Abstract

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Dec 14, 2013 (3 years and 7 months ago)

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1

Abstract


The pivotal role of
nicotinamide adenine dinucleotide (
NAD
)

in cellular metabolic
processes justifies the interest in
nicotinamide mononucleotide
adenylyltransferase
(NMNAT) as an attractive target for the rational design of new drugs.

NMNAT is a
n
indispensable enzyme in both
de novo

and salvage pathways of NAD or deamido
-
NAD
(NaAD) biosynthesis. Significant differences between bacterial and human enzymes in
both substrate specificity and active site conformations have been described that might
le
ad to the rational design of highly selective chemotherapic
agents.
Therefore, the
specific aim of my research was the identification of molecular ligands which could act
as selective inhibitors of NMNAT from
specific

species, useful for the biological
cha
racterization of the enzymes and the discovery of new drugs.

With this purpose in view, two dinucleoside polyphosphate NAD analogs, having a
polyphosphate linker between the nicotinamide riboside function and adenosine
(Np
3
AD and Np
4
AD), that should mimic

the structure of the hypothetical transition state
of the reaction catalyzed by NMNAT, were prepared. Interestingly,

both dinucleosides
polyphosphates

and, in particular

Np
4
AD, exerted a different inhibitory effect toward
the bacterial enzyme with respect

to eukaryotic NMNAT. So, Np
4
AD might represent
the first lead compound for the development of novel potent and selective bacterial
NMNAT inhibitors.

Another part of the research was addressed to the synthesis of
β
-
n
icotinamide and
nicotinic acid
ribosides (
β
-
NAR
and

β
-
NaR, respectively) in ord
er to investigate the
subdomain
of NMNAT that binds the substrates nicotinic mononucleotides
(NMN/NaMN).
Nicotinamide riboside is an intermediate in one biosynthetic pathway
by which nicotinamide is converted into NAD.
It is known that kinetic and structural
studies of both bacterial and human NMNAT require the
β
-
anomer of nicotinic
ribosides as substrates and of monoribotides as products of the NAD degradation
pathways.

-
NAR

and its nicotinic acid analog (

-
NaR) were obtained in high yields by
a stereoselective synthesis
via

glycosylation of the presilylated bases under
Vorbruggen’s protocol and controlled conditions.

In order to obtain novel NAD analogs
as substrate/inhibito
rs of key enzymes involved in the nucleotide biosynthesis, a
β
-
NAR
analog, methylated in 3
-
position of the ribofuranose moiety, was also synthesised.


2

Novel potential inhibitors of NMNAT from different sources were also developed as
product analogs of the enzymatic reaction. Recently, three distinct human NMNAT
isofo
rms, named
h
NMNAT
-
1,
h
NMNAT
-
2, and
h
NMNAT
-
3, have been characterized.
The tissue distribution pattern of the human isoenzymes is different, suggesting that
their expression is differentially controlled at transcription level. Thus, each isoenzyme
may have
a distinct cellular and physiological role; however, seve
ral questions remain
to clarify

and characterize the
specific

roles of these isoenzymes in NAD metabolism
and regulation.

In this respect, the proposed study combines efforts directed toward the des
ign and
synthesis of mechanism
-
based enzyme
-
inhibitors, with
biochemical studies

on targeted
enzymes to better understand the biological function of the human NMNAT isoforms
and define their suitability as specific targets for drug development.

With this
in view, a NAD analog and its deamido derivative were synthesized in
which the adenylyl part of the dinucleotides was modified at the C2’ position of the
furanose ring by introduction of a methyl group (N2’
-
MeAD and Na2’
-
MeAD,
respectively). The effect of
the dinucleotides on the enzymatic activity of NMNAT
resulted in a selective inhibition of

the
human enzyme

with respec
t to the archeal
enzyme, suggesting that the
modification in the ribose moiety of NAD, which induces a
stabilization of the North(
3
T
2
)
-
a
nti

conformation, as detected for both N2’
-
MeAD and
Na2’
-
MeAD, proved to be favourable to the binding at human enzymes.

N2’
-
MeAD showed a significant competitive inhibitory activity toward the
h
NMNAT
-
2, but no activity toward the
h
NMNAT
-
1 isoform.

Surp
r
is
ingly, the deamido dinucleotide Na2’
-
MeAD resulted a non
-
competitive
inhibitor of
h
NMNAT
-
2 and
a selective
comp
etitive inhibitor of
h
NMNAT
-
3 isoform.

A molecular modeling study was also carried out starting from the recently solved
structures of NAD/NaAD
-
bound
h
NMNAT
-
1 and
h
NMNAT
-
3 complexes. Compared
with these complexes, the N2’
-
MeAD/Na2’
-
MeAD
-
bound showed conformational
changes in the binding sites of both

h
NMNAT
-
1
and hNMNAT
-
3 induced by the
modified adenylyl part of the dinucleotides.

The inhibitory
effe
ct of the modified NAD analogs

toward the human enzymes might
be helpful for the rational design of molecules potentially useful as new chemotherapic
agents.


3






Chapter 1.






Nicotinamide mononucleotide adenylyltransferase

(EC 2.7.7.1, NMNAT): a

key enzyme for NAD biosynthesis

















4

1.
1.

Introduction

The development of new strategies for the treatment of infectious diseases involves
the identification of targets mostly represented by enzymes catalyzing metabolic key
-
reactions. The nicoti
namide adenine dinucleotide (NAD) is a cofactor in numerous
enzyme
-
catalyzed redox reactions and plays a fundamental role in cellular metabolic
processes. In fact, NAD homeostasis is critical for cell functioning and it has been
shown that, in many living
organisms including pathogenic bacteria, disturbance of the
nucleotide metabolism causes severe consequences for cell survival. The central role of
NAD or its deamidated form NaAD in cellular metabolic processes justifies a great
focus of investigation tow
ard the biosynthesis of these dinucleotides.

In the five past years, a considerable attention has been devoted to nicotinamide
mononucleotide adenylyltransferase (NMNAT), a central enzyme in the NAD
biosynthetic pathways.

More recently, three different h
uman NMNAT were identified resulting with a
fold
similar to that from bacteria and archaea but limited sequence identity (<20%). They
also differ in quaternary structure, biochemical and enzymatic properties.
At present,
the availability of the structures
of NMNAT from different sources opens up a broad
spectrum for new pharmacological approaches. However, there are many open
questions on the role of the different NMNAT
s
.

On this basis, there is a need for the development of specific inhibitors for the
dif
ferent enzymes.

The focus of the present research was directed on the identification of NMNAT
inhibitors or substrates as lead molecules that could be

useful to better understand the
biological function and physiological role of the different enzymes and t
o define their
suitability as specific targets for drug development.








5

1.2. Nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1.,
NMNAT)


The
evidence

of the numerous functions
carried out

from

nicotinamide adenine
dinucleotide
(
NAD
)

in the ce
llular met
abolism, mostly as co
-
factor and as substrate

of
various enzymes
,

has brought

the

conclusion that
NAD

biosynthesis
has to be

submitted to
a precise regulation, essential to assure the necessary quantity of the dinucleotide to t
he
cell
.

Pyridine d
inucleotides (
NAD and NADP
)

are ubiquitous

co
-
factors involved in
hundreds of redox reactions

essential for the energy transduction and metabolism in

all
living cells.

Together these nucleotides have a direct impact on virtually

every cellular
metabolic pa
thway
s
.
In addition
, NAD serves as

a substrate for the covalent
modification

of a variety

of nuclear proteins by

ADP
-
ribosyltransferases
,
1
-
4
and for the
repair of DNA

by DNA
-
ligase in bacteria
.
5,6

The formation of protein
-
coupled
poly(ADP
-
ribose) relaxes t
he chromatin structure and facilitates DNA regulatory and
repair events under conditions of DNA damage.

Recently, many new

exciting functions have been discovered for
NAD
. These
include its role as co
-
substrate in Sir2
-
mediated

histone deacetylation involv
ed in gene
silencing regulation

and in increasing the lifespan of species ranging from

yeast, to
worm, to certain mammals
.

Indeed, it has been suggested that increasing NAD
biosynthesis enhances Sir2 activity in neurons and may increase the resistance to
n
eurodegenerative

diseases
.

