Charge Deficient Analogues of the Natural Polyamines

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262 Current Pharmaceutical Design, 2014, 20, 262-277
Charge Deficient Analogues of the Natural Polyamines
Janne Weisell1, Mervi T. Hyvönen2, Leena Alhonen3, Jouko Vepsäläinen1, Tuomo A. Keinänen1,4,* Alex R.
Khomutov5
1School of Pharmacy, 3Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, Biocenter Kuopio, University of
Eastern Finland, Kuopio Campus, P.O. Box 1627, FI-70211 Finland. 2Department of Cell and Molecular Biology (CMB), Karolinska
Institutet, P.O. Box 285, Berzelius väg 35, 4Department of Microbiology, Tumor and Cell Biology (MTC), Nobels väg 16, KI Solna
Campus, Karolinska Institutet, Box 280, SE-17177 Stockholm, Sweden. 5Engelhardt Institute of Molecular Biology, Russian Academy
of Sciences, Vavilov Street 32, 119991 Moscow, Russia
Abstract: Mitochondrial dysfunction, either inherited or acquired, is associated with several diseases in humans. Depending on the cell
type and location, cells are prone to multiple types of insults that may compromise their proper function. Generally, these insults are
overcome by defensive mechanisms but sometimes they lead to sustained damage, requiring the action of scavenging and repair machin-
eries to retain the viability of the cells. As a final measure, severely damaged cells are targeted to a controlled cell death pathway in order
to not to compromise the well-being of the whole tissue.
The polyamines, spermine and spermidine are essential cellular constituents, participating in many vital functions such as proliferation
and differentiation, immune response and scavenging of reactive oxygen species. Therefore, dysregulation of polyamine metabolism is
often associated with different pathological states. Polyamine acetylating enzyme spermidine/spermine-N1-acetyltransferase is induced by
inflammation, drugs and by several other environmental insults. Resulting accelerated polyamine acetylation with accompanying poly-
amine biosynthesis induction i.e. activation of polyamine futile cycle generates excessive amount of hydrogen peroxide, hampers cell en-
ergy metabolism and induces mitochondrial dysfunction and biogenesis. Therefore, the drugs inhibiting polyamine metabolism are valu-
able in protecting mitochondria and cell energy metabolism. Here we review the current literature focusing on the applicability of charge-
deficient polyamine analogs as drugs to modulate polyamine metabolism. Alteration of pK of amino group(s) in a respective analog is
a
achieved by fluorine substitution of hydrogen atom, hydroxylamine substitution of methylamine or by reducing the numbers of carbon
atoms between amine groups to two instead of three or four.
Keywords: Cell organelles, charge deficient, ornithine decarboxylase, polyamine, polyamine analog, reactive oxygen species.
INTRODUCTION Generation of reactive oxygen species (ROS) is an integral part
A single living cell harbors all the necessary components for of cell metabolism [16]. Most of the ROS are formed in mitochon-
proliferation and maintaining cell/tissue integrity for up to several dria via escaping electron pairs generating superoxide from molecu-
lar oxygen during oxidative phosphorylation through electron
decades under challenges by various environmental (physiological)
conditions, e.g. stress, inflammation, drugs, and harmful sub- transport chain. In the matrix of mitochondria, Ac-CoA is produced
stances. All these factors are present at variable levels practically from lipid and carbohydrate degradation and through citric acid
cycle; intermediary carbon metabolism is connected to oxidative
throughout the life of an organism. In multicellular organisms, the
cells are specialized through differentiation at the cost of losing phosphorylation producing ATP. Both Ac-CoA and ATP are essen-
most of their pluripotent features. The intra- and cross-tissue com- tial for cell energy- and for polyamine metabolism. Mitochondria-
derived ROS are an integral part of the signaling network including
munication between differentiated cells via hormones and other cell
signaling molecules is very important to the function of the host cytosolic ROS generating oxidative enzymes to maintain cellular
defensive system [1-5]. The most harmful insults are those which redox balance and energy homeostasis under different nutrition
conditions [16, 17]. Cellular redox balance is also controlled by
lead to damage in DNA or defects in protein folding that eventually
result in protective activation of programmed cell death through small molecules like glutathione (GSH) – glutathione disulfide
apoptosis, thus clearing the damaged cells from the body [6, 7]. (GSSG), vitamins C and E, and many other natural antioxidant
compounds [18]. Moreover, enzymes like glutathione reductase
Unsuccessful clearance can lead to different pathophysiological
changes associated with different diseases such as cancer [8-10]. [EC 1.8.1.7] maintain equilibrium between GSH-GSSG and conse-
quent actions by Cu/Zn-dependent superoxide dismutase (SOD1)
Intracellular organelles are specialized subunits within the cells
[EC 1.15.1.1] and catalase [EC 1.11.1.6] neutralize ROS to water
with their own lipid bilayer. Damages in organelles can be dealt
and molecular oxygen [19]. Three different SOD isoforms exist,
with autophagocytosis, regenerative fusion of the mitochondria or
one being specific to mitochondria i.e. SOD2 Mn/Zn isoform [2].
biogenesis of new organelles [11-13]. The regenerative potential of
Altogether, the ROS regulatory network and intracellular signaling
cell organelles is vital, and any dysfunction can cause devastating
involved in its regulation are very complex and vulnerable to excess
effects. Sometimes the consequences of unsuccessful repair of mi-
ROS interfering at many points the overall function and finally
tochondrial or nuclear DNA damage can be inherited by the off-
leading to mitochondrial dysfunction [16]. Therefore, it is not sur-
spring and are associated with hereditary diseases [14, 15].
prising that many diseases have been associated with ROS-induced
dysregulation of cell metabolism [15].
The polyamines, spermidine (Spd) and spermine (Spm) and
their diamine precursor putrescine (Put) are aliphatic and achiral
*Address correspondence to this author at the Department of Microbiology,
organic small molecules that are positively charged under physio-
Tumor and Cell Biology (MTC), Nobels väg 16, KI Solna Campus, Karo-
linska Institutet, Box 280, SE-17177 Stockholm, Sweden; logical conditions. They exist in millimolar concentrations in all
Tel: [phone]; E-mail: [email protected] eukaryotic cells and participate in the regulation of important cellu-
1873-4286/14 $58.00+.00 © 2014 Bentham Science Publishers
Charge Deficient Polyamine Analogues Current Pharmaceutical Design, 2014, Vol. 20, No. 2 263
lar functions, such as proliferation, differentiation and functioning AdoMetDC by inducing stalling of the ribosomes on a small up-
of ion channels. Polyamine homeostasis is tightly controlled via stream open reading frame (uORF) which codes short peptide
complex network consisting of uptake, efflux, biosynthesis, MAGDIS [38, 39].
interconversion and terminal degradation, and disturbances in these
The highly inducible catabolic (interconversion) enzyme sper-
systems have been linked to various pathological conditions such as midine/spermine-N1-acetyltransferase (SSAT, EC 2.3.1.57) is N1-
cancer, acute pancreatitis, diabetes, and decreased immune response acetylating both Spd and Spm, using Ac-CoA as a co-substrate
[8, 20-23].
