Ivan Yanachkov

Synergistic Inhibition of Both P2Y1 and P2Y12 Adenosine Diphosphate Receptors As Novel Approach to Rapidly Attenuate Platelet-Mediated Thrombosis

Objective— Unlike currently approved adenosine diphosphate receptor antagonists, the new diadenosine tetraphosphate derivative GLS-409 targets not only P2Y12 but also the second human platelet adenosine diphosphate receptor P2Y1 and may, therefore, be a promising antiplatelet drug candidate. The current study is the first to investigate the in vivo antithrombotic effects of GLS-409. Approach and Results— We studied (1) the in vivo effects of GLS-409 on agonist-stimulated platelet aggregation in anesthetized rats, (2) the antithrombotic activity of GLS-409 and the associated effect on the bleeding time in a canine model of platelet-mediated coronary artery thrombosis, and (3) the inhibition of agonist-stimulated platelet aggregation by GLS-409 versus selective P2Y1 and P2Y12 inhibition in vitro in samples from healthy human subjects before and 2 hours after aspirin intake. In vivo treatment with GLS-409 significantly inhibited adenosine diphosphate- and collagen-stimulated platelet aggregation in rats. Further, GLS-409 attenuated cyclic flow variation, that is, platelet-mediated thrombosis, in vivo in our canine model of unstable angina. The improvement in coronary patency was accompanied by a nonsignificant 30% increase in bleeding time. Of note, GLS-409 exerted its effects without affecting rat and canine hemodynamics. Finally, in vitro treatment with GLS-409 showed effects similar to that of cangrelor and the combination of cangrelor with the selective P2Y1 inhibitor MRS 2179 on agonist-stimulated platelet aggregation in human platelet-rich plasma and whole blood before and 2 hours after aspirin intake. Conclusions— Synergistic inhibition of both P2Y1 and P2Y12 adenosine diphosphate receptors by GLS-409 immediately attenuates platelet-mediated thrombosis and effectively blocks agonist-stimulated platelet aggregation irrespective of concomitant aspirin therapy.

Modified diadenosine tetraphosphates with dual specificity for P2Y1 and P2Y12 are potent antagonists of ADP-induced platelet activation.

Chang H, Yanachkov IB, Dix EJ, Li YF, Barnard MR, Wright GE, Michelson AD, Frelinger AL 3rd. Modified diadenosine tetraphosphates with dual specificity for P2Y1 and P2Y12 are potent antagonists of ADP‐induced platelet activation. J Thromb Haemost 2012; 10: 2573–80.

A NOVEL THYMIDINE PHOSPHORAMIDITE SYNTHON FOR INCORPORATION OF INTERNUCLEOSIDE PHOSPHATE LINKERS DURING AUTOMATED OLIGODEOXYNUCLEOTIDE SYNTHESIS

A novel thymidine phosphoramidite synthon was synthesized and successfully used for incorporation of primary amino groups, attached through a triethylene glycol linker to the internucleoside phosphates, at desired locations during automated oligodeoxynucleotide synthesis. The synthesized amino-linker bearing oligonucleotides are stable under deprotection conditions and exhibit Watson-Crick base-pairing properties. Covalent labeling of oligonucleotides with carbocyanine near-infrared fluorochromes resulted in 2.5 times higher labeling yields when compared with oligonucleotides containing base-attached aminolinkers. We anticipate that the developed synthetic approach will be useful for nucleotide sequence-specific attachment of single or multiple ligands or reporter molecules.

Antiplatelet Activity, P2Y1 and P2Y12 Inhibition, and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate

Background Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5′,5″″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers. Objective To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma. Methods We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma. Results The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers. Conclusions The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors.

Synthesis of the P1, P2-Methylene Analog of N2- (p-n-Butylphenyl)-2′- deoxyguanosine 5′-Triphosphate: A Non-substrate Inhibitor of DNA Polymerases

Abstract N2- (p-n-Butylphenyl)-2′-deoxyguanosine 5′ - (P1, P2-methylene)- triphosphate (BuPdGMPCH2PP) has been synthesized. Displacement of the mesyloxy group of 5′-MesBuPdG by methanediphosphonate gave 30% of BuPdGMPCH2P, but 68% of 3,5′-cycloBuPdG. Reaction of the nucleoside BuPdG with methanediphosphonate and DCC gave 62% of BuPdGMPCH2PPCH2P, which was hydrolyzed to BuPdGMPCH2P in 77% yield. The title compound was obtained by reacting the imidazolide of BuPdGMPCH2P with orthophosphate. BuPdGMPCH2PP inhibited calf thymus DNA polymerase a with Ki = 9.5 nM, a potency only fivefold weaker than that of BuPdGTP itself.

