model, a number of
other inhibitors of lysyl oxidase in addition to beta-aminopropionitrile
were reported: Carbonyl Reagents [Harris et al., Biochem. Biophys. Acta
341:332-334 (1973); Kagan et al., Biochim. Biophys. Acta 365:223-234
(1974)]; isoniazid [Arem, A. J. and R. L. Misiorowski, J. Med. 7:239-247
(1976)]; Iproniazid [Rucker, R. B. and B. L. O'Dell, Biochim. Biophys.
Acta 22:527-529 (1970)]; and dithiothreitol [Harris et al., Biochem.
Biophys. Acta 341:332-334 (1973)]. More recently, an investigation of
lysyl oxidase inhibition revealed that this enzyme in the presence of
copper ion and the bound carbonyl cofactor was also inhibited by
disulfhydryls, sulfhydryl-amines, and penicillamine in an irreversible
manner. In addition, reversible inhibition was reported using
dithiothreitol, 1,3-dithio-2-propanol, and 1,3-diaminopropane
[Misiorowski, R. L. and N. J. Werner, Biochem. Biophys. Res. Comm.
85:809-814 (1978)].
Within the last few years, however, it was recognized that the lysyl
oxidase enzyme cofactor was not, as previously believed, pyridoxal
phosphate - but instead was pyrroloquinoline quinone (hereafter "PQQ")
[Williamson et al., J. Biol. Chem. 261:16302-16305 (1986); Kagan et al.,
1st International Symposium On PQQ And Quinoproteins, Delft, The
Netherlands, Sept. 5-7, 1988, page 57; and Kagan, H. M., Abstract,
International Congress On Elastin: Chemical And A Biological Aspects,
Universita Della Basilicata, Potenza, Italy, Oct. 10-13, 1988]. This in
turn has generated increased interest within the relevant components of
the scientific community concerning the mode of action of the previously
reported inhibitors of lysyl oxidase. In addition, because many of the
previously reported compositions were not suitable for clinical and/or
therapeutic use in living humans, there remains a long standing and
continuing need for compositions which are effective inhibitors of lysyl
oxidase in-situ, be it for human or for animal therapeutic use.
SUMMARY OF THE INVENTION
A method for inhibiting the enzymatic activity of lysyl oxidase in-situ is
provided, said method comprising the steps of:
obtaining at least one inhibitory analogue substrate composition having the
formula
##STR1##
Wherein a is 0 or 1 and b is at least 1;
Y and Y' individually may be omitted entirely but when present is selected
from the group consisting of hydrogen, a halogen, a hydrocarbon entity,
and a substituted hydrocarbon entity;
Z and Z' individually may be omitted entirely but when present are
individually selected from the group consisting of hydrogen, a halogen, a
hydrocarbon entity, a substituted hydrocarbon entity, and link R when W is
absent;
W may be omitted entirely but when present comprises the number of atoms
necessary to form a saturated or unsaturated cyclic structure;
R may be omitted entirely, but when present is a moiety able to react with
another ligand; and
J may be omitted entirely but when present is any organic moiety joining
the diamine composition to another molecule as a copolymer.
The present invention also includes therapeutic methods for using the
inhibitory analogue substrate composition as a anti-fibrotic agent in-vivo
for therapeutic treatment of living subjects afflicted with fibrotic
diseases, disorders, and pathologies.
DETAILED DESCRIPTION OF THE FIGURES
The present invention may be more easily and completely understood when
taken in conjunction with the accompanying drawing, in which:
FIGS. 1A and 1B are graphs illustrating the pH dependent oxidation of
monoamines and diamines by lysyl oxidase;
FIG. 2 is a graph illustrating the inhibitory activity of cis and trans
isomers of 1,2-diaminocyclohexane upon lysyl oxidase;
FIG. 3 is a graph illustrating the competitive inhibition of lysyl oxidase
activity against n-hexylamine by cis-diaminocyclohexane;
FIG. 4 is a graph illustrating the inactivation of lysyl oxidase by
cis-diaminocyclohexane as a First Order reaction;
FIGS. 5A, 5B and 5C are graphs illustrating the effects of cis-and
trans-isomers of diaminocyclohexane on the absorption spectrum of
pyrroloquinoline quinone;
FIGS. 6A and 6B are graphs illustrating the effect of
cis-diamino-cyclohexane on the adsorption spectrum of lysyl oxidase; and
FIG. 7 is a graph illustrating the effect of oxygen on the inactivation of
lysyl oxidase by cis-diaminocyclohexane.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides a class of highly effective anti-fibrotic
agents; and methods for using this class of anti-fibrotic agents as
effective analogue substrate inhibitors of lysyl oxidase in-situ - that
is, under both in-vitro and in-vivo conditions.