(Fig. 1) [40-42]. Like ODC and AdoMetDC, SSAT has also a short
half-life and complex regulatory mechanism [40, 43, 44]. The N1-
POLYAMINE METABOLISM IN MAMMALS
acetylation of the polyamines Spd and Spm converts them to the
Ornithine decarboxylase (ODC, EC 4.1.1.17) is one of the rate substrates of constitutively expressed N1-acetylpolyamineoxidase
controlling enzymes of polyamine metabolism producing Put by (APAO, EC 1.5.3.13), which catalyzes the oxidation of acetylated
decarboxylating ornithine (Orn) (Fig. 1). Another rate controlling Spm to Spd and acetylated Spd to Put [45, 46]. The most recently
enzyme of polyamine biosynthesis is S-adenosyl-L-methionine discovered enzyme of polyamine catabolism is spermine oxidase
decarboxylase (AdoMetDC, EC 4.1.1.50) that produces decarboxy- (SMO, EC 1.5.3.16) which uses Spm as its substrate, producing
lated S-adenosyl-L-methionine (dcAdoMet), which is consequently Spd, 3-aminopropanal and H O [47-49]. Unlike APAO, SMO is
2 2
used by the aminopropyltransferases spermidine synthase (SpdSy, inducible and is considered to be the major polyamine metabolism-
EC 2.5.1.16) and spermine synthase (SpmSy, EC 2.5.1.22) to pro- based source of ROS in various pathological states [21, 50].
duce Spd and Spm, respectively (Fig. 1) [24]. Both ODC and Ad-
The polyamines can also be terminally deaminated by different
oMetDC are among the most short-living enzymes in the cell,
amine oxidases such as monoamine oxidase, diamine oxidase and
having half-lives of about 20-35 min for ODC [25, 26] and about
serum amine oxidase, but those are not traditionally classified as
35-65 min for AdoMetDC [27-29]. They both are also highly regu-
polyamine metabolic enzymes [51-54]. It is possible that some
lated at all levels of gene expression and possess complex regula-
uncharacterized or unrecognized enzymes that could be involved in
tory mechanisms [30, 31]. The proteasomal degradation of ODC is
polyamine metabolism are still to be found. Alternatively, poly-
triggered by binding of antizyme (Az), a small regulatory protein
amine metabolizing enzymes may possess uncharacterized substrate
which also inhibits the polyamine uptake and induces export [32-
properties toward cellular macromolecules.
34]. Az itself is produced in response to increased polyamine con-
centration, which induces +1 ribosomal frameshift on decoding Az SUBCELLULAR TARGETS OF POLYAMINES IN MAM-
mRNA [35, 36]. Three distinct antizymes have been characterized
MALIAN PHYSIOLOGY
so far and they are regulated by an antizyme inhibitor (AzI; ODC-
Polyamines participate in many cellular processes through ionic
related protein devoid of enzymatic activity) negating Az functions
interactions including hydrogen bonding. In many of these interac-
in the cell [37]. Polyamines also regulate the translation of
Fig. (1). Polyamine metabolism in mammals.
264 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Weisell et al.
tions, polyamines can be replaced with many of their structural to prevent microvascular sprouting in HUVEC cells via deregula-
mimetics or even with mono/bivalent cations like K+,N+,Ca2+, Mg2+ tion of actin cytoskeleton dynamics [91].
[55-60]. Therefore, mimicking physiological ionic conditions in Polyamines are associated with many parts of protein synthesis
polyamine interactions studies is of great importance, because the
machinery [92]. Polyamine depletion reduces general protein syn-
presence of Ca2+/Mg2+ (or other charged molecules) could modulate thesis rate and prevents the synthesis of high molecular weight
polyamine interactions with the target molecules [55, 61, 62]. In glycosylated protein in BHK cells [90, 93]. Recently, it was also
short-term cell culture experiments, many amines are growth sup-
shown that siRNA-mediated knockdown of antizyme inhibitor 2
portive, and the cells are capable of proliferation for few passages mRNA disrupts intracellular vesicle trafficking at trans Golgi net-
by using residual polyamines and their structural mimetics [63-65]. work, retarding the exocytotic release of the detected glycoprotein
Typically the ionic interactions occur in membrane lipid structures.
similarly to (cid:2)-difluoromethylornithine (DFMO) treatment did by
Polyamines also bind tightly with phosphorus-containing molecules depleting polyamines in MCF-7 cells [94]. Posttranslational hy-
including the ribosomes, RNA and DNA and are intimately in- pusine modification of eukaryotic translation initiation factor 5A
volved in transcription and translation [66-68]. Most of the struc-
(eIF5A), where Spd serves as the sole known natural substrate, is
ture/activity studies with the analogues have focused on polyamine among the most specific known polyamine interactions [95]. Hy-
metabolizing enzymes, Az frameshifting or AdoMetDC translation pusinated eIF5A is essential for cell proliferation by being required
i.e. activities of ODC and AdoMetDC [69-72]. Furthermore, the
for the translation of a set of mRNA species in mammalian cells
capability for modulation of ion channels, such as NMDA receptors [95, 96]. Specific deletion of deoxyhypusine synthase (DHS, EC
and inwardly rectifying potassium channels, are among the proper- 2.5.1.46) reduces de novo protein synthesis rate by about 10% [97].
ties tested in vitro for many synthesized analogues [73-76].
Interestingly, the inhibition of hypusinated eIF5A synthesis was
There are still many potentially unrecognized cellular targets of shown to protect against pancreatic (cid:3) cell dysfunction and apoptosis
polyamines and some that are already known are still relatively induced by thapsigargin or streptozotocin [98, 99]. Some of the
poorly characterized, thus validating further analog development, eIF5A target proteins apparently mediate the harmful response of
synthesis and systematic testing. Especially the structural properties these agents, while DHS inhibitors prevent the inflammatory proc-
of substrate/inhibitor recognition of many polyamine metabolizing ess and caspase 3-mediated apoptosis of (cid:3) cells [98, 99]. Thus,
enzymes are still poorly known. For example, S,S-(cid:2),(cid:2)’-Me Spm is DHS and deoxyhypusine hydroxylase (DOHH, EC 1.14.99.29), the
2
an excellent substrate for SMO whereas R,R-(cid:2),(cid:2)’-Me Spm di- enzymes involved in the synthesis of hypusinated eIF5A, represent
2
astereomer is an efficient competitive inhibitor [77]. Likewise, the potential drug targets in type 2 diabetes [100].
structural features of polyamines/polyamine analogs required for Interestingly, an exogenous supply of Spd activates autophagy
the regulation of Az frameshifting or translational regulation of
and can therefore modulate longevity [101]. Autophagy is an inte-
AdoMetDC are yet to be characterized on a wider scale. To facili-
gral part of intracellular organelle maintenance and it is responsible
tate the systematic screening of synthesized polyamine analogs, in for the recycling of damaged proteins [11]. Thapsigargin inhibits
vitro reporter assays have been developed for both key systems [35,
the fusion of autophagosomes with the lysosomes, while low exter-
78, 79].
nal supply of Spd activates autophagosis [102, 103]. Furthermore,
As such, the rate of polyamine biosynthesis and its equilibrium Spd affects the regulation of autophagy through acety-
with the catabolism are of great importance. Many of the currently lases/deacetylases that modulate acetyl lysine levels of acetylpro-
available analytic methods are developed to detect the steady state teome in the nucleus and cytoplasm [104, 105]. Thus, polyamines
levels of the individual polyamines [80]. Many ROS-generating and their metabolizing enzymes play an essential role in protein
insults activate both SSAT and ODC, thus leading to accelerated synthesis and in cellular response against harmful insults.
