Rational Design and Synthesis of Novel Acadesine-Like Modulators of AMPK

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1 . I nt roduc t ion
T he AMP-a c tiva te d prote in kina se , or AMPK for short, is a n e nz yme whose ide ntific a tion
some 30 ye a rs a go ha s brought to inc re me nta l disc ove rie s whic h point to its e sse ntia l role
in the ma inte na nc e of e ne rgy home osta sis in c e ll a nd body [1]. T he initia l disc ove ry tha t
AMPK a c tivity wa s re gula te d by the AMP/AT P ra tio solve d the old c onundrum of how the
c e ll se nse d a nd ma inta ine d its e ne rge tic sta tus quo a nd trigge re d a high le ve l of inte re st
from the sc ie ntific c ommunity, whic h re sulte d in thousa nds of pa pe rs, publishe d ye a rly.
T he se inve stiga te d the role of the AMPK pa thwa y, disc ove re d its c e ntra l role in the he a lth
be ne fits of die t a nd e xe rc ise a nd sugge ste d it a s a nove l ta rge t for dise a se s ra nging from
dia be te s to c e rta in c a nc e r type s a nd e ve n Alz he ime r’s dise a se .
An indire c t c onfirma tion of the c e ntra l role of AMPK is its pe rva sive ne ss in the tre e of life .
Homologue s of AMPK c a n be found throughout the e uka ryotic kingdom a nd its struc ture
ha s re ma ine d la rge ly untouc he d throughout e volution be c a use of its ke y func tion a s a
me ta bolic re gula tor of the c e ll, this role howe ve r ha s e volve d to tha t of a re gula tor of the
whole -body home osta sis in more c omple x orga nisms. In ye a sts, suc h a s Sac c haromy c e s
c e re v isiae the Snf1p kina se is homologue to AMPK a nd c ontrols ge ne e xpre ssion in
re sponse to gluc ose de priva tion, a nd so a re the Snf1p re la te d kina se s (SnR Ks) found in
pla nts whe re the y re gula te c a rbon me ta bolism a nd inhibit synthe sis of isopre noids. Also in
the a nima l kingdom AMPK is highly c onse rve d from C ae norhabditis e le gans ’ a a k-2
AMPK homologue to ma mma lia n AMPK [2,4].
AMPK is a Se rine /T hre onine kina se , the se a re a va st group of e nz yme s me mbe rs of the
tra nsfe ra se e nz yma tic fa mily. W he n a c tiva te d the y phosphoryla te the OH group in Se rine
a nd T hre onine side c ha ins in prote ins c a using the ta rge t prote in to c ha nge its c onforma tion
to a n a c tive or ina c tive sta te . T o fulfill its role a s a c e ntra l e ne rgy re gula tor for the c e ll
AMPK is a c tiva te d by a de c re a se in AT P, trigge re d a s we sha ll se e by a n a lloste ric
a c tiva tion from AMP, whic h se ts off its e nz yma tic a c tivity e xpre sse d by the
phosphoryla tion of se ve ra l downstre a m prote ins. T his ha s the ove ra ll e ffe c t of
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e xtinguishing c e rta in e ne rgy-c onsuming a na bolic pa thwa ys, suc h a s the biosynthe sis of
ma c romole c ule s, c e ll growth a nd prolife ra tion, while e nha nc ing the pa thwa ys of AT P-
produc ing c a ta bolism suc h a s gluc ose upta ke , glyc olysis a nd oxida tion of fa tty a c ids. T his
ma y be through phosphoryla tion of e nz yme s dire c tly involve d in the re gula tion of the
re le va nt pa thwa ys or by re gula ting ge ne e xpre ssion e nsuring tha t the c e ll is not a llowe d to
sta ll a nd sta rve a s a re sult of dwindling AT P re se rve s.
Howe ve r, in more c omple x a nima ls the role of AMPK is not limite d to the c e ll but ra the r it
is now a ppa re nt tha t its role is tha t of a whole -body e ne rgy home osta sis re gula tor.