The most general structural formula for the lysyl oxidase inhibitory
analogue substrates as a class is provided by Formula I as follows:
##STR2##
wherein a is 0 or 1, and wherein b is at least 1;
Y and Y' individually may be omitted entirely, but when present are
individually selected from the group consisting of hydrogen, a halogen, a
hydrocarbon entity, and a substituted hydrocarbon entity;
Z and Z' individually may be omitted entirely but when present are selected
from the group consisting of hydrogen, a halogen, a hydrocarbon entity, a
substituted hydrocarbon entity, and links R when W is absent;
W may be omitted entirely, but when present comprises the number of atoms
necessary to form a saturated or unsaturated cyclic structure;
R may be omitted entirely, but when present is a moiety able to react with
another ligand; and
J may be omitted entirely, but when present is any organic moiety linking
the diamine composition to another molecule as a copolymer.
It will be recognized and appreciated that this inhibitory class of
analogue substrates for lysyl oxidase offer a variety of advantages to the
user, which include:
(a) Synthesis methods for the defined chemical class as a whole which are
conventionally known, relatively simple to perform, and provide the
desired diamine in high yield;
(b) Purification of the diamines conforming to Formula I is easy to perform
using conventionally known techniques and apparatus;
(c) The diamines of Formula I are stable compositions able to be prepared
in a pharmaceutically acceptable manner in both sterile and non-sterile
formats.
(d) The diamines are generally very soluble in aqueous systems at neutral
pH, thereby facilitating their administration in-vivo.
Given the broad class definition of Formula I, the preferred inhibitory
embodiments are those listed in Table I below.
TABLE I
______________________________________
Preferred Linear Diamines
ethylenediamine
1,2-diaminopropane
1,2-diaminobutane
3,4-diaminobutane
1,2-diaminooctane
7,8-diaminooctane
Preferred Branched Diamines
diaminoisopropane
1,2-diaminoisobutane
1,2-diaminoisopentane
Preferred Cyclic Diamines
1,2-diaminocyclohexane
1,2-diaminocyclopentane
1,2-diaminocycloheptane
1,2-diamino, 3-chlorocyclohexane
1,2-diamino, 3-fluorocyclohexane
1,2-diamino, 3-bromocyclohexane
______________________________________
For completeness of description and ease of understanding, each of the
requisite components comprising the inhibitory analogue substrate
composition will be described individually hereinafter. Subsequently, the
various ways of preparing and using the analogue substrates as
anti-fibrotic agents will be reviewed in detail. Finally, a limited number
of laboratory scale experiments will be described to provide empirical
data and results illustrating the variety of therapeutic uses and
applications for the present invention.
The Organic Supporting Structure Comprising At Least Two, Adjacently
Positioned Primary Amine Groups
The chemical structure of the inhibitory anti-fibrotic agent is based upon
the presence of an organic supporting structure - a carbon backbone having
at least two adjacently positioned carbon atoms available for the
attachment of primary amine groups. These organic supporting structures
and their derivatives may comprise saturated and/or unsaturated molecules;
straight and branched linear chains; single and multiple rings including a
variety of heterocyclic ring structures; and any combination of these as
monomers, dimers, and polymers. In addition, each of these organic
supporting structures may also contain substituted hydrocarbons and
organic groups to form derivatized forms.
Each preferred organic supporting structure encompassed by Formula I will
support at least two adjacently positioned primary amine groups in a
manner which permits an enzyme analogue substrate of lysyl oxidase to be
formed. Clearly therefore, at least two adjoining primary amine groups
available for reaction must always be present in these embodiments. In
some instances, three or more primary amine moieties might also be
effectively utilized in more intricate and involved molecular arrangements
and organizational networks. In most embodiments, however, it is more
desirable that the diamine derivative form by employed. To demonstrate the
variety of different organic supporting structures able to employed in
diamine and polyamine derivatized form, a representative but incomplete
listing of supporting backbone structures deemed to be useful in the
present invention is provided by Tables II, III, and IV respectively
below.