polyamine flux, depleting cellular ATP, Ac-CoA and folate levels Polyamine uptake system in mammalian cells has not yet been
and producing H O and reactive aldehydes [81-83]. Mice overex-
2 2 characterized in detail [106, 107]. The induction of Az is shown to
pressing SSAT have activated mitochondrial biogenesis, mitochon- downregulate polyamine uptake in mammalian cells. Az is trans-
drial swelling and decreased cellular ATP levels in white adipose lated in two isoforms, the other being targeted to mitochondria
tissue [82,83]. The induction of SMO has also shown to be associ-
[108, 109]. Its function(s) remain to be established, but it is not
ated with inflammatory response, resulting in formation of H2O2, 3- involved in the regulation of mitochondrial polyamine uptake.
aminopropanal and finally acrolein [84]. The activation of SMO is Polyamines are taken up by mitochondria, and Spm has been shown
detected during carcinogenesis, and metabolites formed via poly- to modulate mitochondrial Ca2+ influx [110, 111]. Mitochondria act
amine catabolism are known to trigger apoptosis [50, 85, 86]. Thus, as active compartments in regulating intracellular Ca2+ homeostasis
polyamine metabolism is an integral part of ROS-affected meta- and trafficking [112]. Insults inducing dysregulation of Ca2+ storage
bolic network in cells. Mitochondria are the main cellular source for
activate a cascade leading to the alteration of mitochondrial mem-
ATP and Ac-CoA. Moreover, ornithine derived from the urea cycle brane permeability, causing swelling of mitochondria and their
is used by ODC for Put biosynthesis. Furthermore, methionine used dysfunction in ATP production and finally cell death through
for polyamine biosynthesis from dcAdoMet is connected through
caspase dependent or independent pathways [113]. Polyamines
AdoMet and homocysteine to GSH metabolism [87]. Therefore, have a dual role in ROS network because Spd and Spm act as ROS
determination of polyamine flux under different cellular conditions scavengers and the oxidized polyamines could act as antipathogen
(health/disease) is needed in order to understand better the connec-
compounds [114-117], but the simultaneous activation of poly-
tions between ROS regulating network and polyamine metabolism amine synthesis (ODC induction) and catabolism (SSAT/APAO
with specific emphasis on mitochondrial biogenesis, regenerative and SMO) generates excess ROS and contributes to tumorigenesis
fusion and their function [12, 21, 84].
in Helicobacter pylori infection [85]. Thus, activation of the poly-
Polyamines influence cell architecture and function through amine metabolism is a part of host defense system against pathogen
interaction with microtubules, which are (cid:2)(cid:3)–tubulin polymers har- infection.
boring anionic C-termini [88]. Microtubule bundles are important
for cell movement and division and comprise an important part of CHARGE DEFICIENT ANALOGUES OF NATURAL
the size-sensing machinery of the cell and cell organelles [89]. In- POLYAMINES
terestingly, polyamine depletion is reported to cause swelling of There are few different methods to alter the pK values of the
a
endoplastic reticulum and medial and trans Golgi cisternae, disrupt amino groups in the polyamine backbone: 1) the substitution of one
actin cytoskeleton structure and bring on disappearance of actin or two hydrogen atoms of the polyamine backbone with electro-
stress fibers [90]. In addition, polyamine depletion has been shown negative fluorines; 2) the substitution of terminal NH CH -group
2 2
Charge Deficient Polyamine Analogues Current Pharmaceutical Design, 2014, Vol. 20, No. 2 265
with NH O- group; 3) the substitution of the three-/four-methylene Fluorinated Analogues of Putrescine, Spermidine and Spermine
2
chains of the polyamine backbone with ethylene group; 4) the sub- Hydrogen atoms in the backbone of Put were substituted to the
stitution of aliphatic chain with conformationally restrained cyclic most electronegative element fluorine (Pauling electronegativity 4)
amines. In order to study the effect of charge on the biochemical
to provide novel diagnostic tools to monitor polyamine accumula-
properties of Spm and Spd the modification of polyamine backbone tion using the radioactive isotope of fluorine (18F) [118, 119]. Fluo-
should not cause major changes in the structure of the parent poly- rination also changes the pK values of the neighboring amino
a
amine analogue. Therefore, the fourth method, destroying the struc-
group(s) (Table 1), which is sufficient to decrease the net charge of
tural similarity with parent polyamine is not discussed further. The the analogue at physiological pH. Furthermore, the introduction of
charge deficient polyamine analogues add a valuable tool to study one fluorine atom converts normally achiral polyamine molecule
the interaction between polyamines and the negatively charged
into a chiral compound. All of the mono-fluorinated analogues
components of the cell. They can also be used to determine how the shown below are mixtures of both enantiomers with unknown ra-
changes in the protonation of polyamines affect the enzymes of tios. It is important to keep in mind that the enantiomers of (cid:2)-
polyamine metabolism. In the following sections, the analogue
methylated analogues of Spd and Spm have different biochemical
effects are described mostly at the enzyme levels. The inhibition of properties [120] and the chiral center in mono-fluorinated poly-
ODC and/or AdoMetDC could be regarded as beneficial for pro- amine analogues may inherit the similar effects.
tecting mitochondria and cell organelles against activated poly-
Both of the fluorinated Put analogues, 2-fluoro- and 2,2-gem-
amine futile cycle, whereas induction of SSAT and/or SMO can be
difluoroputrescine (2-F-Put and 2,2-F -Put, respectively) are good
considered harmful due to the increased hydrogen peroxide produc- 2
substrates of SpdSy and are transformed into 6-fluoro-spermidines,
tion.
(6-F-Spd) and 6,6-gem-difluorospermidine 6,6-F -Spd, respectively
2
Table 1. The structures and the pK values of the fluorinated polyamine analogues.
a
pK
a
Abbreviation Compound structure Net Charge Ref.
1 2 3 4
NH
H N 2
2-F-Put 2 9.75 8.15 1.84 [121]
F
F
NH
2,2-F2-Put H2N 2 9.45 6.80 1.19 [121]
F
NH
H N N 2 10.49 9.0 6.91 2.22† [122]
6-F-Spd 2 H
F 10.40 9.55 7.18 2.37# [123]
F
6,6-F2-Spd H2N NH NH2 1100..324 99..7269 55..1760 22..0001†# [[112223]]
F
F
NH
7-F-Spd H N N 2 10.28 9.00 7.80 2.69 [123]
2
H
F
NH
7,7-F2-Spd H N N 2 10.25 8.24 6.64 2.02 [123]
2 H F
F
NH
2,2-F2-Spd H2N NH 2 10.30 7.30 5.50 1.45 [123]
F
H
N NH
6-F-Spm H2N N 2 10.7 9.7 8.04 6.62 2.95 [122]
H
F
F H
N NH
6,6-F2-Spm H2N NH 2 10.7 9.8 7.74 5.11 2.69 [122]
F
The pK values were determined with †NMR or #potentiometric titration. The net charge at pH 7.4 is calculated from the published pK values using the Henderson-Hasselbach
a a
equation.
266 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Weisell et al.