Hormone s tha t re gula te whole -body me ta bolism by a c ting on the hypotha la mus ha ve be e n
shown to inte ra c t with AMPK. For insta nc e , ghre lin, a pe ptide hormone re le a se d from the
ga strointe stina l tra c t known to re gula te hunge r, se e ms to a c tiva te AMPK in the a rc ua te
nuc le us of the hypotha la mus stimula ting food inta ke , simila r inte ra c tions ha ve be e n
disc ove re d for a dipokine s suc h a s a dipone c tin a nd le ptin. T he se sa me hormone s c a n
a c tiva te or ina c tiva te it e ve n a t the pe riphe ra l le ve l; le ptin for e xa mple ha s be e n shown to
stimula te AMPK in the ske le ta l musc le c a using it to initia te the oxida tion of fa tty a c ids [5].
E ve n the pe riphe ra l e ffe c ts of AMPK a c tiva tion a re worth me ntioning be c a use the y hint to
the unde rlying c omple xity of a c tion of this e nz yma tic pa thwa y. Its e ffe c ts on ske le ta l
musc le a re possibly the most studie d a nd ha ve e ve n unfortuna te ly a ttra c te d unhe a lthy
a tte ntion from the world of doping in sports a nd a lre a dy sc a nda ls c onc e rning the imprope r
use of pre limina ry a c tiva tors of AMPK use d a s “ e xe rc ise in a pill” . Sc a nda ls
notwithsta nding the re sults of the se studie s point to the fa c t tha t the stimula tion of AMPK
through e xe rc ise or a c tiva ting c ompounds, not only c a use s a n imme dia te inc re a se in fa tty
a c id oxida tion a nd possibly gluc ose upta ke , but c hronic stimula tion e na c ts long te rm e ffe c ts
c a use d by a lte ra tions in the tra nsc ription of se ve ra l e xe rc ise -re sponsive ge ne s a nd
upre gula tion of fa tty a c id oxidiz ing e nz yme s. Furthe rmore re c e nt disc ove rie s point a t the
use of AMPK a c tiva tors for tre a tme nt of c e rta in c a nc e rs a nd for c hronic infla mma tion [6].
Our inc re a sing unde rsta nding of AMPK’s role in whole -body e ne rgy ba la nc e ma ke s it a n
a ttra c tive ta rge t for the tre a tme nt of me ta bolic disorde rs suc h a s obe sity, dia be te s a nd
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c a rdiova sc ula r dise a se s. Although the sc ie ntific c ommunity’s inte re st re ma ins high a nd
c la ims to the disc ove ry of nove l a c tiva tors of the e nz yme , both na tura l a nd synthe tic , a re
fre que ntly a nnounc e d, to da te no suc h c la im ha s yie lde d a signific a nt re sult in te rms of
a c tua l ma rke ta ble drugs a nd ma ny promising a c tiva tors a ppe a r to a c t through indire c t
a c tiva tion.
T he obje c tive of this the sis, be side s the a na lysis of the struc ture of the e nz yme , its ta rge ts
a nd the c urre nt c la sse s of a c tiva tors unde r de ve lopme nt, is to inve stiga te the synthe sis of
nove l modula tors of AMPK.
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2 . AM PK: St ruc t ure and R egulat ion
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2 .1 St ruc t ure
AMPK is a he te rotrime ric e nz yme c omprise d of a n α , β a nd γ subunit. W ith the α subunit
be ing the kina se doma in a nd β a nd γ the re gula tory doma ins (Fig. 1).
Figure 1
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In huma ns, the α subunits a re e nc ode d by two ge ne s PR KAA1/PR KAA2 e nc oding α 1/α 2;
the β subunit by two ge ne s PR KAB 1/PR KAB 2 e nc oding β1/ β2; a nd the γ subunit by thre e
diffe re nt ge ne s PR KAG1/PR KAG2/PR KAG3 e nc oding the thre e γ 1/ γ 2/ γ 3. T hus the re a re
12 possible c ombina tions of the subunits whic h ma ke a s ma ny isoforms of the e nz yme ,
though the ir diffe re nt e xpre ssion in the va rious tissue s is still unde rgoing study [7]. W ha t is
known though is tha t the α 1 subunit e xpre ssion is c ytopla smic a nd though it is ubiquitous
in the body it is te rme d “ he pa tic isoform” be c a use it a c c ounts for 94% of c a ta lytic a c tivity
of the α subunit in the ra t live r. Me a nwhile the α 2 subunit is loc a te d in both the c ytopla sm
a nd nuc le us a nd is mostly e xpre sse d in the ske le ta l musc le a nd re la tive ly le ss in he a rt a nd
live r [8]. Simila rly to the α isoforms the two β isoforms ha ve a slight diffe re ntia l e xpre ssion
in tissue s, with β1 more pre se nt in the live r a nd β2 in ske le ta l musc le . So too the γ
isoforms, though γ 1 is e xpre sse d ubiquitously γ 2 a nd γ 3 a re spe c ific a lly distribute d in he a rt
a nd ske le ta l musc le re spe c tive ly [9].