TABLE II
__________________________________________________________________________
ORGANIC STRUCTURE
BACKBONE STRUCTURE
DIAMINE DERIVATIVE
__________________________________________________________________________
ethane
##STR3##
##STR4##
butane
##STR5##
##STR6##
heptane
##STR7##
##STR8##
2-pentene
##STR9##
##STR10##
##STR11##
2,4-hexadiene
##STR12##
##STR13##
__________________________________________________________________________
TABLE III
__________________________________________________________________________
ORGANIC SUPPORT
BACKBONE STRUCTURE
DIAMINE DERIVATIVE
__________________________________________________________________________
cyclopropane
##STR14##
##STR15##
cyclohexane
##STR16##
##STR17##
dicyclohexane
##STR18##
##STR19##
1,4-cyclopentadiene
##STR20##
##STR21##
4,6-cyclohexadiene
##STR22##
##STR23##
__________________________________________________________________________
TABLE IV
__________________________________________________________________________
ORGANIC SUPPORT
BACKBONE STRUCTURE
DIAMINE DERIVATIVE
__________________________________________________________________________
phenyl ring and phenyl derivatives
##STR24##
##STR25##
napthalene and napthalene derivatives
##STR26##
##STR27##
##STR28##
quinoline and quinoline derivatives
##STR29##
##STR30##
##STR31##
indole and indole derivatives
##STR32##
##STR33##
phenanthrene and phenanthrene derivatives
##STR34##
##STR35##
##STR36##
##STR37##
__________________________________________________________________________
Table II illustrates some of the saturated and unsaturated organic support
structures and their respective diamine derivatized forms wherein R is
selected from the group consisting of hydrogen, an inorganic entity, and
an organic moiety. It will be appreciated that the two primary amine
groups are always positioned on adjacent carbon atoms when employing any
of the alkyl support structure regardless of the total number of carbon
atoms in the molecule as a whole.
Similarly, when unsaturated moieties form part of the organic support
structure, it is most desirable that the molecular configuration assume a
cis orientation rather than a trans orientation in order that the primary
amine groups be aligned in the same stereochemical plane. It will be
recognized by ordinary practitioners in this art that the saturated
organic support structures as a class offer a greater number of rotational
degrees of freedom in comparison to unsaturated, olefin support
structures. Nevertheless, straight and branched linear chains comprising
hydrocarbons or substituted organic saturated molecules will provide many
useful and operative support structures as backbone components in the
present invention.
Table III illustrates a variety of saturated and unsaturated cyclic
molecular arrangements and diamine derivatives. Clearly, the number of
degrees of rotational freedom provided by cyclic structures is more
limited in comparison to linear and branched chain arrangements having the
same number of carbon atoms. It is desirable also that each attached
primary amine group lie in a cis orientation rather than a trans
orientation with respect to at least one other adjacently positioned
primary amine group in the overall structure. For descriptive purposes,
however, the primary amine groups of Table III are attached to adjacently
positioned carbon atoms without orientational specificity in each
instance. In addition, the positioning of primary amine groups on adjacent
carbons within a ring structure is an absolute requirement. A variety of
alternative arrangements are illustrated by Table IV in which the organic
supporting structures are all cyclic ring structures in nature and are
deemed to represent all aromatics and/or aryl derivatives without
limitation. It will be appreciated that while the two primary amine groups
are attached to adjacent carbon atoms in the single phenyl ring structure,
the napthalene and quinoline structures individually permit attachment of
adjacently positioned primary amine groups at different locations on the
ring structure. Furthermore, the 3-ring structure of phenanthrene allows
the user to position the two primary amine groups upon adjacent carbon
atoms at various positions within the multi-ring organization.
The requisite principle and sole limiting factor is that the organic
supporting structure provide at least one pair of adjacently positioned
carbon atoms for the attachment of two primary amine groups such that a
discrete, identifiable diamine composition is formed. The true formula and
sterochemical arrangement of the supporting structure, the placement of
the two adjacently positioned amine groups within the support structure,
and the true total number of primary amine groups within the composition
are not paramount or decisive; rather, these are secondary and tertiary
factors which affect the ease of preparation, effective use concentration,
and inhibitory efficacy of the resulting substrate analogue.
It will be recognized and appreciated also that the chemical structure of
Formula I is defined broadly to encompass all possible useful sizes,
structures, moieties, constituents, and chemical formulations of carbon
and other atoms which are believe able to demonstrate at least some
measurable inhibitory effect on the enzymatic activity of lysyl oxidase,
given the presence of its co-factor, pyrroloquinoline quinone (PQQ),
in-situ. For these reasons also, the stated definitions of Y, Y', Z, Z',
W, R and J are given broadly, rather than narrowly, to insure that the
many structural variations, formulations, and derivative forms known for
organic structures are included.