Thus, i
t would be of great interest to investigate the

molecular mechanism of NAD biosynthesis regulation and its

impact on aging and
longevity in mammals.
7
-
9


Moreover, several
NAD
derivatives
such as
cyclic ADP
-
ribose and nicot
inic acid
adenine dinucleotide phosphate (
N
a
ADP) w
ere found to be potent intracellular

calcium
-
mobilizing agents and are involved in a variety

of Ca
2+
-
signaling pathways,
including
the regulation of intracellular insulin levels and its secretion, T
-
cell ac
tivation
or
catecholamine secretion
.
10,11

T
hese processes may d
eplete cellular NAD pool which
could
determine
a cellular energy crisis in cell
. Thus,
i
t is crucial that NAD biosynthesis
is actively regulated and proper NAD levels are

maintained.

The disco
very of NAD
-
dependent regulatory mechanisms has also led to the
realization that NAD(P) biosynthesis has to be an ongoing process to fuel these

6

pathways. That is, while the metabolic redox reactions are not accompanied by any net
consumption of pyridine nu
cleotides, their participation in signaling pathways requires a
steady resynthesis to maintain a

stable cellular concentration.
These recent
developments brought a significant amount of additional interest to the investigation of
cellular NAD biosynthesis
and regulation
.
Besides
,

NAD(P) is used reversibly in
catabolic reactions such as glycolysis, fatty acid oxidation, nitrogen metabolism, one
-
carbon metabolism, and in anabolic reactions such as gluconeogenesis, and fatty acid,
amino acid, cholesterol and n
ucleotide synthesis.

The synthesis of NAD was first described by Arthur Kornberg
12

who

was on a quest
to investigate the synthesis of nucleic acids starting

with

a simple dinucleotide. He
discovered the crucial step of NAD
-
synthesis

in 1954 by detecting a
n enzymatic activity
in yeast extracts that catalysed

the condensation of ATP with nicotinamide
mononucleotide to form

NAD.
NAD is synthesized via a multi
-
step
de novo
pathway or
via a pyridine salvage pathway
.
13

All the known biochemical pathways converge

to the
reaction

catalyzed by NMN adenylyltrans
ferase (EC 2.7.7.1, NMNAT).
13


NMNAT is an ubiquitous enzyme,
member of nucleotidyltransferase
α
/
β

phosphodiesterases superfamily.
Members of this family share the same basic catalytic
mechanism, involving dir
ect nucleophilic attack upon an
α
-
phosphate

followed by the
release of pyrophosphate, whereas the enzyme

provides stabilization of the transition
state prior to the formation

of a new phosphodiester bond.

The enzyme

was found

to be present in different org
anisms ranging from archaea to

eubacteria to yeasts to man. R
ecently
it was demonstrated

that NMNAT exists

in
multiple forms
, characterized by different subcellular localiz
ation and enzymatic
properties.
14
-
1
8

While the enzymes from
archaea
, eubacteria and
human (
h
NMNAT
-
1)
were known, two other human NMNATs (named
h
NMNAT
-
2 and
h
NMNAT
-
3) were
cloned and partially characterized
when my thesis was in progress
.
However, the roles
of these new isoenzymes in NAD biosynthesis and NAD
-
dependent signal trasduction
pa
thways remained unclear.

On the basis of
t
he protein structure of
h
NMNAT
-
1 and
h
NMNAT
-
3

available in the
Protein Data Bank
, my research work

was

directed toward

the identification of ligand
inhibitors of NMNAT isoforms

with the purpose to define

their in
dividual functions. At

7

this moment, NMNAT has been suggested to represent an interesting target for the
rationale design of new chemotherapic agents.



1.
2.1.

Enzymatic activity of NMN adenylyltransferase


Nicotinamide 5’
-
monophosphate adenylyltransferase
is an indespensable central
enzyme in the NAD biosynthetic pathways catalyzing the transfer of the adenylyl group
from ATP to NMN or nicotinic acid mononucleotide (NaMN) to form NAD or NaAD
(Figure 1).



Figure 1.

NMNAT catalyzed reaction
.



8

The reaction p
roceeds via a nucleophilic attack by the 5'
-
phosphate of the
mononucleotide

(NMN or NaMN)

on the
α
-
phosphate of ATP, thus releasing the
pyrophosphate and
NAD or NaAD
.


Although NAD biosynthesis occurs with
many

differences between prokaryotes and
eukaryotes, the NMNAT catalyzed reaction is common to both the
de novo
and salvage
routes (Figure

2).
13


Figure 2.

NMNAT catalyzed reaction in the NAD biosynthetic pathway
s.



The nicotinic acid mononucleotide (NaMN) adenylyltransferase (
NaMN
AT, EC
2.7.7.18) catalyzes

the conversion of ATP and NaMN to nicotinic acid adenine

dinucleotide (NaAD)
that is directl
y processed to NAD

by NAD synthetase. The
nadD
gene, encoding NaMN
AT, was

the first enzyme demonstrated to be essential for NAD
biosynthesis

and bacterial cell survival by both the
de novo
and

salvage pathways. A

9

number of enzymes demonstrating
in

vitro
ad
enylyltransferase activity for NaMN and
NMN have

been identified in eu
karya, archaea, and bacteria
.
13

Adenylyltransferases encoded

by the
nadD
gene prefer the nicotinic acid containing
NaMN

over NMN as a substrate by a factor that ranges from 6:1 to

2000:
1
. Eubacteria
also contain enzymes that demonstrate

higher specificity for the nicotinamide
-
containing

NMN. This group includes the products of the
nadR
gene,

which in addition
to its regulatory role in NAD biosynthesis

also displays NMNAT activity
.
19

The
eukaryotic and archeal

NMNAT
, such a
s those from human
,
20

Methanococcus

jannaschii
,
21

and
Methanobacterium thermoautotrophicum
,
22

either demonstrate higher
specificity for

NMN as a substrate as compared with NaMN, or show little

pre
ference
for either subst
rate
.
13


Following are reported the structures of NMNATs from various species. The ribbon

r
e
presentation
s

of the NMNATs
were drawn using

the coordinates deposited in Protein
Data Bank and the program PyMol.
23































10

1.2.2. Prokaryoti
c NMN adenylyltransferase
s



NMNAT from archea and eubacteria and other enzymes play important functions in
the control of NAD homeostasis in some bacteria.



1.2.2.1.
Archaeal NMN
a
denylyltransferases


NMNAT from the
archaeon

Methanococcus jannaschii

has
been the fi
rst

adenylyltransferase to be structurally characterized

(Figure 3)
.
21

It is
extremely
thermophilic:

in fact

show a continuo
u
s increase in activity at temperatures ranging
from 37 to 97°
C.

Co
2
+

and
Ni
2+

resulted
to

be the most effective cofactor
s

for the
M.
jannaschii
enzyme
.
24













Figure
3
.

Ribbon representation of the hexamer of NMNAT
M
.

jannaschii
(1F9A) in complex
with NAD

as produced using the program PyMol.
2
3


The crystal structure in comple
x

with the substrate ATP has been the fir
st str
ucture
reported for this enzyme.
21,25
The result has been complemented b
y

the resolution of the
structure of the
Methanobacteriu
m termoautotrophicum

NMNAT in complex with
NAD (Fig. 4) and NMN.
2
2
The analyses revealed a hexameric assembly

and a common


11

overall fold, highly similar to the members of the nucleotydyl tr
ansferase family
,
allowing the identification of NMNAT as a novel member of the family. The structural
results suggest that catalysis does not rely on the formation of a covalent adenylyl
-
NM
NAT intermediate, but it is carried out via direct attack of ATP by NMN, in
agreement with the
17
O
-
NMR studies conducted on the enzyme catalyzed reaction.













Figure 4
.

Ribbon representation of the hexamer of NMNAT
M. termoautotrophicum
)

in
comple
x with NAD

(1M8G)

as produced using the program PyMol.
23



1.2.2.2.
Eubacterial NMN
a
denylyltransferase (NadD)


NMNAT activity in bacteria has been first reported

in 1961
26

as a

partially purified
preparation of the enzyme from
E
.
coli
extracts.
Unlike the

yeast and mammalian
enzyme,
E
.
c
oli

NMNAT resulted to be extremely specific for the deamidated form of
the pyridine nucleotides, being the pyrophosphorolysis of NAD almost undetectab
le
.
26

Like the archaeal and eukaryotic counterparts, eubacterial NMNATs b
elong to the
nucleotidyltransferase
α
/
β
phosphodiesterases family of proteins containing the
conserved (T/H)XGH motif.