[124]. The elongation of the polyamine chain shows selectivity Polyamine Analogues with Altered Chain Length and Number
because neither 7-fluorospermidine (7-F-Spd) or 7,7-gem- of Amino Groups
difluorospermidine (7,7-F2-Spd) could be detected [121, 124]. 2-F- The reduction of the carbon chain length between two amino
Put shows high affinity to SpdSy (K 12 (cid:2)M) and the singly
m groups causes a significant increase in the electrostatic repulsion
charged Put analogue, 2,2-F2-Put, has as high affinity to the enzyme [134]. The repulsion decreases when the carbon chain is elongated
as the natural substrate Put. However, 2,2-F2-Put shows mixed type to propylenic and becomes irrelevant with a butylenic or longer
inhibition of SpdSy in concentrations above 25 (cid:2)M. While both 2-
carbon spacer. The studies of the pK values of various diamines
a
F-Put and 2,2-F2-Put are able to substitute Put in the biosynthetic with different lengths of the aliphatic carbon chain between the
pathway, 2-F-Put cannot support cell growth [125]. On the other amino groups demonstrate this phenomenon [135]. Some of the pK
a
hand, 2,2-F -Put was reported to be able to support the growth of
2 values of the polyamine analogues of this family are atypical for
chick embryos after administration of DFMO [124]. amino groups and only a few of the analogues are isosteric to natu-
Put is a known activator of eukaryotic AdoMetDC. The modu- ral polyamines (Table 2). The biochemical studies with these ana-
lation of the charge in both 2-F-Put and 2,2-F -Put causes decreased logues concentrate on the research of different features that are
2
affinity towards the enzyme [126]. Both 2-F-Put and 2,2-F -Put needed for the polyamines to support cell growth, be efficiently
2
accumulate to HTC cells and the accumulation is increased with the taken up to cells and to condense DNA.
treatment of cells with DFMO, which also accelerate the metabo- The pK values of the analogues of this family were determined
a
lism of the Put analogues to respective higher polyamine analogues
with potentiometric titration. However, when the protonation of the
[121]. Both fluorinated Put analogues also accumulate to normal
only unsymmetrical analogue, SpmTrien, is studied with NMR
and tumor tissues but the accumulation of 2-F-Put is more than [136] the protonation does not seem to occur in a classical way. The
twice as high as that of 2,2-F -Put. 2-F-Put is first converted to 6-F-
2 protons seem to be able to drift between the secondary amines of
Spd, which is further elongated to 6-F-Spm in HTC cells and in
the molecule at physiological pH. The later computational studies
mouse tissues, and the 2,2-F2-Put behaves almost identically. showed that there are many major populations of tautomers at the
When the mono and difluorospermidines were studied as sub- charge states three (+3) and four (+4), which are the relevant ones
strates of HTC cell SpmSy, 6-F-Spd had similar affinity as Spd but at physiological pH [137]. The major populations of tautomers of
6,6-F -Spd displayed about 5 times weaker affinity [123]. The af- SpmTrien apparently enable various interactions. The pK values of
2 a
finities of 7-F-Spd and 7,7,-F -Spd towards SpmSy are increased other polyamine analogues have not been studied with 1H-15N -
2
eight and over 100-fold, respectively. Interestingly, 7-F-Spd and coupled NMR method.
7,7-F -Spd show substrate inhibition of the enzymes with reversed
2 Most members of this family have been studied for their ability
Kivalues compared to the Km values; 7-F-Spd had much higher Ki to replace the functions of the natural polyamines in restoring
than 7,7-F2-Spd. 2-F-Put highly resembles Put and behaves nearly growth after DFMO-induced growth arrest of cells, for uptake
identically in vitro and in vivo. Hence,it turned possible to monitor
competition and for their influence on cell migration (Table 3).
the polyamine metabolic flux (acknowledging some considerations Even though all of the analogues can mimic at least some functions
[127]) using ornithine analogue 4-fluoro-ornithine, which is me- of natural polyamines [139] and also interact with DNA quite effi-
tabolized by ODC to 2-F-Put and consequently to higher polyamine
ciently in vitro [60, 133]they have not been systematically studied
analogues [81, 125]. These studies also confirmed that 6-F-Spd and in polyamine metabolism. The only exceptions are the newest ana-
6-F-Spm are substrates of SSAT. There are also some indirect find- logue belonging to this family, SpmTrien, which is isosteric to
ings indicating that 6,6-F -Spd may also be a substrate of SSAT,
2 Spm, and the molecule from which SpmTrien was derived, namely
but no studies with purified SSAT enzyme have been presented by Trien, which is isosteric to Spd. Trien is an efficient and selective
far [128]. When the gem-difluorospermidines were tested as sub- copper chelator and used as an orphan drug to treat Wilson’s dis-
strates of hepatic acetyltransferase activity from desalted super-
ease [140]. Moreover, it has advanced to clinical trials in alleviating
natant fraction of rat liver, both 7,7-F2-Spd and 2,2-F2-Spd were 1.8 cardiac hypertrophy associated to type 2 diabetes [141, 142]. It is
times better as substrates than Spd; also 6,6-F2-Spd showed good reasonably well tolerated and has potential in other therapeutic
substrate properties (1.4 times better) [129]. It is important to note
fields and thus could be regarded a promising representative of this
that the acetylating enzyme of the hepatic fraction is not known and class of polyamine analogues [143-145]. The ability of the ana-
the Vmax for each of the acetylation was under 100pmol/min/mg logues from this group to cause B-to-Z transition in DNA confor-
soluble liver protein. Furthermore, 7,7-F -Spd is converted to two
2 mation correlates with their ability to overcome the DFMO-induced
acetylated metabolites whereas only one metabolite has been ob- growth arrest [60]. Interestingly, Trien was found to restore cell
served for the other two analogues. growth after DFMO-induced short term growth arrest in DU145
6-F-Spd and 6,6-F -Spd inhibit DHS (IC 48 (cid:2)M and 14 (cid:2)M cells [136] while it was ineffective in IEC-6 cells [139]. Trien and
2 50
respectively) whereas 7,7-F -Spd display no inhibition suggesting SpmTrien both were found to compete with [14C]-Put for transport
2
that both of the primary amines should be adequately protonated for but the competition with 14C-labelled Spd and Spm was low sug-
the recognition by the enzyme [130]. In the absence of eIF5A and gesting a weak affinity towards polyamine transport [65]. Neverthe-
in the presence of the excess of Put, DHS can catalyze a reverse less, the intracellular concentration of SpmTrien was found to be as
reaction with the formation of homospermidine [131]. However, high as that of the natural polyamines, suggesting the presence of
neither 6-F-Spd, 6,6-F -Spd nor 7,7-F -Spd acted as a donor of another uptake mechanism for the molecule. Also a copper chelat-
2 2
4-aminobutanal group in either direct or reverse reactions [131]. ing isosteric charge deficient analogue of norspermine, 1,11-
Difluorospermidines have different substrate profiles with glu- diamino-3,6,9-triazaundecane (2-2-2-2), has been synthesized but it
tathionylspermidine synthetase of E.coli and 2,2-F -Spd is a supe- has not been studied as polyamine even though it has been studied
2
rior substrate of the enzyme when compared to the natural substrate in stem cell differentiation and it clearly affects CD34 and CD38
Spd or other fluorinated Spd analogues [132]. expression levels [146]. 2-2-2-2 has been used in phase I/II clinical
The affinity of 6,6-gem-difluorospermine (6,6-F -Spm) to con- trials for transplantation of umbilical cord blood-derived hema-
2
topoietic stem cells grown ex vivo [147].
dense calf thymus DNA is greatly decreased as compared to the
parent Spm [133]. One probable reason behind this phenomenon is Both Trien and SpmTrien are substrates of mouse recombinant
the decrease in the pK values of the amino groups of the analogue SSAT with V of around 1.2 ((cid:2)mol/min)/mg of enzyme for both
a max
that lead to a drop in the net charge of the molecule at physiological of the analogues and K values of 144 and 106 (cid:2)M, respectively.
m
pH. While Trien or its acetylated metabolites are not substrates of either
of the FAD-dependent polyamine oxidases, SMO and APAO, N12-
Charge Deficient Polyamine Analogues Current Pharmaceutical Design, 2014, Vol. 20, No. 2 267
Table 2. The structures and the pKa values of the polyamine analogues with altered chain lengths.
pK
a Net
Compound structure Abbr. Ref.