Moreover, the subject matter as a whole comprising the present invention is
provided as a discrete, integral, preformed enzyme substrate analogue
which is intended to be prepared in advance and expected to be used as an
antifibrotic agent in-vivo. For this reason, the preferred embodiments are
simple chemical structures and formulas. However, the simpler forms may be
joined to many different ligands obtained from diverse sources and origins
if more complex embodiments are desired. The intended manner of covalently
attaching the diamine composition of Formula I is via an organic moiety
identified as "R" in the most general definitions of the present
invention; and preferably are the entities provided by Table V in the
majority of instances.
TABLE V
______________________________________
CHEMICAL STRUCTURE
"R" ENTITY
______________________________________
COOH Carboxyl
N.sub.3 Azide
##STR38## N-Hydroxysuccinimide esters
NH.sub.2 Amine
OH Hydroxyl
SH Thio
NCS Isothiocyanate
CN Nitrile
##STR39## Halogens (fluoro, chloro, bromo, iodo)
______________________________________
It will also be recognized and appreciated that the organic supporting
structure comprising at least two adjacently positioned primary amine
groups, regardless of the formula and internal structure of the molecule,
may be usefully employed in a variety of different formats--that is, as a
monomer, a dimer, or a polymerized molecule. For illustrative purposes
only, this discussion will focus upon and be limited to a branched
saturated composition having two primary amine groups such as is provided
by Table VI below.
TABLE VI
______________________________________
FORMAT POLYAMINE STRUCTURE
______________________________________
monomer
##STR40##
dimer
##STR41##
polymer
##STR42##
______________________________________
wherein R.sub.1 -R.sub.8 individually are selected from the group
consisting of hydrogen, a hydrocarbon moiety, a halogen, and an organic
group;
J is a linking molecule; and
q and t individually are zero or a positive integer.
The monomer format of Table VI follows and includes all the examples
previously given by Table II (and inherently Tables III and IV as well).
The dimer format of Table VI is a covalent linking of two monomer units
which may or may not be identical in composition. The reactions and
reagents employed in preparing the dimer format are conventionally known
in the scientific literature and are easily adapted to link any
combination of individual monomer units. The polymer format of Table VI
envisions and intends the use of a cross-linking agent identified
generally as "J"; and also recognizes that each copolymer may itself be
prepared as a homopolymer prior to cross-linking with the other copolymer.
In addition, it is intended that each prepared homopolymer be useful in
its own right without further cross-linking to another copolymer--if this
is either desirable or necessary for the user. Methods, reactions, and
reagents are conventionally known for preparing both homopolymers, and
cross-linking individual copolymers. All of these conventionally known
formats and techniques may be freely employed as desired when preparing a
particular organic support structure comprising a plurality of primary
amine groups for use in the present invention.
Therapeutic Applications
The inhibitory analogues substrates of Formula I are deemed to provide
therapeutic benefits in-vivo when administered to living subjects, humans
and animals, afflicted with a pathological fibrotic state. These
inhibitory analogue substrates can serve as chemotherapeutic agents to
prevent, lessen, and/or control the development of fibrosis in such
diseases as: lung fibrosis, atherosclerosis, keloid, liver fibrosis, scar
formation, diabetes, tumor development, and post-operatively in procedures
such as radial keratotomy where scar formation is counterproductive to the
purposes of the surgery.
Routes Of Administration In-Vivo
Compounds embodying Formula I of the invention can be administered in any
appropriate carrier for oral, topical, inhalatory, or parenteral
administration. They can be introduced by any means that effects
conditions of fibrosis in living humans or animals. The dosage
administered will vary and be dependent upon the age, health, and weight
of the recipient; the kind of concurrent treatment, if any; the frequency
of treatment; and the nature of the therapeutic effect desired. Generally,
the expected daily dosage of an inhibitory substrate analogue will be from
about 0.01 milligrams/kg to 1.0 milligrams/kg, the true dosage and best
route of administration to be established after thorough assessment of
potential toxicity, inhibitory potency, and efficacy. Normally, from 0.5
to 50.0 milligrams per day, in one or more administrations per day, is
expected to be therapeutically efficacious to yield the desired beneficial
results.
If the inhibitory substrate analogues prepared in accordance with Formula I
are to be applied topically, they can be admixed in a pharmacologically
inert topical carrier such as a gel, an ointment, a lotion, or a cream;
and include such carriers as water, glycerol, alcohol, propylene glycol,
fatty alcohols, triglycerides, fatty acid esters, or mineral oils. Other
possible topical carriers are liquid petrolatum, isopropylpalmitate,
polyethylene glycol ethanol 95%, polyoxyethylene monolaurate 5% in water,
sodium lauryl sulfate 5% in water, and the like. Materials such as
anti-oxidants, humectants, viscosity stabilizers, and the like may be
added, if necessary.