The crystal structures of
E. coli
NaMN adenylyltransferase
and its complex with deamido
-
NAD (NaAD)

revealed that ligand binding causes la
rge
conformational changes in sev
eral loop regions around the active site (Figure 5).
27
The
enzyme specifically recognizes the deamidated pyridine nucleotide through interactions
between nicotinate carboxylate with several protein main chain amides and a p
ositive

12

helix dipole. Comparison of
E. coli
NMNAT with th
at

from archaeal organisms
,

revealed extensive differences in the active site architecture, enzyme
-
ligand interaction
mode, and bound dinucleotide conformations. The bacterial NaMN adenylyltransferas
e
structures provide a foundation for structure
-
based design of specific inhibitors that may
have therapeutic potential.












Figure
5
.

R
ibbon representation of the homo
trimer of NMNAT
E
.

coli
in complex with NaAD
(
1K4M
)


as produced using the prog
ram PyMol.
23



The
nadD
gene has been also identified in
Bacillus subtilis
.
2
8

It codes for a 189
residues protein which has been obtained in recombinant form in
E. coli
and purified
(Figure 6)
.
2
8

A detailed kinetic study on the forward and reverse reaction

revealed a
clear preference of
Bs
NadD
for the deamidated nucleotides.
2
8

Thus, it appears that the

sequence of the
B. subtilis

enzyme is more closely related to the

group of eubacterial
enzymes that prefer NaMN as a substrate

than to the archeal or eukary
otic enzymes that
show little

preference among the substrates or prefer N
MN over NaMN.
29
,
30








13















Figure
6
.

Ribbon representation of the dimer of NMNAT
B. subtilis

in complex with NaAD


(
1KAM
)

as produced using the program PyMol.
23



1.2.2.
3. NadR Proteins


Haemophilus influenzae
NadR protein (
hi
NadR) has been shown to be a bifunctional
enzyme possessing both

NMN adenylytransferase and ribosylnicotinamide

kinase
(RNK,

EC 2.7.1.22) activities.
Two enzymatic steps are required to convert
ribos
ylnicotinamide to NAD in the cytoplasm: a ribosylnicotinamide

kinase to catalyze
the phosphorylation

of nicotinamide riboside to produce NMN
,

and N
a
MN

adenylytransferase
to link NMN and the

AMP moiety of ATP to generate NAD.

In
H.
influenzae
, the NadR prot
ein encodes the
only N
M
NAT

activity in the organism. The
RNK activity encoded in NadR

was also shown to be of central importance for the NAD
generation

in
H. influenzae
.
31
,
32

For this reason,

i
ts

function is essential for the growth
and survival of

H. infl
uenzae
and thus may present a new highly specific

anti
-
infectious
drug target.

The NMNAT activity of the NadR proteins has been experimentally
confirmed

both

in
E. coli
, and more recently

in the
H
.

influenzae
.
19
,
33

The crystal
structure of NadR protein has

been solved in complex with NAD resulting as tetrameric
one (Figure 7).
34





14












Figure 7.

Ribbon representation of the tetramer of NMNAT
H.

influenzae

in complex with
NAD
(
1LW7
)

as produced using the program PyMol.
23























15

1.2.3. E
ukaryotic NMN

a
denylyltransferase
s


Since the detection in yeast autolysate
35
,
36

of an enzymatic activity able to catalyze
adenylylation of both NMN and NaMN, NMNAT has been observed in several
eukaryotic organisms
.

The eukaryotic NMNATs

h
ave

been purified

to homogeneity and
extensively characteri
zed

in their molecular and kinetic properties.



1.2.3.1.
Yeast
NMN

a
denylyltransferases


The first homogeneous NMNAT preparation was obtained from the yeast
Saccharomyces cerevisiae
.
36
,
37

Yeast NMNAT (yNMNAT
-
1) wa
s found to

be an
oligomeric glycoprotein.

After cloning and expression in
E.

coli
cells, the pure
recombinant yNMNAT
-
1 showed molecular and kinetic properties undistinguishable
with respect to the wild
-
type enzyme.
38

Subsequently,

has been
found
a second i
soform
of yeast NMNAT
, named yNMNAT
-
2 that
has been expressed in
E.

coli
cells

and
kinetically characterized
.
39



1.2.3.2. Human

NMN

a
denylyltransferase
s


Human NMNAT has been studied for more than 50 years by assuming that it was
coded by a single gene an
d that the protein was exclusiv
ely located within the nucleus
.
13

However,
recently,

two new variants

of the human enzyme
, called
h
NMNAT
-
2
1
8,40
and
h
NMNAT
-
3,
41

were identified. T
hese new isoforms, localized in the cytoplasm, have
been kinetically and struct
urally characterized with respect to the nuclear NMNAT
protein, thus renamed
h
NMNAT
-

1.

In humans,
NMNAT

has been hypothetically linked to neurologi
cal disorders such as
epilepsy, Alzheimer disease and Huntington disease because its substrate, quinolinate
, is
a very well known endogenous excitotoxic agonist of N
-
methyl
-
D
-
aspartate (NMDA)
receptors and thereby may modulate the effects of excitotoxins
.
14
-
16



16

In human, the
de novo
NAD biosynthesis proceeds via

several enzymatic steps to

transform tryptophan
into NaMN (Figure 8
)
. Quinolinate phosphoribosyltransferase
(QaPRT) (EC 2.4.2.19) catalyzes the conversion of quinolate and 5
-
phospho
-
α
-
D
-
ribose
1
-
diphosphate (PRPP) to nicotinate mononucleotide (NaMN), pyrophosphate (PPi) and
CO
2
. NaMN

is then converted i
nto deamido
-
NAD (NaAD) via the

action of the
ubiquitous enzyme NMNAT, and into NAD by

means of NA
D synthetase.
13

Alternatively, the nicotinamide

or nicotinamide ribose from degradation of NAD or
from the

extracellular medium can be re
-
used and adenylated b
y

NMNAT to form NAD
directly, potentially bypassing the last

amidation step catalyzed

by NAD synthetase
.





Figure
8
.

Schematic

NAD metabolic pathways in human. P
roposed salvage pathways for
utilization of exogenous nicotinamide and nicotinamide ribose
(
NmR
), and anabolic activation
of
the antitumor agent
tiazofurin (
TR
).





17

Human

NMNAT displays unique dual substrate specificity toward both

NMN and
NaMN
41
-
44

and is thus capable of participating in both
de novo
and salvage pathways of
NAD generation.

Intr
acellular

levels of nicotinamide
are
modulated by the action of
nicotinamide deamidase

(EC 3.5.1.19), nicotin
amide phosphoribosyltransferase
(NamPRT)

(EC 2.4.2.12),
and nicotinate phosphoribosyltransferase (NaPRT)
(EC
2.4.2.11)
, enzymes involved in nicotin
amide salvaging
. Surprisingly, no in
-
depth
studies have bee
n carried out on the characteri
zation of these enzymes in humans
despite their evident importance in niacinamide utilization.

Moreover, human NMNAT catalyzes the rate
-
limiting

step of the metabolic

conversion of the anticancer agent tiazofurin

(2
-
β
-
D
-
ribofuranosylthiazole
-
4
-
carboxamide) to its active

metabolite

adenine dinucleotide (TAD)
45

(Figure

9
).
















Figure
9
. Metabolic conversion of tiazofurin to TAD.


TAD as a NAD analog,
is a potent inhibitor of inosine monophosphate
dehydroge
nase

(IMPDH)
,

a rate
-
limiting enzyme in the
de novo
biosynthesis of guanine
nucleotides, including GTP and dGTP. Inhibition of
IMPDH

produces an overall
reduction

in guanine nucleotide pools, which leads to the interruption of

DNA and RNA
synthesis, and co
mpromises the ability of G

proteins to function as transducers of

18

intracellular signals.
46

The development of tiazofurin

resistance has been shown to
relate mainly to a decrease

in
h
NM
NAT
-
1

activity.
47


The different human NMNAT differs with regard to cel
lular localization, tissue
-
specificity of expression, and molecular properties, supporting the idea that the three
enzymes might play distinct physiological roles in NAD homeostasis.



1.2.3.2.1.
Human

NMNAT
-
1


The first isoform of a human NMNAT (
h
NMNAT
-
1)

was cloned in 2001
17,20

and
soon thereafter 3D structure was reported by three indipendent groups reviewed by
Rizzi
et al
.
48

The purification of
h
NMNAT
-
1 to homogenei
ty

h
as been first obtained
from placenta
43
and very recently the cDNA

has been isolated,
cloned, and the
recombinant enzyme has been characterized as to its major m
olecular and kinetic
properties
.