A more de ta ile d a na lysis of the thre e subunits shows tha t:
1. T he α subunits c onsist of two doma ins, a typic a l kina se doma in (α -KD) a t the N-
te rminus a nd imme dia te ly a fte r it a n a uto inhibitory doma in (α -AID) (Fig.2).
Figure 2
T he la tte r is so c a lle d be c a use the ba c te ria l [α -KD - α -AID] c omple x is le ss a c tive
tha n the α -KD a lone , a nd c urre nt e vide nc e indic a te s tha t the α –AID e na c ts its
inhibitory role whe n AMP is not bound in γ subunit le a ving the e nz yme in a low
a c tivity sta te . T his pa rtia lly ina c tive sta te is e xpla ine d by the fa c t tha t the α -AID
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c omple x, ma de up of thre e short α -he lic e s, binds on the opposite surfa c e of the
c a ta lytic c le ft of the α -KD. More so, upon the binding of AMP a “ re gula tory spine ”
of four hydrophobic re sidue s, una ligne d in the ina c tive c onforma tion, be c ome s
sta c ke d in a lignme nt a llowing the a c tive site to be c orre c tly dispose d for a c tivity.
T he α -AID c onne c ts to the C -te rmina l doma in (α -C T D) through a n α -linke r re gion
tha t wra ps a round the γ subunit a nd is e sse ntia l for the a c tiva tion me c ha nism. T he
α -C T D is a sma ll globula r doma in, ric h in Se rine /T hre onine 50-re sidue se que nc e s
te rme d ST loops, a nd it inte ra c ts with the c ore of AMPK, the β-C T D whic h bridge s
the α a nd γ subunits.
2. T he β subunit c onta ins two highly c onse rve d doma ins: the c e ntra l c a rbohydra te
binding module (β –C B M) a nd the β-C T D. T he β-C B M is a me mbe r of a fa mily of
c a rbohydra te -binding module s (C B M) found in isoa myla se s a nd glyc oge n-
bra nc hing e nz yme s. In AMPK, it a llows a portion of the c e llula r e nz yme to bind to
glyc oge n pa rtic le s, e spe c ia lly in the c a se of isoforms with the β2 subunit whic h
displa ys highe r a ffinity for glyc oge n tha n β1 a nd is e xpre sse d in ske le ta l musc le
whe re glyc oge n c onte nt is ve ry high. E ve n so, only a portion of the e nz yme is
bound to the c a rbohydra te , whic h in the c a se of β1 de pe nds on a n a uto
phosphoryla tion of T hr148 in the β-C B M, by the e nz yme whic h pre ve nts binding.
T he role e na c te d by the β-C B M is still unc e rta in though sinc e glyc oge n syntha se is
a physiologic a l ta rge t of AMPK it ma y be use d to c o-loc a liz e AMPK with its
substra te . Inte re stingly the c le ft be twe e n β-C B M a nd the N-te rminus α -KD is a
binding site for the c la ss of a c tiva tors of AMPK inc luding A-769662 a nd
sa lic yla te s.
3. Fina lly, the γ subunit c onta in a t the ir C -te rmina l e nd four ta nde m re pe a ts known a s
c ysta thione β syntha se (C B S 1 to 4) tha t in most othe r C B S-c onta ining prote ins
a sse mble two by two in struc ture s known a s B a te ma n doma ins whic h c a n bind
liga nds c onta ining a de nosine , suc h a s AMP, ADP, AT P or S-a de nosylme thionine .
T he pe c ulia rity of AMPK is tha t inste a d of the norma l two C B S re pe a ts tha t
a sse mble to ma ke one B a te ma n doma in it ha s four thus forming two B a te ma n