In contrast, administration by inhalation will utilize pressurized gases,
propellants, and emulsifiers. Also, the inhibitory substrate analogue
composition may be disposed within devices placed on, in, or under the
skin; such devices include patches and implants which release the active
material into the skin or body either by diffusion or by an active release
mechanism.
Similarly, if the inhibitory substrate analogues of Formula 1 are to be
given parenterally, they will be prepared in sterile form; in multiple or
single dose formats; and dispersed in a fluid carrier such as sterile
physiological saline or 5% dextrose solutions commonly used with
injectables.
To demonstrate the utility, enzyme specificity, and efficacy of the
therapeutic methodology comprising the present invention, a variety of
experiments and empirical data will be described hereinafter. It will be
expressly understood, however, that these experiments and empirical
results are merely descriptive of the present invention as a whole; and
serve to merely illustrate some situations in which the present invention
may be usefully employed. None of the experimental modes, empirical data,
or conclusions are deemed to be restrictive of the invention in any form
or use; to the contrary, it will be recognized and appreciated that these
experiments merely demonstrate the variety of applications and the range
of effective parameters one may expect to be in effect when employing the
present invention.
Empirical Experiments
A. Materials and Methods
Reagents
Homovanillic acid, horse radish peroxidase, n-hexylamine,
1,6-diaminohexane, ethylenediamine, 1,5-diaminopentane, and n-propylamine
were obtained from Sigma Corp. The cis and trans isomers of
1,2-diaminocyclohexane were obtained from Alfa Products. [.sup.3
H]NaCNBH.sub.3 (10 Ci mmole.sup.-1) was a product of Amersham Corp.
Pyrroloquinoline quinone was obtained from Fluka Corp.
Enzyme Purification
Lysyl oxidase was purified from bovine aorta as previously described.
[Williams, M. A. and H. M. Kagan, Anal. Biochem. 113:336 (1985)]. The
resulting preparation consists of a copurified mixture of four ionic
variants of lysyl oxidase. Each of these enzyme variants has a molecular
weight of 32,000 in sodium dodecylsulphate; peptide maps of proteolytic
digests of the individual variants are very similar; and the substrate
specificities and inhibition profiles of the variants appear to be
virtually the same. This indicates that the catalytic mechanism is likely
to be the same for each of these enzyme forms [Sullivan, K. A. and H. M.
Kagan, J. Biol. Chem. 257:13520-13526 (1982)].
The purified lysyl oxidase was assayed against an insoluble elastin
substrate prepared from chick embryo aortas which had been pulsed in organ
culture with L-[4,5-.sup.3 H]lysine [Kagan, H. M. and K. A. Sullivan,
Methods Enzymol. 82A:637-649 (1982)]. Enzyme assays include 125,000 cpm of
the elastin substrate in 0.1M sodium borate, 0.15M sodium chloride at pH
8.0 in a total volume of 750 ul and were incubated for 2 h at 37.degree.
C. Tritiated water formed during the incubation was isolated by vacuum
distillation and quantified by liquid scintillation spectrometry of 0.5 ml
aliquots of the distillates. All activities were corrected for enzyme-free
controls and were within the linear range of this assay (100 to 1200 cpm
released per 2 h). One enzyme unit was defined as 1 dpm of .sup.3 H
released by enzyme action in 2 h. Functional active site content was
quantified by comparing the specific activity of each enzyme preparation
against the elastin substrate to the theoretical, maximum value of
4.times.10.sup.6 u mg.sup.-1 previously estimated as the value for the
fully functional enzyme [Williamson et al., Biochem. J. 235:597-605
(1986)].
The purified lysyl oxidase enzyme was also assayed against alkyl monoamines
and alkyl diamines using the reported peroxidase-coupled fluorescence
method at 55.degree. C. [Trackman et al., Anal. Biochem. 113:336-342
(1981)]. The reaction mixtures contained 40 ug horseradish peroxidase, 0.7
mM homovanillic acid, 1.2M urea, 0.02M potassium phosphate/borate buffer,
at pH 8 (or at varying values of pH as indicated) in a total volume of 2
ml. Assay solutions were adjusted to an ionic strength of 0.04 using
potassium chloride. Lysyl oxidase was then added to initiate the reaction.