17

h
NMNAT
-
1 uses both pyridine mon
o
nucleotides, NMN and NaMN, with the same
efficiency, thus participating in both
de novo
and sal
vage pathways of N
AD synthesis
.
17

Like other eukaryotic NMNATs
,
h
NMNAT
-
l catalyzes the synthesis of NADH from
NMNH, while
α
-
NMN is ineffective both as an

inhibitor and as a s
ubstrate, suggesting
that the adenylyl

trans
fer is strongly stereospecific
.
43

The human NMNAT
-
1 gene has
been isolated
and
mapped to chromos
ome 1p36.2
,
17

and its entire stru
cture

has been
determine
d (Figu
re 10).
49

h
NMNAT
-
1

appears to

be localized to the nucleus

and its

activity has been reported to correlate with DNA synthesis

during the cell cycle
.

Further
analys
is of

h
NMNAT
-
1 mRNA expression among a panel of cancer lines indicated
that,
with the exceptio
n of Burkitt’s

lymphoma and chron
ic myelogenous leukemia, the
enzyme

is weakly expressed

in tumor cells.
17

This result in

agreement with the
previously reported data concern
ing a

reduced
h
NMNAT
-
1 activity afte
r cancerous
transformation,

makes this enzyme a

p
otential chemotherapi
c targe
t
.
50






19













Figure 1
0
.

Ribbon representation of the hexamer of
h
NMNAT
-
1
in complex with NAD

(
1KKU
)

as produced using the program PyMol.
23


Recently, it has also been found that

human nuclear
NM
NAT
-
1

may play an
impor
tant role in delaying the Wallerian

degeneration
1
5

in injured axons and synapses:
the causative

mutation in the slow Wallerian degeneration mouse is found to

be a
chimeric gene
Ube4b/NMNAT
encoding NMNAT
-
1 fused

with the N
-
terminal region of
the ubiquitina
tion factor E4B
.
51,52



1.2.3.2.2.
Human

NMNAT
-
2


In 2002, Magni
et al
.
1
8
have

identified

of
h
NMNAT
-
2 as
a new human member of
the

NMNAT family, based on its similarity with the nuclear

enzyme

(
h
NMNAT
-
1).
KIAA0479 protein as a new putative NMNAT isoenzyme

was identified
.
53

T
he
corresponding

cDNA has been isolated, cloned,
expressed in E.
coli,
and th
e purified
recombinant protein
h
NMNAT
-
2 was

characterized as to its molecular and functional
properties.

Yalowitz
et al
.
40

have cloned a novel

human NMNAT
-
2
cDN
A

from human brain.
h
NMNAT
-
2 shared only

3
4
% amino acid sequence homology with the
h
NMNAT
-
1
, but
possessed enzym
at
ic activity comparable

with
h
NMNAT
-
1.

However,
h
NMNAT differs

20

from 1 isoform with regard to chromosomal and cellular localization.
N
orthern

bl
ot
analysis revealed highly restricted expression of
h
NMNAT
-
2, which contrasts with the
wide

tissue expression of
h
NMNAT
-
1
.

I
mmunohistochemical

analysis of sections of
normal adult human pancreas revealed that

h
NMNAT
-
2 protein was markedly
expressed in the

islets of Langerhans.

However, the pancreatic exocrine cells exhibited
weak

expression of
h
NMNAT
-
2 protein. Sections of pancreas from

insulinoma patients
showed strong expression of
h
NMNAT
-
2

protein in the insulin
-
producing tumour cells,
whereas acinar

ce
lls exhibited relatively low expression of
h
NMNAT
-
2 protein.

These
data suggest that the unique tissue
-
expression patterns

of
h
NMNAT
-
2 reflect distinct
functions for the isoforms in the

regulation of NAD metabolism
, but t
he functional

significance of
this

isoform within a given tissue remains to be

elucidated.



1.2.3.2.3. Human
NMNAT
-
3


In 2003, a third human isoform (
h
NMNAT
-
3) has been discovered, for which the X
-
ray structure has been solved, and localized outside the nucleus. The protein, consisting
of
252 amino acid residues,
is 50

and 34% identical to
h
NMNAT
-
1 and
h
NMNAT
-
2,
respectively. Its cDNA has been cloned and the purified recombinant protein
catalytically and structurally characterized.
41

Compared with the nuclear isoform, the overall expression

levels of
h
NMNAT
-
3 are
lower, being the major sites of expression lung and spleen, even though mRNA blots
show a weak signal also in placenta and kidney. Furthermore,
h
NMNAT
-
3 has been
reported to be expressed at very low quantities in tumor cells.
41

The
structure of
h
NMNAT
-
3 has been solved in its apo form and in complex with either the ATP analog
AMP
-
CPP or NMN, with either NAD (Figure 11) or NaAD, and with NMN and AMP
-
CPP together. The solved structure reveals small conformational changes induced by
the

presence of the ligands with respect to the apo form. This in contrast to the more
drastic changes occuring upon substrate binding in the
E.

c
oli

and
B.

subtilis

NMNATs.





21










Figure 1
1
.

Ribbon representation of the tetramer of
h
NMNAT
-
3
in complex

with NAD

(
1NUU
)

as produced using the program PyMol.
23


In particular, it’s very important the role

developed by a molecule of water

"dual
specificity” displayed by the enzyme toward both nicotinamide and nicotinic acid
substrates. Indeed, this water mole
cule is able to act alternately as a hydrogen acceptor
when hydrogen
-
bonded to the amide group of nicotinamide and as a hydrogen donor
when interacting with carboxylate oxygen of nicotinic acid, thus contributing the
neutralization of its negative charge.
T
he existence of a

cytosolic and mitochondria
l

h
NM
NAT
-
3 strongly suggests that

final steps of NAD biosynthesis may occur outside
of the nucleus.

All of the three characterized human
NM
NAT isoforms

display dual
substrate specificity toward either NMN or Na
MN

and thus are flexible to participate in
either
de novo

or salvage/recycling pathways via either intermediate. The nuclear

h
NM
NAT
-
1 is undoubtedly involved in the NAD regeneration

in the nucleus, but its
role in NAD
de novo
biosynthesis is

less obvious.
It i
s tempting to speculate that
h
NM
NAT
-
3 may be

one of the key participants in NAD biogenesis outside of the

nucleus. Further experiments are required to revisit the early

dogma on the cellular
NAD biosynthesis, which was based

solely on the localization
of the major
NM
NAT
activity inside

nuclei
.

All three
h
NM
NAT isoenzymes

are expressed in cancer cells at much lower levels,
which are

assumed to be close to the critical level for cell survival.
54
Consequently,
NM
NAT has been regarded as a potential target

for anticancer chemotherapy, because

the minimal inhibitory

concentration lethal to cancer cells is not likely to cause serious

damage to normal cells.
5
5


22

1.2.3.3.
Subcellular
c
ompartmentation of
human NMNATs


One

exciting result of the

discovery of multip
le NMNAT isoforms is their
differential

subcellular localization. Although
h
NMNAT
-
1 was found to

be localized
in

the nucleus
,
17

the other two isoforms

appear to be cytosolic,
18,40

and
h
NMNAT
-
3 might
also reside

within mitochondria.
41

The
c
ompartmentation

o
f the human NMNAT
isoforms has been depicted in Figure 12 with respect to NAD biosynthesis.
The reasons
for such a compartmentation

of NAD synthesis
are unknown
.



Figure 12.

Compartmentation of NAD(P) biosynthesis and major NAD(P)
-
mediated signalling
pathw
ays in eukaryotic cells. Abbreviations: CAC, citric acid cycle; cADPR, cyclic ADP
-
ribose; ER, endoplasmic reticulum; NAADP, nicotinic acid adenine dinucleotide phosphate;
NADase, bifunctional NAD glycohydrolase/ADP
-
ribosyl cyclase; NMNAT, nicotinamide
mono
nucleotide adenylyltransferase; ox. p
hos., oxidative phosphorylation.





ATP + NMN

h
NMNAT
-
3

NAD
+

CAC

NA
DH

Ox.Phos
.