Charge
1 2 3 4 5
H
H N N NH2 2-3 10.21 9.17 6.10 2.03 [134]
2
H H
N N 2-3-2 10.08 9.26 6.88 5.45 2.23 [134]
H N NH
2 2
H
N NH
H N N 2 3-2-3 10.53 9.77 8.30 5.59 2.90 [134]
2
H
H
N NH
H N N 2 Trien 9.74 9.07 6.59 3.27 1.97 [134]
2
H
H
N NH
H N N N 2 SpmTrien 10.3 9.5 8.5 6.3 3.3 2.99 [136]
2
H H
H
N
H N N N NH 3-2-2-3 10.72 10.50 8.94 7.89 3.96 3.73 [134]
2 2
H H
H
H N N NH
2 N N 2 2-2-2-2 9.70 9.14 8.05 4.70 2.92 2.80 [134]
H H
The net charge at pH 7.4 is calculated from the published pK values using the Henderson-Hasselbach equation. Table adopted from [138].
a
Table 3. The ability of the charge deficient analogues with the altered chain length to replace the functions of natural polyamines.
Abbreviation Uptakea Cell growthb Cell migrationb Ref.
2-3 No Yes Yes [139]
2-3-2 No Yes Yes [139]
3-2-3 No Yes Yes [60, 139]
NoIEC-6 Cells
Trien No Yes [65, 136, 139]
YesDU145 cells
SpmTrien No Yes N.D. [65, 136]
3-2-2-3 N.D. Yes N.D. [60]
aCompetition with [14C]Spd, bgrowth recovery after short term PA depletion with DFMO; N.D. Not determined. Table adopted from [138].
Ac-SpmTrien is a good substrate of APAO, producing Trien as the may thus be regarded as multitarget drug although its main use has
oxidation product. This provides additional information about the earlier been as selective copper chelator. Moreover, acetylated
differences between substrate recognition of these polyamine oxi- Trien could be spontaneously rearranged to novel metabolites
dases. SMO seems to need adequate protonation of the central part which may contribute to biological activity of Trien [148]. Further
of the substrate polyamines while APAO is not so rigorous about studies with both Trien are SpmTrien are warranted to develop
the charge state of the substrate molecule(s), which is not surprising more efficient drugs against peripheral tissue damage associated to
because SMO has narrower substrate specificity than APAO. diabetes, inflammation and cancer. These studies should also in-
SpmTrien downregulates ODC and AdoMetDC activity and also volve preformed metal complexes of Trien, SpmTrien and their N-
moderately induces SSAT activity while Trien does not cause acetylated metabolites [149, 150]. Regulation and activities of hy-
marked changes in the enzyme activities. Interestingly, mono- and poxia inducible factor 1-alpha, mitochondrial/cytosolic SOD and
diacetylated metabolites of Trien inhibit human recombinant SMO polyamine metabolism are among interesting drug targets. Moreo-
[65]. Trien is primarily N-acetylated by thialysine acetyltransferase ver, the differences in Trien metabolism, if any, between human
but when SSAT is induced it may markedly contribute to the me- and rodent animal models (mouse, rat) should be clarified to prop-
tabolism of Trien [145, 148]. Trien and its mono- and diacetylated erly interpret the biological role of mono- and diacetylated Trien in
metabolites could interfere at many sites of polyamine metabolism the mechanism of Trien action [148, 150-152].
e.g. uptake, eIF5A synthesis and stability and SMO activity and
268 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Weisell et al.
Hydroxylamine-containing Analogues of Putrescine ODC, 2-F-APA turned out to be an irreversible inhibitor of the
Pyridoxal-5’-phosphate (PLP) dependent ODC is the rate- enzyme [175], but the mechanism for irreversible binding has not
controlling enzyme of polyamine biosynthesis and the most investi- been studied further. Me-APA CGP54169A and cyclohexyl-
containing hydroxylamine CGP52622A are as active as APA (Ta-
gated enzyme of polyamine metabolism. Many different inhibitors
of ODC are known, including enzyme-activated irreversible inhibi- ble 4). Specificity of APA towards ODC is confirmed by low activ-
tor DFMO, which was synthesized in 1978 [153] and at present is ity of APA (IC50 about 0.1 m(cid:4)) towards PLP-dependent ornithine
transaminase and AdoMetDC from E.coli [168]. Moreover, APA-
still the main and definitely most investigated inhibitor of ODC
both in vitro and in vivo [154, 155]. Moreover, it is used as a drug related hydroxylamines CGP54169A and CGP52622A (Table 4)
to cure African sleeping sickness and for facial hirsutism i.e. exces- specifically inhibit ODC of recombinant P.falciparum ODC-
AdoMetDCbifunctional protein [181].
sive men type hair growth in women [156-159]. DFMO has also
advanced to clinical trials with promising results as a cancer che- All efficient and specific hydroxylamine-containing inhibitors
mopreventive agent among populations with an elevated risk for of PLP-dependent ODC [168, 171, 175], L-glutamic acid decar-
specific epithelial cancers [160]. In general cancer (hyperprolifera- boxylase [182], (cid:3)-aminobutyric acid transaminase [183], aspartate
tive disease) treatment, ODC inhibition is recognized as a possibil- aminotransferase [184], 1-aminocyclopropane-1-carboxylate syn-
ity but by far the consequent activation of polyamine uptake facili- thase [185] (cid:3)-cystathionase [186] and pyruvate-dependent
tating polyamine supply from gut bacteria and diet has hampered its AdoMetDC [180, 187, 188], are structurally similar either to the
clinical applicability [161-163]. However, by limiting dietary poly- enzyme substrate or resemble the product of the enzymatic reaction.