Fluorescence was continuously monitored at an excitation wavelength of 315
nm and an emission wavelength of 425 nm. The enzyme-dependent production
of hydrogen peroxide was quantified by reference to standard plots
relating nanomoles of hydrogen peroxide added to fluorescence units.
Assays carried out at a pH which occurred within the overlapping buffering
ranges of phosphate and borate resulted in the same value of Vmax
regardless of the presence of the phosphate, of the borate, or of both
buffering species. Kinetic constants were derived from initial rate assay
data by using the Fortan program of Cleland [Meth. Enzymol. 63:103-138
(1979)] which determines Vmax and Km values from data of Michaelis-Menten
experiments using a least squares fitting procedure applied to the
Michaelis-Menten equation.
Spectral Studies
Absorption spectra were recorded in 1 cm cuvettes with a Hewlett Packard
Diode Array spectrophotometer, with temperatures controlled by circulating
water through the cuvette holder. Mixtures were made anaerobic by purging
buffers and stock solutions in rubber-capped tubes with high purity
nitrogen for 15 min, and then injecting aliquots of the solutions into a 1
cm semi-micro cuvette fitted with a rubber septum and flushed with
nitrogen before addition of reagents. Once in the capped cuvette, each
solution was further exposed to a stream of nitrogen passed over the
surface of the solution and out of the capped cuvette through 21 guage
needles for 5 min. just prior to spectral recording. Each perturbation
described was reproducible in three or more trials.
NMR spectra were obtained on a Bruker AC 200 spectrometer. Solutions for
NMR studies were prepared in D.sub.2 O and the pH was adjusted to 8.0 with
NaOD. Solutions containing 10 mM PQQ were prepared in the presence or
absence of 9 mM ethylenediamine or of 9 mM concentrations of the cis or
trans isomers of 1,2-Diaminocyclohexane or "DACH". The presence of 40 mM
sodium borate did not affect the spectra. The HOD peak was suppressed by
homonuclear decoupling. Chemical shifts are referenced to sodium
3-trimethylsilyl[2,2,3,3-.sup.2 H ]propionate as the internal standard.
B. Synthesis of [1,2-.sup.3 H]-1,2-Diaminocyclohexane
[1,2-.sup.3 H]-1,2-diaminocyclohexane was synthesized by reductive
amination of 1,2-cyclohexanedione according to the method of Borch et al.,
[J. Am. Chem. Soc. 93:2879-2903 (1971)]. A solution of
1,2-cyclohexanedione (5 mmol), ammonium acetate (100 mmol), and [.sup.3
H]NaCNBH.sub.3 (7 mmol; 230 uCi mmole.sup.-1) in 30 ml of absolute
methanol was stirred at 25.degree. C. for 48 h. The pH was then adjusted
to pH 1.5-2 with 12N HCl; the solution brought to dryness; and the residue
was taken up in 10 ml of water and extracted with three 20 ml portions of
ether. The aqueous layer was then adjusted to pH 10-11 with KOH; solid
NaCI was added to saturation; and the solution was then extracted with
several 15 ml portions of ether. The combined ether extracts were dried
over anhydrous magnesium sulfate, filtered, and evaporated in vacuo. The
crude product was crystallized and then recrystallized from
ethanol-ethylacetate mixtures, with 300 mg of the purified product finally
obtained. Thin layer chromatography of the resulting product using
methanol:water (9:1) as the developing solvent revealed one
ninhydrin-positive spot with an Rf of 0.65-identical to that of a mixture
of authentic cis- and trans-DACH. The product melted at
38.degree.-40.degree. C. (literature 37.degree.-42.degree. C.). This
product also perturbed the absorption spectrum of pyrroloquinoline quinone
or "PQQ" in a manner identical to that of authentic DACH.
C. Empirical Results
Experimental Series 1: Reaction of Lysyl Oxidase with Monoamine and Diamine
Substrates
The pH-dependency of the Vmax for the oxidation of a series of diamines and
monoamine substrates by lysyl oxidase was first explored. As shown by FIG.
1, the pH optimum shifts to more acidic values as the chain length of
diamine substrates is decreased from 6 to 5 to 3 carbons. Moreover, the
Vmax values at the pH optima decrease with decreasing chain length and
thus the substrate potential decreases with decreasing chain length.
Ethylenediamine or "EDA", the shortest of the diamines, proved to be an
effective competitive inhibitor of lysyl oxidase activity against
n-hexylamine with a K.sub.I of 2.times.10.sup.-6 M. In contrast to the
diamines, the pH optimum remains at approximately 8.5 for monoamines of
different chain length as shown in FIG. 1. The Vmax decreases only
slightly with decreasing monoamine chain length, contrasting with the
marked decrease in Vmax seen with the diamine substrates.