ATP + NMN

h
NMNAT
-
2

NAD
+

Ca
2+

Ca
2+

Ca
2+


A
A
T
T
P
P


+
+


N
N
M
M
N
N


h
NMNAT
-
1

N
N
A
A
D
D
+
+


NAD
+

kinase

NADP
+

NADase

ADP
-
ribosyl


cADP
R

NAAD
P

Nucleus

Golgi

ER

Mitochondria

Lysosome

C
C
y
y
t
t
o
o
p
p
l
l
a
a
s
s
m
m
i
i
c
c


m
m
e
e
m
m
b
b
r
r
a
a
n
n
e
e



23

1.2.3.4.
Expression of NMNAT isoforms in human tissues


Human nuclear
NM
NAT
-
1 was shown to

be
express
ed

at high levels in

heart,
skeletal muscle and, to a lesser extent, in kidney and

liver.
17

h
NMNAT isoforms 2 and 3
exhibit a rather specific tissue distribution.
h
NMNAT
-
2 is predominantly found in
brain
,
1
8

hearth, and skeletal muscle.
h
NMNAT
-
2 was found to be strongly expressed in
pancreas and in the islets of Langerhans
.
40

h
NMNAT
-
3 is

more strongly expressed in
tissues from which
h
NMNAT
-
2 is nearly absent,
i.e.

kidney, lung and spleen.


Table 1.

Expression of NMNAT isoforms
in human tissues and cell lines;

(+), very weak
expression; ++, strong expression. ND, not determined.



h
NMNAT
-
1

h
NMNAT
-
2


h
NMNAT
-
3

Tissues

Brain

(+)

++

_

Hearth

++

+

(+)

Skeletal

muscle

++

+

(+)

Liver

+

_

(+)

Kidney

++

_

+

Lung

(+)

_

++

Spleen

_

_

++

Thymus

(+)


(+)

Pancreas

+

+

(+)

Colon

(+)

_


Placenta

+

(+)

+

Cell lines

HeLa

+

+

(+)

HEK293

+

+

+

K562

+

_

ND

Raji

+

_

(+)


24

Surprisingly, the oligomeric state of the three human NMNAT isoforms appears to
differ considerably. As deduced from the crystal structures
,
h
NMNAT
-
1 is a
homohexamer
,
2
5,44,56

whereas
h
NMNAT
-
3 is a
homotetramer
.
41

Recombinantly
expressed
h
NMNAT
-
2 has be
en suggested to be a homodimer
.
1
8

Only
h
NMNAT
-
1 has
been extensively studied with regard to physical and kinetic properties.
24,43


Given the rather different tertiary structures of the proteins, their
functional
characteristics could also differ substantially. Analyzing the presence of mRNA in two
human cell lines it was also found that all three isoforms can be expressed
simultaneously.
41

Therefore, the existence of three NMNAT isoforms appears to be
c
onsistent with important compartment
-
specific functions rather than to reflect simple
functional redundance.

The variability among the NMNAT isoforms regarding substrate

specificity may also have important implications.

Recently
, it

has been shown that th
e three
human isoform
s are able to synthesize
NA
DH beginning from NMNH and ATP. So far, NAD biosynthesis has been described
as a pathway generating the oxidized form, NAD, which can then be reduced in a
dehydrogenase reaction. This new evidence

suggests th
e possibility that the NAD
doesn't be synthesized in the oxidized form and then reduced by the
dehydrogenase
.

To determine and compare the affinities (
K
m

values) of
all three human NMNATs
57

for their substrates, NMN and ATP, the coupled photometric assay w
as used.

h
NMNAT
-
1

exhibited the highest affinity for these substrates as well as the highest
specific activity.
h
NMNAT
-
3 has not been kinetically characterized so far. Both
h
NMNAT
-
1 and
-
3 showed no preference with regard t
o the pyridine mononucleotides
NM
N and NaMN
, whereas
h
NMNAT
-
2 preferred NMN

over NaMN.

h
NMNAT
-
1 is
known to also catalyze the reverse reaction. That is, it

ge
nerates ATP and NMN

from
NAD and pyrophosphate.
T
his capacity was also tested for
h
NMNAT
-
2 and
-
3.









25

Magni
et al
.
58

desc
r
ibed
the three
-
dimensional structure of NMNAT activity
possessing enzymes.


Table 2.
S
olved structures of enzymes endowed with NMNAT activity
.

a
Both NMN
and
NaMN substrates are efficiently utilized
.









26

1.2.3.5.
Mechanisms for the
d
ual
s
ubstrate
s
pecificit
y of
hs
NMNAT and
i
ts
i
mplications


The more relaxed substrate specificity of human NMNAT isoforms contrasts with
the stringent substrate preference of bacterial and archaeal NMNATs.
E. coli
and many
other bacterial NMNATs strongly pref
er the deamidated NaM
N to NMN,
whereas
archaeal NMNAT prefers the amidated NMN to NaMN by about 2 orders of magnitude.
T
he structural mechanisms underlining the different substrate

specificities

has been
detected

f
rom the structural comparisons
of NMNATs from human,
E. coli,
a
nd
archaeal species and their

complexes with respective ligands. In
E. coli
NMNAT, an
anion
-
binding pocket is responsible for the recognition of the

negatively charged
carboxylate group of NaMN. The electrophilic

nature of this pocket is further enhanced
b
y the surrounding

hydrophobic residues, which would increase its ability to
discriminate

between a carboxylate and a carboxamide. In archaeal

NMNAT, the
pyridine
-
binding site is substantially different from

those in
E. coli
and human enzymes,
and the bound

pyridine

nucleotide adopts a rather different conformation than those in

E. coli
and human NMNAT
-
ligand complexes.
59,60

The specific

interactions with the
nicotinamide include hydrogen bonding

between both amide and carbonyl groups of the
Ile
-
81 main chai
n

and th
e carboxamide groups of NMN
.
22
These interactions

clearly are
more favorable for the binding of a carboxamide than

a carboxylate.


Compared with the bacterial and archaeal NMNAT active

sites, one prominent
feature of the human NMNAT pyridine

bindin
g

site is the presence of several conserved
water molecules

(ω1
-
4) and a negatively charged Asp residue (Asp
-
173).

Based on the
structural comparison of
h
NMNATs complexed

with NAD or NaAD, has been
proposed that human NMNAT recognizes

both NMN and NaMN rea
dily without the
need for any

significant conformational changes. The presence of Asp
-
173

and several
conserved water molecules could change the subtle

electrostatic distributions of the
binding site and enable the

enzyme to accommodate substrates of diffe
rent electrostatic

properties.
61
-
63

This intrinsically relaxed substrate specificity

makes human NMNAT
flexible in balancing different NMN/NaMN metabolic fluxes for NAD generation,
which may be

required to satisfy the requirement for NAD in nuclei.



27

The di
stinct substrate specificity patterns of NMNATs from

different organisms are
indicative of significant variations in

the metabolite fluxes in the cell that lead to the
NAD production.

The unique dual specificity evolved by
h
NMNATs has

clear
biological impl
ications with respect to possible NAD biosynthetic

pathways in human,
which include both types of

inte
rmediates, NaMN and NMN
. The relative importance

of these pathways in different types of tissues and in

different physiological states
remains to be studi
ed. In the

majority of bacterial pathogens, such as
Staphylococcus
aureus
,

Streptococcus
pneumoniae
,
and

Helicobacter pylori
, the preferred

intermediate
in NAD biosynthesis is NaMN. It correlates

with a strong preference of bacterial
NMNAT (NadD

family) t
oward this form

of pyridine mononucleotide
. Significant

differences between bacterial and human enzymes in

both substrate specificity and the
active site conformations, as

revealed in this study, are of crucial importance for the
design

of highly selective

NMNAT inhibitors with anti
-
infectious

potential.






















28









Chapter 2.




2.1. Polyphosphate NAD analogs as NMNAT inhibitors based on
"transition
-
state concept"



2.2. Stereoselective synthesis of
β
-
nicotinamide/nicotinic acid ribosides
a
nd of a ribose
-
modified analog



2.3.
Synthesis, conformational analysis and biological evaluation of
dinucleotide NAD analogs modified at ribose adenylyl part as NMN
adenylyltransferase inhibitors. Influence of the methyl group on
enzyme affinity and mole
cular modeling investigation






29

2.1. Polyphosphate NAD analogs as NMNAT inhibitors based on
"transition
-
state concept"


The study carried out on nicotinamide mononucleotide adenylyltransferase
(NMNAT) has proved to be an extremely interesting one: as desc
ribed above, this is an
indispensable enzyme catalyzing the central step of NAD biosynthesis pathways by
linking the AMP moiety of ATP with NMN or NaMN to form NAD or NaAD.