amines and/or using polyamine transport inhibitors, the feasibility Such a similarity ensures high affinity of the inhibitor to the en-
of ODC inhibition–mediated chemotherapy could become evident zyme active center, while the properly positioned aminooxy group
[164]. The same therapeutic areas could be possible for any effi- forms the oxime with PLP or pyruvate residue. Structural similarity
cient polyamine biosynthesis inhibitor described below just keeping of these E-I complexes (oximes) with “external aldimine” (the first
in mind the general tolerability (therapeutic window) of a given intermediate) of all PLP-dependent transformations of amino acids
compound in humans. was confirmed by X-ray investigations of corresponding E-I com-
plexes of aspartate aminotransferase [189], GABA transaminase
The first step of ornithine decarboxylation is the formation of
Schiff base between PLP and the substrate ornithine. Therefore, [190], 1-aminocyclopropane-1-carboxylate synthase [185], and also
AdoMetDC [188]. In the case of ODC–APA complex, the proper
carbonyl reagents (derivatives of hydrazine, hydroxylamine, semi-
carbazide etc.) can be used to inhibit ODC. (cid:2)-Hydrazinoornithine is binding of the inhibitor to the enzyme active center promotes the
effective (IC 0.5 - 2.0 (cid:2)(cid:4)) [165, 166], while canaline ((cid:3)- cleavage of (cid:2)=N-double bond of “internal aldimine” formed by
50
aminooxy-(cid:2)-amino butyric acid) and aminooxyacetic acid are much carbonyl group of the coenzyme and (cid:5)-amino group of Lys-69 and
rotation of the plane of the pyridine ring. However, E-I complex
less active (IC 1.0 and 0.1 m(cid:4)) [167]. Simple carbonyl reagents
50
do not offer required specificity and efficacy for the inhibition of remains “frozen” with carbonyl group of PLP being at the distance
of ~ 3Å from aminooxy group [173]. Hence, the structure of ODC–
ODC. However, by using 1-aminooxy-3-aminopropane (APA), a
APA complex in crystal is not the same as in solution, where the
structural hydroxylamine-containing mimetic of Put, efficient inhi-
bition is readily achieved at nM concentrations as shown in Table 4. oxime of the enzyme is formed. There is at least one example re-
lated to PLP-enzymes, confirming that the conformation in crystal
Ornithine does not protect the enzyme from APA, and the activity
and in solution may be different. In solution 3-aminomethyl-
of the inhibited ODC could not be restored upon addition of the
excess of the substrate. APA inhibits ODC pseudo-irreversibly aminopropane phosphinic acid and S-ethyl-Cys are substrates of
methionine-(cid:8)-lyase, while in crystal both inhibitors bind to the en-
since the activity of the enzyme can be restored by gel-filtration of
zyme active center but PLP still forms “internal aldimine” with (cid:5)-
ODC-APA complex (elution buffer contained PLP) [168]. Like-
wise, APA dramatically decreases ODC activity in undialysed amino group of Lys [191].
L1210 cell extracts, but the activity of ODC increases several fold (cid:9)(cid:10)(cid:9) is the simplest charge deficient analog of Put, existing at
when it is determined in dialysed extracts [169]. These observations physiological pH in single cationic form. Since the terminal amino
are in good agreement with the reversible character of PLP binding group of Put must be deprotonated in SpdSy active center before
to the apoenzyme (the affinity of PLP to rat liver ODC is 0.25 (cid:2)(cid:4)) reacting with dcAdoMet, APA may be considered as an analogue of
[170]. the intermediate (transition) monocationic form of Put. Respec-
tively, APA is a potent competitive inhibitor (K 2.3 (cid:6)M) of homo-
Carbonyl group of PLP forms “internal aldimine” with (cid:5)-amino i
group of Lys-69 in ODC active center that dramatically increases geneous bovine brain SpdSy [171] and in the case of SpdSy from
its reactivity towards hydroxylamines when compared with the P.falciparum APA has IC50 of 84 (cid:6)M [192].
reaction rate of non-conjugated carbonyl group of PLP with hydra- The activity of APA in different cell cultures has been studied
zines and O-substituted hydroxylamines [177-179]. The high reac- and in all cases the inhibitor penetrated inside cells by passive dif-
tivity of internal aldimine is the reason why inhibition of ODC by fusion that has been confirmed using 3H-labelled APA [193]. APA
APA and related compounds is not evolved in time. In contrast, the is unstable in cell culture media and is degraded by aminoguanidine
inhibition of pyruvate-dependent AdoMetDC (the enzyme has a sensitive oxidase(s) [194, 195]. Degradation of APA begins either
free carbonyl group in the active center) with hydroxylamine- from aminooxy-, or amino terminus since introduction of a single
containing analogue of dcAdoMet is time-dependent [180]. Pre- methyl group in (cid:11)-position to amino group, or aminooxy group of
formed oxime of 2-F-APA with PLP has relatively low inhibitory APA does not prevent the degradation [194]. However, the oximes
activity towards ODC (IC 5.2 (cid:6)M, [175]) when compared with of APA with pyridoxal (PL) and pyridoxal-5’-phosphate (PLP),
50
inhibition of ODC by 2-F-APA, confirming that 2-F-APA reacts APA-PL and APA-PLP, respectively, are much more stable than
with PLP in the enzyme active center. APA in the cells, but APA-PLP is slowly dephosphorylated [194].
The ODC inhibitory potency of an aminooxy analogue of ca- Growth inhibition activity of APA, as well as 2-OH-APA, and
daverine 1-aminoxy-4-aminobutane (ABA) is one tenth of that of 2-F-APA have been investigated in different tumor cell lines and
APA. Non-protonated bis-aminooxy derivatives are not active even these compounds are active at 0.01-0.1 mM medium concentrations
at 1 (cid:6)M concentration at the physiological pH ((cid:7)able 4). Introduc- [169, 174, 175, 196-199]. In all cases the studied drugs deplete
tion of hydroxyl group into APA structure (2-OH-APA) has no Put/Spd pools and cause accumulation of dcAdoMet that are typical
effect on the inhibitory potency of the analogue, and the activities for the inhibitors of ODC. Moreover, cell growth is completely
of (R)- and (S)-isomers of 2-OH-APA are similar (Table 4). Con- restored upon the addition of exogenous polyamines to the media.
trary to APA, which is a pseudo irreversible binding inhibitor of Interestingly, APA-PL and APA-PLP completely prevent the DNA
Charge Deficient Polyamine Analogues Current Pharmaceutical Design, 2014, Vol. 20, No. 2 269
(cid:2)able 4. Inhibition of ODC by hydroxylamine-containing analogues of putrescine.
Compound structure Abbreviation. Source of the enzyme Inhibition Ref.
Mouse liver IC 2(cid:2)10-8(cid:3) [168]
50
Mouse kidney IC 3.2(cid:2)10-8(cid:3) [171]
50
H N O NH2 L. donovani Ki 1(cid:2)10-9(cid:3) [172]
2
APA Human recombinant ODC K 1.4(cid:2)10-9(cid:3) [173]
i
P. falciparum K 2.7(cid:2)10-9(cid:3) [174]
i
Rat liver IC 1.5(cid:2)10-8(cid:3) [175]
50
O
H N NH ABA Mouse liver IC 2(cid:2)10-7(cid:3) [168]
2 2 50
O O
H N NH Mouse liver IC >(cid:2)10-6(cid:3) [168]
2 2 50
OH
O O Rat liver IC 6.7(cid:2)10-4(cid:3) [175]
50
H N NH
2 2
OH
H N O NH2 2-OH-APA Rat liver IC50 3.9(cid:2)10-8(cid:3) [175]
2
OH
O NH (R)-2-OH-APA Rat liver IC 4.9(cid:2)10-8(cid:3) [175]
H N 2 50
2
OH
O NH (S)-2-OH-APA Rat liver IC 3.3(cid:2)10-8(cid:3) [175]
H N 2 50
2
F
O NH 2-F-APA Rat liver IC 1.4(cid:2)10-8(cid:3) [175]
H N 2 50
2
Cl
H N O NH2 2-Cl-APA Rat liver IC50 9.3(cid:2)10-8(cid:3) [175]
2
H2N O NH2 CGP54169A P.falciparum IC50 2.5(cid:2)10-8(cid:3) [176]
P.falciparum K 7.9(cid:2)10-9(cid:3) [174]
i
NH
2 P.falciparum IC50 6.3(cid:2)10-8(cid:3) [176]
CGP52622A
H2N O P.falciparum Ki 2.4(cid:2)10-8(cid:3) [174]
synthesis of BHK cells at 0.01 mM concentrations, i.e. are as effec- with APA, AMA, and 5-fluorouracil reduced colon cancer cell sur-
tive as APA [194]. Likewise, 2-OH-APA-PLP and 2-F-APA-PLP vival more potently than treatment with 5-fluorouracil alone [201].