The possibility was then explored that the inhibitory effects of diamines
as expressed maximally with EDA may involve the interaction of both of the
amino functions of the diamine with an enzyme constituent(s). Since such
enzyme groups would likely be specifically oriented relative to each
other, it seemed possible that opposite configurational isomers of a
sterically restricted diamine might have different reactivities with the
enzyme. It is known that benzylamine and its analogues can serve as
substrates of lysyl oxidase [Williamson P. R. and H. M. Kagan, J. Biol.
Chem. 262:14520-14524 (1987)]; and thus it seemed possible that DACH, a
saturated cyclic diamine for which the cis and trans isomers are
available, possibly could also be accommodated at the active site.
Accordingly cis and trans isomers of DACH were used to probe this
possibility. As shown in FIG. 2, cis-DACH is revealed to be a potent
inhibitor of lysyl oxidase, with an I.sub.50 of 1.5 .mu.M in assays of
n-hexylamine oxidation while trans-DACH is at least 10,000-fold less
inhibitory with an (extrapolated) I.sub.50 >10 mM. The differential
sensitivity of lysyl oxidase to these agents was virtually the same in
assays of [.sup.3 H]elastin oxidation (not shown). A Lineweaver-Burk plot
of the initial rate assays for the mode of inhibition by the cis compound
resulted in a series of lines intersecting at the 1/v axis as shown by
FIG. 3, indicating that cis-DACH competes with the amine substrate for
interaction at the active site. The K.sub.I was calculated from the slopes
of this plot and found to be 3.8.times.10.sup.-7 M. The inhibition by
cis-DACH was determined to be irreversible rather than reversible since
the lysyl oxidase enzyme (which had been preincubated with 0.05M cis-DACH
at 37.degree. for 15 min and then dialyzed to remove the substrate
compound) was catalytically inactive.
In addition, the time-dependency for the expression of irreversible
inhibition was assessed by initially preincubating lysyl oxidase at
37.degree. in the presence or absence of various concentrations of
cis-DACH; and then diluting aliquots of these individual reaction mixtures
into peroxidase-coupled assays for n-hexylamine oxidation - thereby
reducing the cis-DACH concentrations to non-inhibitory levels within the
lysyl oxidase assays. The rates of loss of enzyme activity were found to
increase with increasing cis-DACH concentrations, with the inactivation of
lysyl oxidase following apparent first order kinetics. This is illustrated
by FIG. 4. The limiting apparent first order rate constant for
inactivation determined from the intercept of a secondary reciprocal plot
of these data was calculated as K.sub.inact =18 min.sup.-1. The
possibility that the inactivation derived from the removal of the metal
ion cofactor of the enzyme by the cyclic diamine was then assessed by
atomic absorption spectroscopy. This method of analysis yielded the same
values for protein-bound copper in aliquots of purified lysyl oxidase
which had been incubated in either the presence or absence of 1 mM
cis-DACH and then dialyzed extensively against metal-free buffer. This
data reveals that cis-DACH did not remove copper from the active site in
order to cause irreversible inhibition of lysyl oxidase.
Experimental Series 2: Spectral Studies
This series of experiments evaluated the possibility that the adjacently
positioned primary amino groups of cis-DACH may be interacting with the
ortho-carbonyl functions of PQQ in lysyl oxidase. Spectral effects seen
with authentic pQQ appeared to support this possibility. As seen in FIG.
5B, the spectrum of PQQ is markedly perturbed by cis-DACH in a
time-dependent fashion; in contrast, as seen in FIG. 5A, there is
relatively little change generated in the PQQ spectrum by trans-DACH under
the same test conditions. This is analogous to the markedly different
sensitivities of lysyl oxidase to these isomers individually. Incubation
of PQQ with monoamines, including 5 mM cyclohexylamine or 5 mM
n-hexylamine, did not perturb the spectrum of PQQ measurably under
corresponding conditions (not shown).
FIG. 5C shows that the perturbation of the spectrum by cis-DACH was
dependent upon the presence of oxygen as noted by the relatively small
spectral changes induced in the PQQ spectrum under anaerobic conditions.
