6 Aptamers for Targeted Therapy
137
Tagx-0004 was screened from a DNA library containing artificial hydrophobic
base 7-(2-thiophene) imidazole [4, 5-b] pyridine (Ds). DNA aptamer containing Ds
has a unique 3D structure since the Ds bases cannot be complementary. In addition,
the high hydrophobicity of Ds bases may contribute to significantly improve the
binding affinity between the aptamers and their target proteins. The results prove
that the affinity of TAGX-0004 (K D = ∼2.2 ± 0.9 nm) was 16 times higher than
that of ARC1779 (K D = 35.5–1.5 nm). It is more remarkable that TAGX-0004 had a
superior inhibitory effect on vWF which was comparable to that of caplacizumab (a
TTP therapeutic drug approved by FDA in 2019). Shahid M. Nimjee et al. obtained
30 nt-modified (2
-OMe, 2
-F) DNA aptamer DTRI-031 (K D = ∼1.6 nM) and
designed detoxifying oligonucleotide (AO) to rapidly reverse its antiplatelet aggregation activity. DTRI-031 was able to prevent platelet-rich thrombosis in the mice
carotid artery injury model, then complementary AO was administrated to reverse
the activity of DTRI-031 rapidly (<5 min) in mice great saphenous vein hemorrhage
model. In addition, toxicological studies in mice showed that the DTRI-031 and AO
were well tolerated which provided a novel reversible aptamer/OA system for the
antiplatelet agent.
6.3.4 Aptamers Targeting Thrombin
Thrombin is a serine protease produced by the prothrombin hydrolysis which is
formed by the complex of coagulation factors such as FXa, FVa, and Ca
2+ . Thrombin
catalyzes coagulation-related reactions to amplify the coagulation pathway, including
activating thrombomodulin, promoting platelet activation and aggregation and other
coagulation-related events [70], which ultimately promotes thrombosis and reduces
blood loss [71]. Thrombin has two active sites, fibrinogen recognition exosite
(Thrombin Exosite I) and heparin binding exosite (Thrombin Exosite II) [72]
(Fig. 6.2). Thrombin Exosite I is positively charged and has a hydrophobic region [73]
that binds fibrinogen, clotting factor V, VIII, XI, XIII, thromboregulatory protein,
heparin cofactor II, and par-1 platelet receptors [74–76]. Thrombin Exosite II binds
thrombin factors V and VIII, heparin, thrombin factor F2, platelet GPIb-IX-V, and
Beta2 glycoprotein I (beta2GpI).
At present, aptamers with thrombin activity inhibitory effect are mostly designed
for targeting exosite. In 1992, Bock et al. [38] obtained a 15 nt unmodified DNA
aptamer for human thrombin named HD1, which can form stable G-quadruplex structure. HD1 binds to Thrombin Exosite I with high affinity and specificity, producing a
dose-dependent inhibition of thrombin. In order to extend the half-life [78], HD1 was
optimized and reconstructed by researchers, and four thymidine pyrimidines were
replaced with 4-thiodeoxyureic acid (4dU) in HD1, which surprisingly enhanced the
inhibition capacity for thrombin activity [79]. An improved RNA aptamer R9D-14
bound to Thrombin Exosite I was identified, which prolongs the TCT aPTT and
PT significantly, and can be reversed by its complementary sequence quickly and
permanently in vitro [80].
137
Tagx-0004 was screened from a DNA library containing artificial hydrophobic
base 7-(2-thiophene) imidazole [4, 5-b] pyridine (Ds). DNA aptamer containing Ds
has a unique 3D structure since the Ds bases cannot be complementary. In addition,
the high hydrophobicity of Ds bases may contribute to significantly improve the
binding affinity between the aptamers and their target proteins. The results prove
that the affinity of TAGX-0004 (K D = ∼2.2 ± 0.9 nm) was 16 times higher than
that of ARC1779 (K D = 35.5–1.5 nm). It is more remarkable that TAGX-0004 had a
superior inhibitory effect on vWF which was comparable to that of caplacizumab (a
TTP therapeutic drug approved by FDA in 2019). Shahid M. Nimjee et al. obtained
30 nt-modified (2
-OMe, 2
-F) DNA aptamer DTRI-031 (K D = ∼1.6 nM) and
designed detoxifying oligonucleotide (AO) to rapidly reverse its antiplatelet aggregation activity. DTRI-031 was able to prevent platelet-rich thrombosis in the mice
carotid artery injury model, then complementary AO was administrated to reverse
the activity of DTRI-031 rapidly (<5 min) in mice great saphenous vein hemorrhage
model. In addition, toxicological studies in mice showed that the DTRI-031 and AO
were well tolerated which provided a novel reversible aptamer/OA system for the
antiplatelet agent.
6.3.4 Aptamers Targeting Thrombin
Thrombin is a serine protease produced by the prothrombin hydrolysis which is
formed by the complex of coagulation factors such as FXa, FVa, and Ca
2+ . Thrombin
catalyzes coagulation-related reactions to amplify the coagulation pathway, including
activating thrombomodulin, promoting platelet activation and aggregation and other
coagulation-related events [70], which ultimately promotes thrombosis and reduces
blood loss [71]. Thrombin has two active sites, fibrinogen recognition exosite
(Thrombin Exosite I) and heparin binding exosite (Thrombin Exosite II) [72]
(Fig. 6.2). Thrombin Exosite I is positively charged and has a hydrophobic region [73]
that binds fibrinogen, clotting factor V, VIII, XI, XIII, thromboregulatory protein,
heparin cofactor II, and par-1 platelet receptors [74–76]. Thrombin Exosite II binds
thrombin factors V and VIII, heparin, thrombin factor F2, platelet GPIb-IX-V, and
Beta2 glycoprotein I (beta2GpI).
At present, aptamers with thrombin activity inhibitory effect are mostly designed
for targeting exosite. In 1992, Bock et al. [38] obtained a 15 nt unmodified DNA
aptamer for human thrombin named HD1, which can form stable G-quadruplex structure. HD1 binds to Thrombin Exosite I with high affinity and specificity, producing a
dose-dependent inhibition of thrombin. In order to extend the half-life [78], HD1 was
optimized and reconstructed by researchers, and four thymidine pyrimidines were
replaced with 4-thiodeoxyureic acid (4dU) in HD1, which surprisingly enhanced the
inhibition capacity for thrombin activity [79]. An improved RNA aptamer R9D-14
bound to Thrombin Exosite I was identified, which prolongs the TCT aPTT and
PT significantly, and can be reversed by its complementary sequence quickly and
permanently in vitro [80].