A Novel Agent Effective against Clostridium difficile Infection

ABSTRACT N2-(3,4-Dichlorobenzyl)-7-(2-[1-morpholinyl]ethyl)guanine (MorE-DCBG, 362E) is a synthetic purine that selectively inhibits the replication-specific DNA polymerase of Clostridium difficile. MorE-DCBG and its analogs strongly inhibited the growth of a wide variety of C. difficile strains. When administered orally in a hamster model of C. difficile-specific colitis, 362E was as effective as oral vancomycin, the current agent of choice for treating severe forms of the human disease.

Synthesis and biochemical study of N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-(alpha,beta-imido)triphosphate (BuPdGMPNHPP): a non-substrate inhibitor of B family DNA polymerases

BuPdGMPNHPP was synthesized and assayed as a non-incorporable inhibitor of B family DNA polymerases. The derivative was synthesized by preparation of the imidophosphorane of BuPdG followed by reaction with orthophosphate using the imidazolide method. BuPdGMPNHPP inhibited human DNA polymerase alpha and T4 DNA polymerase 10 and 3.5-times more potently than BuPdGTP, respectively, and was not a substrate for either enzyme. BuPdGMPNHPP acts as an active site affinity probe that could find use in co-crystallization trials of B family DNA polymerases.

GLS-409, an Antagonist of Both P2Y1 and P2Y12, Potently Inhibits Canine Coronary Artery Thrombosis and Reversibly Inhibits Human Platelet Activation

Dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) P2Y12 receptor antagonist reduces ischemic events in patients with acute coronary syndrome. Previous evidence from our group, obtained in a preclinical model of recurrent platelet-mediated thrombosis, demonstrated that GLS-409, a diadenosine tetraphosphate derivative that inhibits both P2Y1 and P2Y12 ADP receptors, may be a novel and promising antiplatelet drug candidate. However, the salutary antiplatelet effects of GLS-409 were accompanied by a trend toward an unfavorable increase in bleeding. The goals of this study were to: 1) provide proof-of-concept that the efficacy of GLS-409 may be maintained at lower dose(s), not accompanied by an increased propensity to bleeding; and 2) establish the extent and kinetics of the reversibility of human platelet inhibition by the agent. Lower doses of GLS-409 were identified that inhibited in vivo recurrent coronary thrombosis with no increase in bleeding time. Human platelet inhibition by GLS-409 was reversible, with rapid recovery of platelet reactivity to ADP, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. These data support the concept that GLS-409 warrants further, larger-scale investigation as a novel, potential therapy in acute coronary syndromes.

Prodrugs of herpes simplex thymidine kinase inhibitors

Background Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. Methods Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. Results Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N2-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. Conclusions Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo.

Nucleoside carbonyl(di- and triphosphates)

The first examples of nucleoside di- and triphosphates containing the electrophilic and potentially reactive carbonyl group in place of a phosphoanhydride oxygen are reported, using the DNA polymerase inhibitors N2-(4-butylphenyl)-2′-deoxyguanosine 5′-triphosphate (BuPdGTP) and N2-(4-butylphenyl)-2′-deoxyguanosine 5′-diphosphate (BuPdGDP) as platforms. The P2,P3-carbonyltriphosphonate, BuPdGMPPCOP, was obtained by reaction between the phosphoro-N-methylimidazolidate of the monophosphate BuPdGMP and carbonyldiphosphonic acid (PCOP), and it was isolated by preparative reverse phase chromatography. The carbonyldiphosphonate analogue, BuPdGMPCOP, was obtained by displacement of the 5′-mesyl group of the corresponding 5′-mesylnucleoside with carbonyldiphosphonate. While BuPdGMPCOP was stable in aqueous solutions, BuPdGMPPCOP hydrolyzed to BuPdGMP and PCOP with a half-life of 3 hours. Both BuPdGMPPCOP and BuPdGMPCOP were potent, competitive inhibitors of human DNA polymerase α.