Further, as shown in FIG. 6A, the spectrum of lysyl oxidase is also
perturbed upon the addition of the cyclic diamine. Increased absorption
occurs in accordance with an increase in optical density occurring in the
region of 250 nm; a broad peak also develops with a maximum absorbance at
350-355 nm upon incubation of 47 uM cis-DACH with 4.25 uM of functional
active sites in lysyl oxidase as shown in FIG. 6B. The absorption peak at
350-355 nm correlates quite well with a peak generated at the same
position in the spectrum of PQQ by its interaction with cis-DACH. In
contrast, the spectrum of lysyl oxidase was not altered significantly by
cis-DACH under anaerobic conditions.
Experimental Series 3: Dependency of the inhibition Upon Oxygen
In view of the apparent inhibiting effect of anaerobic conditions on the
induction by cis-DACH of changes in the spectra of PQQ and lysyl oxidase,
the possibility was evaluated that oxidation of the diamine substrate
analogue may be involved in the development of irreversible inhibition of
lysyl oxidase. In this regard, the formation of H.sub.2 O.sub.2 was not
detectable within the limits of sensitivity (ca. 1 nanomole of H.sub.2
O.sub.2) if cis-DACH was incubated with catalytic quantities (2-4 ug) of
lysyl oxidase in the peroxidase-coupled assay for H.sub.2 O.sub.2 release.
This argues against significant catalytic turnover with the cyclic
diamine. Nevertheless, as illustrated by FIG. 7, the development of
irreversible inhibition was strongly dependent upon the presence of oxygen
since the rate of inactivation was negligible under anaerobic conditions.
Although cis-DACH did not appear to be a productive substrate, one might
question whether indications of cis-DACH oxidation by the free cofactor or
the purified enzyme might be obtained at levels stoichiometric with PQQ or
lysyl oxidase active sites. Toward that end, evidence was sought for the
release of H.sub.2 O.sub.2 upon the reaction of cis-DACH with limiting
amounts of PQQ or of relatively large quantities (although limiting
relative to cis-DACH) of pure lysyl oxidase. As given by Table E1,
hydrogen peroxide formation was seen under these conditions with the
maximum levels of H.sub.2 O.sub.2 formed essentially equal to the amount
of the limiting reactant in both cases, i.e., free PQQ or lysyl oxidase,
respectively.
TABLE E1
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Oxidation of cis-DAH: Release of H.sub.2 O.sub.2.sup.a/
Nanomoles H.sub.2 O.sub.2 Released
Ratio
Oxidant of Oxidant
(nanomoles) H.sub.2 O.sub.2 /Oxidant
______________________________________
PQQ 13.2 14.2 1.08
26.4 24.6 0.93
52.8 51.2 0.97
79.2 74.9 0.95
.sup.-- X = 0.98 .+-. 0.07
Lysyl Oxidase
0.71 1.0 1.40
1.78 1.70 0.96
2.20 1.52 0.69
.sup.-- X = 1.02 .+-. 0.29
______________________________________
.sup.a/ Hydrogen peroxide as assayed in the peroxidasecoupled fluorescenc
assay using 1 mM cisDACH as the sole source of amine substrate and
containing lysyl oxidase or PQQ at the indicated amounts. Incubations wer
continued until the release of H.sub.2 O.sub.2 was maximal in each case.
The quantity of lysyl oxidase used is expressed in the Table in terms of
functional active site content. The concentration of PQQ was determined
from the A.sub.257,
using 19,122 as the molar extinction coefficent.
TABLE E2
______________________________________
Oxidation of [1,2-.sup.3 H]DACH: Release of .sup.3 H.sup.+a/
Nanomoles .sup.3 H.sup.+ Released
Ratio
Oxidant of Oxidant (Nanomoles)
.sup.3 H.sup.+ /Oxidant
______________________________________
PQQ 2.1 5.1 2.43
4.0 9.0 2.30
6.0 13.0 2.17
7.5 17.2 2.29
.sup.-- X = 2.3 .+-. 0.11
Lysyl Oxidase
0.50 0.98 1.96
1.24 2.23 1.80
3.72 6.95 1.87
.sup.-- X = 1.88 .+-. 0.07
______________________________________
.sup.a/ The indicated amounts of PQQ or functional active sites of lysyl
oxidase were incubated with [.sup.3 H]DACH (1 mM; 0.13 mCi/mmole) in 50 m
sodium borate, pH 8, at 37.degree.C. and aliquots of the incubation
mixture were removed at intervals, distilled in vacuo and the isolated
[.sup.3 H]H.sub.2 O was quantified by liquid scintillation spectrometry.
The values shown are the maximum levels accumulated in these assays and
are corrected for minor background
effects in the absence of oxidant.
Toward similar ends [1,2-.sup.3 H]DACH was synthesized and used to
determine whether the re | | |