inhibit T24 bladder carcinoma cell growth as efficiently as their non In the case of BHK cells being treated with APA, but not with
PLP conjugated parent drugs [175, 200]. Treatment of Ehrlich as- DFMO, the disturbances of Golgi apparatus are found [90]. 2-F-
cites-tumour cells with APA greatly stimulate the synthesis of APA is slightly more effective than APA in inhibiting human blad-
AdoMetDC, as well as that of ODC – in the latter case enzyme der carcinoma T24 cells growth [175]. 2-F-APA (at a dose of 1/20
accumulate in the inhibited form [198]. Exposure of L1210 cells to of the maximal tolerated dose) is as effective as DFMO (at a dose
APA greatly decreases ODC activity in undialysed cell extracts, but of 1/5 of the maximal tolerated dose) in its antitumour activity in
does not decrease AdoMetDC activity – thus confirming the speci- female CD-1 nude mice bearing human bladder carcinoma T24
ficity of APA action. Activities of both enzymes are increased sev- [200]. These results suggest that compounds like 2-F-APA might
eral fold by APA treatment when activity is measured in dialyzed have therapeutic value as cytostatic drugs. To our knowledge, the
extracts [197]. Treatment of human colon cancer cells (Caco-2 and only published in vivo data about the tolerability and the anticancer
HT-29) with APA and AMA (S-(5’-deoxy-5’-adenosyl)- efficiency of any APA related structure in mouse is published in
methylthioethylhydroxylamine – irreversible inhibitor of [200] and for rat some tolerance data are found from patent [202]. It
AdoMetDC) in combination (0.1 mM of each) reduced ODC and seems likely that no conjugation of primary amino terminus of APA
AdoMetDC activities to undetectable levels within 24 hr and intra- structure is tolerated but aminooxy derivatives with PL or PLP
cellular polyamines to 8–23% of control. Simultaneous treatment retain growth inhibitory activity and they are metabolically stable
270 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Weisell et al.
[175, 194, 200]. Thus, the chemical reactivity of aminooxy group Hydroxylamine-containing Analogues of Spermine and Sper-
and rapid catabolism of APA, hampering in vivo usability, might be midine
controlled by using PL/PLP or other oximes of APA. Furthermore, The introduction of the aminooxy or oxa-group to the poly-
by altering the APA oxime structure, one may expect to regulate
amine backbone causes minimal steric disturbances as compared
chemico-biological properties of the parent drug e.g. APL-PL with parent NH CH -group. Unlike the fluorinated polyamines,
2 2
oxime inhibits PL-kinase and may compete with PL for cellular these analogues have no tendency to form strong hydrogen bonds
uptake (unpublished T. Keinänen) [203, 204]. In further develop-
that is typical for fluorine atom. Depending on the analogue struc-
ment of this series of compounds, the characterization of enzyme(s) ture, the pK value of terminal aminooxy groups are between 3.5
a
responsible for their rapid catabolism as well as studying their and 4.5 (Table 5) that is roughly five orders of magnitude lower
chemical reactivity as carbonyl reagents toward cellular carbonyl
than that of an amino group. This causes a drop of one unit in the
compounds is of importance. total net charge of the analogues at physiological pH when com-
Trypanosomatids causing malaria, leishmaniasis, African sleep- pared to their parent polyamines and opens a possibility to investi-
ing sickness, etc. requires Spd for growth and survival. Therefore, gate the impact of charge on the biochemical activity of the ana-
enzymes involved in Spd biosynthesis and utilization including logue(s).
ODC are considered as promising targets for drug development
Hydroxylamine analogues of Spd AOE-PU and (cid:5)(cid:6)-(cid:5)(cid:6)(cid:5) (Ta-
[205]. APA and its derivatives 3-Me-APA (CGP54169A) and 1- ble 5) inhibit ODC and AdoMetDC only at millimolar concentra-
aminooxy-4-aminohexane (CGP52622A) exhibit plasmodicidal
tions [129]. The interaction of AOE-PU and AP-APA with SpmSy
activity in vitro (IC 1-3 at (cid:4)M), and are much more active in
50 is of special interest, since AP-APA may be considered as a mi-
comparison with DFMO under the same conditions [174, 176]. The metic of intermediate doubly (N1- and N4-amino groups) protonated
decrease of Put and Spd pools in parasite and the reversal of the
form of Spd, which is formed after the binding of the substrate in
inhibition with exogenous polyamines demonstrate that compounds
the enzyme active center. In contrast, AOE-PU is a mimetic of dou-
of APAs’ family exert their antimalarial activity due to the inhibi- bly (N4- and N8-amino groups) protonated Spd, and is not a mimetic
tion of bifunctional ODC/AdoMetDC of P.falciparum [174].
of transition form of Spd in SpmSy reaction. Therefore, AP-APA is
APA is a potent inhibitor of L.donovani promastigote growth a 100 times more effective competitive inhibitor of SpmSy than
with an IC of 42 (cid:4)M, reducing Put, Spd and trypanothione levels, AOE-PU (K 1.5 (cid:4)M and 186 (cid:4)M, respectively [129]). Moreover,
50 i
but after addition of Put/Spd the antiproliferative effect of APA is AP-APA, as an unusual mimetic of the transition form of Spd, has
abolished [172]. Surprisingly, APA is more effective against amas- better affinity to SpmSy than Spd, having K of 27 (cid:4)M [129].
m
tigotes in the macrophage model, having IC value of 5 (cid:4)M, i.e. is
50 The differences in the reactivity of the terminal aminooxy- and
10-fold more effective than DFMO [172]. However, forms of para- amino groups of hydroxylamine-containing analogues of Spd and
sites that are overexpressing ODC exhibit resistance to both APA Spm [214] towards ketones was the reason to use these compounds
and DFMO, indicating that the effect of APA is primarily a result of
as building blocks to construct bisubstrate inhibitors of SSAT con-
inhibition of ODC [172]. APA passively penetrate inside parasites, taining acetone linker. The most active is the oxime with AP-APA,
but in the case of L.donovani it was attempted to convert APA into having IC of 1 (cid:12)M [216], while earlier conjugates of CoA with
50
actively transported inhibitor/proinhibitor 1-guanidinooxy-3-
Spm, nor-Spd or their symmetric homologues containing amide
aminopropane (GAPA), which is an agmatine isoster with terminal linker inhibit purified enzyme with IC 0.5–5 (cid:12)M [217]. Recently,
50
guanidinooxy-group having pKa 6.71 [206]. GAPA itself inhibit the conjugates with amide linker were used for X-ray investigation
recombinant L.donovani ODC with K 60 (cid:4)M, while IC towards
i 50 of SSAT [42].
amastigotes is already 9.0 (cid:4)M [206]. The decrease of Put and Spd
Among other data on the interaction of aminooxy analogues of
levels in GAPA-treated L.donovani and the ability of exogenous
polyamines with the enzymes of polyamine metabolism it is worth
Put and Spd to reverse the growth of the parasite indicates that the
to note the good substrate properties of N1-(cid:5)(cid:13)-AOE-PU in (cid:5)(cid:6)(cid:5)(cid:7)-
cellular target of GAPA is closely related to metabolism of poly-
reaction (k 0.54 sec-1) [218] and substrate properties of A(cid:7)-Spm
amines [206]. However, GAPA, on the contrary with APA and cat
((cid:14)(cid:15)ble 5) towards SSAT (colon cancer (cid:16)(cid:15)(cid:13)(cid:17)-2 cells) – K is 2.2
DFMO proved to be effective even against sodium antimony gluco- m
mM [196]. Both AOE-PU and AP-APA are substrates of crude
nate-resistant forms of the parasite, overexpressing ODC, but the
acetylase activity from BHK cells extracts [129], but in BHK cells
molecular reasons of this effect have not been yet investigated
only AOE-PU is efficiently acetylated [219]. AP-APA and AOE-
[207].
