Functionalized congeners of P2Y1 receptor antagonists: 2-alkynyl (N)-methanocarba 2'-deoxyadenosine 3',5'-bisphosphate analogues and conjugation to a polyamidoamine (PAMAM) dendrimer carrier.
The P2Y(1) receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y(1) receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y(1) receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of an intermediate amide group revealed high affinity of carboxylic congener 8 (K(i) 23 nM) and extended amine congener 15 (K(i) 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended epsilon-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y(1) receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y(1) receptor modeling and ligand docking. Attempted P2Y(1) antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM dendrimer to produce a multivalent conjugate exhibiting a desired biological effect, i.e., antithrombotic action.
de Castro
Sonia
S
Molecular Recognition Section and Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, 20892-0810, USA.
Maruoka
Hiroshi
H
Hong
Kunlun
K
Kilbey
S Michael
SM
Costanzi
Stefano
S
Hechler
Béatrice
B
Brown
Garth G
GG
Gachet
Christian
C
Harden
T Kendall
TK
Jacobson
Kenneth A
KA
D050091:Dendrimers / Q000737:chemistry*; D003838:Deoxyadenine Nucleotides / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D011073:Polyamines / Q000737:chemistry*; D058919:Purinergic P2 Receptor Antagonists; D013237:Stereoisomerism
D050091:Dendrimers; D003838:Deoxyadenine Nucleotides; C531249:Poly(amidoamine); D011073:Polyamines; D058919:Purinergic P2 Receptor Antagonists