The reaction





NMN + ATP = NAD + PPi


represents the final step in the biosy
nthesis of NAD.

In the context of this project, the cooperation with Prof. Magni team of the
Università Politecnica delle Marche (Italy), enabled me to obtain good results in the
identification of NMNAT inhibitors.

In the first part of the research, my at
tention was focused on the identification of NMN
adenylyltransferase inhibitors based on "transition
-
state concept".

With this purpose in view,
two
dinucleoside polyphosphate NAD analogs were
prepared, consisting of two nucleoside moieties, nicotinamide ri
boside and adenosine,
joined through their ribose 5'
-
position by a linear polyphosphate chain consisting of 3 or
4 phosphate groups (Np
3
AD and Np
4
AD
, respectively
).
69


The rationale underlying the production of these molecules arises from the
hypothesis th
at they
should have a structure resembling the putative transition state
analog of the reaction catalyzed by
NMNAT and hence compete with the catalytic site.
This approach could prove to be of great importance. In fact, it is well known that the
natural di
nucleoside polyphosphates (DNPs) are ubiquitous compounds involved in a
number of intra
-

and extracellular processes, even though the importance of their
physiological role in these events, fundamental for cellular survival, remains to be
understood.


The

synthesis of the nucleoside polyphosphates, P
1
-
(adenosine
-
5’)
-
P
3
-
, and P
4
-
(nicotinamide
-
riboside
-
5’)tri
-
, and tetraphosphate (Np
3
AD,
1
, and Np
4
AD,
2
,


30

respectively) and their effect on the enzymatic activity of NMNAT series, were
described.


2.1.1. Synthe
sis of Np
3
AD and Np
4
AD


The dinucleotides Np
3
AD (
1
) and

Np
4
AD (
2
) were prepared using the method
reported
by Franchetti
et al.
64

(
Scheme 1). The yield of the final products was optimized
by a different purification step.

ADP or ATP as sodium salt and nicot
inamide riboside monophosphate (NMN)
commercialy available (Sigma
-
Aldrich) were used as starting compounds.

The synthesis of
1

and
2

was

carried out by CDI
-
catalyzed coupling reaction of the
activated
nicotinamide ribo
side monophosphate (
8
)

with ADP

or AT
P

as mono
-
tributylammonium salts

(
5

and
6
, respectively)
.
The electrophilic nicotinamide riboside
monophosphate imidazolide (
8
) was obtained by reaction of NMN with 1,1’
-
carbonyldiimidazole (CDI). ADP and ATP salts
(
5

and
6
, respectively)

were obtained
sta
rting from the free acid
3

and
4
of the commercial sodium salts by treatment with
n
-
tributylamine in MeOH. Coupling of
5

and
6
with the NMN imidazolide
8

(molar ratio
1.5:1) was performed in anhydrous N,N
-
dimethylformamide for two days. Nucleotides
1

and
2

were obtained as triethylammonium salts after purification by a DEAE
-
Sephadex column (HCO
3
-
).

The structure of Np
3
AD and Np
4
AD was determined by mass spectrometric analysis
which confirmed the expected
m/z
ratio of 743.4 and 823.3, respectively. The str
ucture
was also confirmed by nuclear magnetic resonance
1
H
-
, and
31
P
-
NMR

in D
2
O.










31


32

2
.
1.2.

Biological
e
valuation


The effect of the polypshosphates
Np
3
AD and Np
4
AD

on NMNAT activity was
evaluated on both microbial and human recombinant enzymes, in
cluding NMNAT from
M.

jannaschii
,
21,65
yeast
S. cerevisiae
,
38

and human NMNAT
-
1
,

17,20

h
NMNAT
-
2
18
and
h
NMNAT
-
3 isoforms.
41


The influence of the NAD polyphophates was determined by the measure of
NMNAT activity in the direct reaction to form NAD with both
substrates ATP and
NMN.

Figure 13 showed the transferase activity (ATT) in the presence of different
concentration of
Np
4
AD

as the most active compound, toward NMNAT from
M.

jannaschii
. The activity was expressed as mU/ml: one Unit was defined as the amoun
t
of enzyme catalyzing the formation of 1 mmol of NAD per minute at 37 °C.





















33


A)


B)


Figure
1
3.

ATT (mU/ml) (NMNAT
M. jannaschii
)

versus
A
) [ATP], and
B
) [NMN] plot in
the presence and in the absence of a fixed concentration (0
-
100 μM) of the competitive inhibitor
Np
4
AD.






34


Figure

14 showed the competitive inhibition of
Np
4
AD against the
NMNAT from
M.
jannaschii
.



A)














B)












Figure

14.

1/V Versus A) 1/[ATP] or B) 1/[NMN] plot in the presence and in the absence of
different fixed concentrations of the competitive inhibitor Np
4
AD against NMNAT from
M.
jannaschii
.



35


The

inhibition data of the tested compounds

Np
3
AD and Np
4
AD

were also
determined.
The values were

expressed as
K
i

and IC
50

(
Table
3)
.

IC
50

values

are

the concentrations
required to inhibit 50% of enzyme activity related to enzyme catalyzing the formation
of NAD.












36







37


2.1.3. Stability of
Np
3
AD and Np
4
AD


The pre
sence of the negatively charged phosphorus in the polyphosphates
Np
3
AD
and Np
4
AD

was expected to induce an instability of these molecules.
It was already
speculated that the stability of these dinucleotides might play a critical role in
determining the fin
al concentration of both compounds in the biological medium.
So, to
verify the stability of

Np
3
AD and Np
4
AD

in

enzymatic

condition, the dinucleotides (100
μM concentration) were mantained in
150
μ
l of 35 mM Tris buffer (pH 7.5).

NMN and
ATP at 100 μM concentration were added. Under these conditions both polyphosphates
were stable during the entire enzymatic assay (1 h), as was detected by HPLC analysis.



2
.
1.4.

Result and Discussion


Influence of the dinucleotides
Np
3
AD and Np
4
AD

on the activity of eubacterial and
human NMNAT isoenzymes was determined by
measuring

of enzymatic activity in the
presence of NMN and ATP substrates.

The NMNAT activity
was calcu
lated

as
the
amount of formed NAD with or without the
Np
3
AD and Np
4
AD
.

The polyphosphates
1

and
2

proved to be inhibitors toward
the enzyme

from
M.

jannaschii

and human

isoforms

(Figures

13, 14
)

exhibiting a competitive
-
type inhibition
toward both NMN and

ATP substrates
.
However, both the compounds exerted a
different inhibitory effect toward the b
acterial enzyme with respect to
eukaryotic
NMNAT.

In particular,
Np
4
AD
showed a selective potency toward both ATP and NMN
substrates against the
M.

jannaschii

en
zyme (
K
i

10
μM and 21

M
, respectively
)
as
compared to that against the other enzymes.

In the case of Np
3
AD,

the
K
i
value toward NMN

against NMNAT from
M.

jannaschii

resulted similar to that of
h
NMNAT
-
1 and 2 isoforms.

Futhermore,
IC
50
values of

Np
3
AD and in particul
ar of
Np
4
AD resulted in a
discriminating activity between
M.

jannaschii

and human enzymes. So, Np
4
AD might
represent a lead

compound

for the development of potent and selective inhibitors
of
bacterial NMNAT

from different sources.



38

2.2. Stereoselective sy
nthesis of
β
-
nicotinamide/nicotinic acid ribosides
and of a ribose
-
modified analog


In the last five years, a number of publications have schematized NAD biosynthesis.
Recently, a convergence of the flux to NAD from
de novo

synthesis, nicotinic acid
import, and nicot
inamide salvage at NaMN was also depicted.
66


Bieganowski P. and
Brenner C.
67

showed

that nicotinamide riboside which was known to be an NAD
precursor in bacteria such as
Haemophilus influenza
, is also a NAD precursor in a
previously unknown but apparently

conserved eukaryotic NAD biosynthetic
pathways.
68

The accepted view of eukaryotic NAD biosynthesis, that all anabolism flows through
nicotinic acid mononucleotide, was challenged experimentally and revealed that
nicotinamide riboside is an unanticipated N
AD precusor in yeast. However,
nicotinamide riboside was discovered as a nutrient in milk, suggesting that nicotinamide
riboside is a useful compound for evaluation of NAD levels in humans and could
protect some tissues by diluting the antitumor tiazofurin

into the NAD precusor.
68

Nicotinamide riboside (
β
-
NAR
) is an intermediate in one biosynthetic pathway by
which nicotinamide is converted into NAD. In fact, nicotinamide or nicotinamide
riboside derived from degradation of NAD can be reused by nicotinamide
phosphoribosyl transferase or ribosylnicotinamid
e kinase, respectively, to form the
NMN that is adenylated to NAD by NMNAT. Kinetic and structural studies of both
human and bacterial NMNAT require the
β
-
anomer

of nicotinamide monoribotide as a
substrate and of nicotinamide riboside as a product of the N
AD degradation pathway.
The stereospecificity of NAD
-
mediated reactions is determinant in all living organisms
because the pyridine ribotide moiety of NAD reacts only in the
β
-
configuration.