PU display moderate inhibition on cell proliferation in dose de-
DFMO is known to exhibit a fungicidal activity that was first
pendent manner [219], but they are much more active in the L1210
demonstrated in mid-80s for Uromyces phaseoli [208]. Both APA
cells as compared with BHK cells [197]. Interestingly, AOSpm
and DFMO were tested against phytopathogenic fungus Pyricularia does not have any impact on polyamine levels or proliferation in
oryzae Cav, causing rice blast disease in almost every rice-growing
Caco-2 cells [196] or BHK cells, while it was shown to moderately
country, and found to inhibit its growth at 0.1-1.0 mM, while the
inhibit growth of rapidly proliferating L1210 cells [197].
inhibition vanish with the exogenous Put supplementation [209].
A small family of oxa-analogues of Spm and Spd has also been
Surprisingly, APA, on the contrary with DFMO, bleaches colonies
synthesized [212, 214, 215]. 1,8-Diamino-4-aza-5-oxaoctane (5-
by blocking the biosynthesis of pentaketide aromatic precursors of
oxa-Spd, (cid:14)able 5) may be considered as an isoster of double proto-
melanine. The bleaching effect can be reversed by Put, while the
nated form of Spd, but it is a poor inhibitor (IC 0.38 mM) of DHS
combined application of APA and DFMO results in the arrest of the 50
[212]. SMO was discovered only about 10 years ago [47, 220] and
fungus pigmentation in additive manner [209]. Scitalone, one of the
is now considered to be the key enzyme of Spm catabolism. At the
precursors of melanine, also reduces fungus pigmentation indicat-
moment, only one powerful inhibitor of the enzyme is known - cys-
ing that the arrest of melanine biosynthesis might take place at one
3,8,13,18,23,28,33,38,43,48-decaazapentacontene-25, having IC
of the earlier stages, presumably at the level of multienzyme com- 50
of 10-7 M [221]. It was assumed that 5-oxa-Spm, being an isoster of
plex performing polycondensation of Ac-CoA and malonyl-CoA
the intermediate triply protonated form of Spm ((cid:14)able 5), might
[209]. These data are the first indication on the involvement of Put
have a good inhibitory potency towards SMO as such, or due to its
in the regulation of the pentaketide biosynthetic pathways in fun-
oxidation into corresponding oxime directly in the enzyme active
gus.
center if redox potential of the FAD-FADH system would allow the
Charge Deficient Polyamine Analogues Current Pharmaceutical Design, 2014, Vol. 20, No. 2 271
(cid:3)(cid:4)ble 5. The structures of the isosteric aminooxy and –oxa analogues of natural polyamines and the pK values of the aminooxy-
a
and oxa-group [138].
Compound structure Abbreviation pK values Ref.a
a
O NH
H N 2 APA 4.2 [210]
2
NH
O NH
H N N 2 GAPA 6.71 [206]
2
H
O
H2N NH2 ABA 4.5 [211]
O NH
H N N 2
2 AOE-PU 3.25 [210]
H
NH
H N N O 2
2 AP-APA 4.2 [210]
H
O NH
H N N 2
2 5-oxa-Spd --- [212, 213]
H
H
O N NH
H N N 2 AO-Spm 3.2 [214]
2
H
H
O N NH
H N N O 2 BAO-Spm 3.2 [214]
2
H
H
O N NH
H N N 2 5-oxa-Spm 2.7 [215]
2
H
aThe reference for the first paper that describes the synthesis of the analogue
transformation [215]. However, these assumptions have not yet larly to Spm [196]. Under these conditions Spd is less effective
been proven, and oxa-analogues of Spm and Spd have not found a when compared with Spm [114] and ROS-protecting activities of
proper application in polyamine biochemistry despite of their obvi- AOE-PU and AP-APA are about the same as that of Spd. Hence,
ous potential. the charge deficiency of AOE-PU, AP-APA and AO-Spm does not
practically alter their ROS-protecting properties when compared
Interactions of Putrescine, Spermidine and Spermine Aminooxy with Spd and Spm.
Analogues with Nucleic Acids Aminooxy analogues of Spm, Spd and Put are not only charge-
Polyamines bind to the major and/or minor grooves of the DNA deficient isosters of polyamines, but also typical carbonyl reagents
double helix, interacting with nucleic acid bases and neutralizing like other O-substituted hydroxylamines. This property has been
the charges of the phosphate groups [66, 222]. Polyamines can used to introduce reactive amino groups via 3’-oxidized end of
affect the local conformation of DNA, and due to their ability to RNA [229]. This reaction requires only 10-fold molar excess of
stabilize highly charged structures they promote the transition from hydroxylamine to proceed with practically quantitative yield at
right-handed B-DNA to either left-handed Z-DNA or A-DNA [223- room temperature and pH 5 [230]. Later, the same reaction was
225] and also promote DNA bending [61, 226]. used to determine the amount of apurinic sites in DNA using
chemiluminescent acridinium hydroxylamine [231], being N-
Polyamine-induced condensation of DNA is one of the simplest
substituted derivative of aminooxy analogue of cadaverine – ABA
models to evaluate the effectiveness of the interaction of Spm and
(Table 5).
Spd with nucleic acids. The ability of polyamines to condense DNA
decreases in the order: pentamine> tetramine>triamine [227]. In- Hydroxylamine and (cid:2)-methylhydroxylamine were widely used
vestigation of the interaction of AOE-PU, AP-APA and AO-Spm in the 70-80s’ to modify cytidine bases of nucleic acids [232]. This
(Table 5) with DNA demonstrated that only AP-APA and AO-Spm, reaction proceeds with a good velocity only when high concentra-
where the two positive charges were separated with trimethylene tions (0.1-1.0 (cid:8)) of hydroxylamines are used at 37°(cid:9) and at (cid:10)(cid:11) 5,
moiety, but not AOE-PU, are able to induce DNA condensation. i.e. only highly water soluble hydroxylamines are suitable for this
AO-Spm induces DNA condensation at physiological pH, whereas purpose. The aminooxy analogue of cadaverine – ABA (Table 5) is
AP-APA is effective only at low pH values [228]. among small family of highly soluble functionally substituted esters
of hydroxylamine and it was applied to introduce reactive amino
Spm is known to protect DNA from the ROS-induced strand
groups to tRNA molecule via cytidine residues [229, 230]. This
breakage by either altering DNA structure and making it less sus-
reaction was also successfully used to modify deoxyoligonucleo-
ceptible to attack, or by directly scavenging free radicals and inacti-
tides to prepare hybridization probes after introduction of biotin
vating ROS [114]. AO-Spm has been shown to protect DNA from
oxidative damage (ROS were generated in Cu2+/H O system) simi- [233] or fluorescent label [234].
2 2