In order to develop the research of ribonucleoside and nucleotid
e analogs as
chemotherapic agents,

such as tiazofurin
-
related drug,
69

it was important

to synthesize
pyridinium ribosidic derivatives

as precursor of NAD analogs
.

From a survey of the literature,
was

found

that nicotinamide and nicotinic acid ribosides
co
uld be obtained from three e
ssentially different pathways: 1)
enzymatic degradation
of NAD;
69

2) condensation of 1
-
amino sugar with N
1
-
(2,4
-
dinitrophenyl)
-
3
-
amin
ocarbonylpyridinium halogenides;
70

3) condensation of peracylated halo sugars

39

with nicotinamide
.
71

However, most of the reported procedures gave the nicotinamide
riboside as an
α
/
β

anomeric mixture. In addition, a simple but non
-
stereoselective
chemical synthesis of
β
-
NAR starting from 1,2,3,5
-
tetra
-
O
-
acetyl
-
β
-
D
-
ribofuranose was

reported by Tanimori

et al.
72


At first, this synthetic strategy was repeated, but all attempts to isolate the NAR were
unsuccessful. Thus was decided to reinvestigate the above methodology making
changes in some parameters to set up a stereoselective synthesis of both nicoti
namide
and nicotinic acid
β
-
ribosides (
β
-
NAR
9
and
β
-
NaR
10
, respectively).
73




2.2.1 Synthesis of
β
-
NAR

(
9)

and

β
-
NaR
(10)


The key elements of the synthesis involved the formation

of silylated nicotinamide as
starting material, and

then its coupling with per
acylated sugars in th
e presence
of
trimethylsilyl trifluoromethanesulfonate. However,

was found that when a large excess
of trimethylsilyl chloride

in hexamethyldisilazane was used to silylate the

nicotinamide,
a poor yield of the desired nucleoside was obtained and the purifi
cation of 3
-
(carbamoyl)
-
1
-
(2,3,5
-
tri
-
O
-
acetyl
-
β
-
D
-
ribofuranosyl) pyridinium triflate

(
1
7
) was very
difficult. In addition, the amount of

TMSOTf proved to be crucial in this reaction.

A stereoselectivity in the synthesis of NAR

was obtained
by condensation of
nicotinamide with both 1,2,3,5
-
tetra
-
O
-
acet
yl
-
β
-
D
-
ribofuranose (
1
5
) and 1
-
O
-
acetyl
-
2,3,5
-
tri
-
O
-
benzoyl
-
β
-
D
-
ribofuranose (
1
6
) using carefully controlled

conditions
(Scheme
2
). In this procedure was used 2

equiv of

TMSCl to silylate the dry
nicotinamide under reflux.

The intermediate
1
2

was directly
coupled with the protected

sugars (1 eq
u
iv) under Vorbruggen

s
74
conditions

in the presence of a catalytic amount
of TMSOTf, to give only
β
-
anomers

of the protected N
-
nucleosides

1
7

and
1
8

in high
yield. Finally,
β
-
NAR (
9
) was obtained by

basic hydrolytic
deblocking of compounds
1
7

or
1
8

carried out at
-
5 °C to minimize

cleavage of the glycosidic linkage. In the case
of compound

1
8
, for removal of benzoyl groups, two days were

required under
methanolic ammonia condition. The nucleoside
9

was then purified b
y chromatography
on activated charcoal

and isolated as a white solid.


40

In order to validate the above stereoselective procedure,

we investigated the
ribosylation of 3
-
nicotinic acid using

both the sugars
1
5

and
1
6
. In this case, for the
O
-
silylation

of
1
3
,
1 equiv of TMSCl was used. Both the glycosylation reactions

provided
the tri
-
O
-
acylated ribo
nucleo
sides
1
9

and

2
0

uniquely as
β
-
anomers. Nucleoside
1
0

wa
s
obtained
as a white solid after deprotection of
1
9

or
2
0

as reported for
9
.






























41







Scheme
2































42

2
.
2.2.

Structure determination of
β
-
NAR and
β
-
NaR


Anomeric purity of
β
-
NAR and
β
-
NaR was revealed to
be 100% by reverse phase
HPLC and the structure of

these nucleosides was
confirmed by mass spectrometric
analysis. Unambiguous assignment of the configuration

of ribonucleosides was best
accomplished by
1
H
-
NMR

data and proton nuclear Overhauser enhancement

(NOE)
effects. It is worth noting that the
1
H
-
NMR

spectrum of compound
9

was different from
that reporte
d

by Tanimori et al.,
73

but similar to that described

by Jarman and Ross
75

for
the corresponding chloride.

Mor
eover, the coupling constant
J
1’,
2’
and the proton

chemical shift for NAR (
9
) corresponded to values reported

by Oppenheimer and
Japlan.
76


The pr
oton spectrum

of both the protected compounds
1
7
-
2
0

and

nucleosides
9

and
1
0

contained a unique doublet,

which was attrib
uted to the anomeric proton (H1’
) of

the
ribose moiety. The absence of a second doublet in

this region provided strong evidence
for the

essential

anomeric purity of the nucleosides. The
β
-
configuration

was confirmed
thr
ough the coupling constants (
J
1’,
2’
)

value and by proton NOE data. Because of the
overlapping

of the proton signals of H2’
, and H4


in the deprotected

ribosides
9

and
1
0
,
the NOE spectrum was

performed in the corresponding
protected nucleosides.

In the
one
-
dimension
al NOE spectrum, irradiation of
the anomeric protons of
1
7
-
2
0

showed
an enhancement

of H2’ and H4’

in the ribose moiety, while the intensity

enhancement
of the H3’

signal was zero, supporting a

spatial arr
angement

where H1’ and H4’

protons
are

on the same side
of the

ribofuranose, as would be

in

the case

of

β
-
configuration.
Moreover an enhancement of H2

and H6 signals in the pyridine ring was also observed.

The correct struct
ure of
β
-
NAR was also confirmed
by the
13
C
-
NMR spectrum on the
basis of reported

values for NMN and NAD,
77,78

and through a C
H

COSY diag
ram
in
D
2
O.









43

2.2.3. Glycosylation mechanism of pyridinic

ribonucleosides
β
-
NAR and
β
-
NaR


The stereoselectivity of the synthesis of
9

and
1
0

may be explained in

the following
way (Figure 15
). In the first step, the TMSOTf

catalyst converts peracylate
d sugars
1
5

and
1
6

into the corresponding

1,2
-
acyloxonium salts as a unique electrophilic

sugar
moiety.
Under these reversible and thus
thermodynamically controlled conditions, the
nucleophilic

silylated bases
1
2

and
1
4

can attack only the furanose

cations

from the top
(the
β
-
side) to afford the

β
-
nucleosides
1
7
-
2
0
. Only the silylated bases will react

with
the electrophilic sugar cations to form the final

products; since an excess of TMSCl
blocks the
N
1

of

nicotinic bases, a slow and poor reactivity was observed

when more
tha
n 2

equiv of TMSCl were used.
















44

2.2.4 Synthesis of
β
-
nicotinamide
-
3’
-
C
-
methylribofuranoside
(
NA
-
3’
-
MeR,
27)


A
modified
-
ribose
methylated in 3

position
was also used t
o obtain a new class of
nicotin
amide riboside derivatives using the above
-
de
scribed

methodology. Thus,
compound
N
-
pyridinium 3
-
(carbamoyl)
-
1
-
(3

-
C
-
methyl)
-
β
-
D
-
ribofuranose (NA3

-
MeR) was prepared using
1,2,3
-
tri
-
O
-
acetyl
-
5
-
O
-
benzoyl
-
3
-
C
-
methyl
-
β
-
D
-
ribofuranose

(
21
)
.
The sugar
21

(mixture of
α

and
β
anomers) was obtained using the
Ong method
79

with modifications.
8
0

The commercial 1,2
-
O
-
isopropylidene
-
α
-
D
-
xylofuranose was used
as starting compound (Scheme 3).

Coupling of
2
1

with silylated nicotinamide
1
2
, followed by direct

basic deblocking
of nucleoside
26
, gave nucleoside
27

(
Schem
e

4)
. The structure of
27

was determined
by
1
H
-
NMR and

mass spectrometric analysis and
β
-
configuration of this

nucleoside was
confirmed by proton NOE.

NA
-
3’
-
MeR as the first ribose
-
modified pyridinium nucleoside represents a model
for the development of co
nformational study of this type of nucleosides.



45






Scheme 3



























46














Scheme
4











































47

2.3. Synthesis, conformational analysis and biological evaluation of
dinucleotide NAD ana
logs modified at ribose adenylyl part as NMN
adenylyltransferase inhibitors. Influence of the methyl group on
enzyme affinity and molecular modeling investigation


Most enzymes have some degree of flexibility in the backbone and especially the
side chains
to accommodate different ligands. However, it is known that there are cases
where adding a

methyl group to a drug molecule can significantly decrease

bioactivity
or change selectivity; so, there are limits

to how much an enzyme can adapt its shape to
achie
ve affinity to different ligands
.
8
1

In fact, it is known that nucleosides bearing
modifi
ed
-
ribose moiety can be used as tools for studying the enzyme modulation
processes, as well as for preparation of bioanalogous systems.


On this basis,
the effect of me
thylation at the ribose of

adenosine moiety on the
conformational properties of

N
AD

has been
investigated.
Being the purpose, I prepared
novel dinucleotide NAD analogs modified at adenylyl part of the molecule.

The interest in compounds that interfere wit
h NAD activity, was previously triggered
by Franchetti's group in the design, synthesis and conformational studies of NAD
analogs, modified at nicotinic part, as inhibitors of enzymes that use NAD as
cofactor.

69, 82

Furthermore, nucleoside monophospha
tes whose pyridine base of NAD was
substituted by heterocycles such as thiophen and selenophen, proved to be competitive
substrates of
M. jannaschii
NMN adenylyltransferase in the enzymatic reaction
(Franchetti
et al
. Manucript in preparation).

Several w
orks have been made

on NAD analogs as active metabolites of nucleosides
endowed with antitumor and antiviral potency. However, all of these were modified at
pyridine part of the dinucleotide. On the other hand, very little is known on the
biological effect

caused by the modification of the adenylyl part of NAD molecule.

A
number of C
-
branched
-
chain sugar nucleosides has been reported to display several
promising biological activity such as antimicrobial, antiviral and antitumor activity.

83
-
85


Among ribos
e modifications in ribonucleosides, replacement of hydrogen atoms of
the ribose ring with a methyl group such as in adenosine and adenosine analogs,
afforded compounds with various biological activities.
80



48

Sin
c
e 1998, Franchetti'
s

team initiated a
program

aimed toward developing some
purine and py
rimidine nucleosides and nucleotides methylated at the carbon in 2'
-
position of ribofuranose as bioorganic tools and as potential chemotherapic agents.
82,86


Thus, in order to investigate the biological effects in
duced in the NMNAT
-
catalyzed
reactions by the modified "non
-
functional" part of the NAD structure, novel
dinucleotides as NAD analogs modified at the adenylyl ribofuranose moiety

were
prepared
.

The dinucleotides in which
the adenosine ribofuranose was repl
aced by a 2'
-
C
-
methyl
-
ribofuranose
(N2’
-
MeAD and Na2’
-
MeAD)
were synt
hesized and evaluated as
inhibitor molecules toward the NMNAT from
different

sources.
8
7




2
.3.
1

Synthesis of P
1
-
[5’
-
(2’
-
C
-
methyl
-
β
-
D
-
ribofuranosyl)adenine]
-
P
2
-
[5’
-
(
β
-
D
-
ribofuranosyl)nic
oti
namide]diphosphate (N2’
-
MeAD,
28), and P
1
-
[5’
-
(2’
-
C
-
methyl
-
β
-
D
-
ribofuranosyl)
adenine]
-
P
2
-
[5’
-
(
β
-
D
-
ribofuranosyl)nicotinic

acid]diphosphate
(Na2’
-
MeAD,
29)


The dinucleotid
es

N2’
-
MeAD and Na2’
-
MeAD

were

prepared by coupling of
nicotinamide

or nicotinic a
cid

riboside
5’
-
monophosphate
s

with 2’
-
C
-
methyl
-
adenosine
5’
-
monophosphate

(2’
-
MeAMP) (Scheme 6)
.

The synthesis of 2’
-
MeAMP starting from the synthesis of the protected 2’
-
C
-
methyl
ribofuranose was described (Scheme 5).



2.
3.
1
.1.
Synthesis
of 2’
-
C
-
m
eth
yladenosine
5’
-
monophosphate
(2’
-
Me
AMP, 38
)


Several synthetic methodologies have been
reported

toward 2

-
β
-
C
-
branched
nucleosides,
such as convergent approaches
88
-
90

and a linear approach
starting from t
he
unmodified nucleoside.
91

Compared with the linear approach, the convergent

approach
is potentially more flexible since a variety of

nucleobases can be coup
led to the
modified sugar.

A short route toward 2’
-
C
-
branched ribonucleoside synthesis was

49

reported
Franchetti, P.
et

al
.
87

A similar procedure was carried out

to prepare 2’
-
MeAdo

(Scheme 5)
.

The
1,2,3,5
-
tetra
-
O
-
benzoyl
-
2
-
C
-
methyl
-
β
-
D
-
ribofuranose for the
synthesis of 2’
-
MeAdo

was obtained starting
from commercially available

1,3,5
-
tri
-
O
-
benzoyl
-
α
-
D
-
ribofuranose
using the Wolfe method
.
92

1,3,5
-
tri
-
O
-
benzoyl
-
α
-
D
-
ribo
se

(
30
)

was
oxidized to ketone
31

with Dess
-
Martin periodinane reagent.
91

Treatment of
31

wit
h
MeMgBr/TiCl
4

gave compound
32

as well as its transesterified isomers

33

as a mixture
of anomers
.
All
three
products were useful to
obtain
the tetrabenzoylated derivative
34

(
β
/
α

ca.
4:1)

upon treatment

with benzoyl chloride and DMAP in the presence of
tr
iethylamine
.

2’
-
C
-
Methyladenosine was obtained by a convergent synthesis.

Coupling of 1,2,3,5
-
tetra
-
O
-
benzoyl
-
2
-
C
-
methyl
-
β
-
D
-
ribofuranose

(
34
)
with

6
-
chloro
-
purine (
35
)

was carried out using
trimethylsilyl

trifluoromethanesulfonate

(TMSOTf) as Lewis acid
i
n acetonitrile in

the presence of DBU
.

Nucleophilic
displacement of 6
-
chlorine atom in protect compound
36

and deprotection with liquid
ammonia gave 2’
-
MeAdo.

2’
-
C
-
Methyladenosine
87

(
37
)
was converted into

5’
-
monophosphate

38

by the
procedure of
Yoshikawa

et al.
95

using trimethyl phosphate and POCl
3
.
2’
-
C
-
MeAMP
was purified by chromatography on DEAE Sephadex (HCO
3
-

form) column
. The
mononucleotide
38

was then treated with Dowex 50x
8 (H
+
form)

to obtain the
corresponding free
acid which was converted in
to th
e
n
-
tributylammonium

salt

39

by
reaction with tributylamine in DMF.

2’
-
C
-
Methyladenosine was physically and spectroscopically identical to previous
report
86

providing unambiguous proof of its regio
-

and stereochemical assignments.








50



51

2.
3.
1.2.


Synth
esis of

N2’
-
MeAD and Na2’
-
MeAD


N
-
2’
-
MeAD (
28
) and Na
-
2’
-
MeAD (
29
) were obtained by coupling of nicotinamide
riboside 5’
-
monophosphate (NMN) or nicotinic acid riboside 5’
-
monophosphate
(NaMN) as imidazolide derivatives (compounds
8

and
41
, respectively), w
ith the mono
n
-
tributylammonium salt of 2’
-
MeAMP (
39
)

(Scheme 6).

The electrophilic nicotinamide riboside monophosphate imidazolide (
8
) and
nicotinic acid mononucleotide analog

(
41
) were obtained by treatment of
7

or
40

with