Cyclic dinucleotides (CDNs) are second messengers that bind to the stimulator of interferon genes (STING) and trigger the expression of type I interferons and proinflammatory cytokines. Here we evaluate the activity of 3′,3′-c-di(2′F,2′dAMP) and its phosphorothioate analogues against five STING allelic forms in reporter-cell-based assays and rationalize our findings with X-ray crystallography and quantum mechanics/molecular mechanics calculations. We show that the presence of fluorine in the 2′ position of 3′,3′-c-di(2′F,2′dAMP) improves its activity not only against the wild type (WT) but also against REF and Q STING. Additionally, we describe the synthesis of the acyloxymethyl and isopropyloxycarbonyl phosphoester prodrugs of CDNs. Masking the negative charges of the CDNs results in an up to a 1000-fold improvement of the activities of the prodrugs relative to those of their parent CDNs. Finally, the uptake and intracellular cleavage of pivaloyloxymethyl prodrugs to the parent CDN is rapid, reaching a peak intracellular concentration within 2 h.
The cGAS-STING (cyclic GMP-AMP synthase−stimulator of interferon genes) pathway plays a crucial role in inducing an antiviral and antitumor immune response. We studied the effects of synthetic STING agonists on several immune populations and related cytokine production. In comparison with the toll-like receptor 7 (TLR7) agonist, STING agonists induced secretion of a broader proinflammatory cytokine spectrum. Unlike the TLR7 agonist, the structurally diverse STING agonists partially depleted B and NK cells and completely depleted CD14+ monocytes via induction of apoptosis. The TANKbinding kinase 1 inhibitor efficiently prevented interferon alpha (IFNα) secretion and cell depletion, suggesting their possible dependence on the cGAS-STING pathway activation. Finally, IFNα, tumor necrosis factor alpha, interleukin 6, and interleukin 1 beta secretion and CD14+ monocyte apoptosis were primary responses to STING agonists, whereas IFNγ was secreted secondarily. These findings bring new insights into the cGAS-STING pathway immunomodulation that is of future therapeutic importance.
9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and its cyclic form were selected for further evaluation as potential drug candidates against poxvirus infections. To increase bioavailability of these compounds, synthesis of their structurally diverse ester prodrugs was carried out: alkoxyalkyl (hexadecyloxypropyl, octadecyloxyethyl, hexadecyloxyethyl), pivaloyloxymethyl (POM), 2,2,2-trifluoroethyl, butylsalicylyl, and prodrugs based on peptidomimetics. Most HPMPDAP prodrugs were synthesized in the form of monoesters as well as the corresponding cyclic phosphonate esters. The activity was evaluated not only against vaccinia virus but also against different herpes viruses. The most potent and active prodrugs against vaccinia virus were the alkoxyalkyl ester derivatives of HPMPDAP, with 50% effective concentrations 400-600-fold lower than those of the parent compound. Prodrugs based on peptidomimetics, the 2,2,2-trifluoroethyl, the POM, and the butylsalicylyl derivatives, were able to inhibit vaccinia virus replication at 50% effective concentrations that were equivalent or ∼10-fold lower than those observed for the parent compounds.
While noncanonic xanthine nucleotides XMP/dXMP play an important role in balancing and maintaining intracellular purine nucleotide pool as well as in potential mutagenesis, surprisingly, acyclic nucleoside phosphonates bearing a xanthine nucleobase have not been studied so far for their antiviral properties. Herein, we report the synthesis of a series of xanthine-based acyclic nucleoside phosphonates and evaluation of their activity against a wide range of DNA and RNA viruses. Two acyclic nucleoside phosphonates within the series, namely 9-[2-(phosphonomethoxy)ethyl]xanthine (PMEX) and 9-[3-hydroxy-2-(phosphonomethoxy)propyl]xanthine (HPMPX), were shown to possess activity against several human herpesviruses. The most potent compound was PMEX, a xanthine analogue of adefovir (PMEA). PMEX exhibited a single digit µM activity against VZV (EC50 = 2.6 µM, TK+ Oka strain) and HCMV (EC50 = 8.5 µM, Davis strain), while its hexadecyloxypropyl monoester derivative was active against HSV-1 and HSV-2 (EC50 values between 1.8 and 4.0 µM). In contrast to acyclovir, PMEX remained active against the TK– VZV 07–1 strain with EC50 = 4.58 µM. PMEX was suggested to act as an inhibitor of viral DNA polymerase and represents the first reported xanthine-based acyclic nucleoside phosphonate with potent antiviral properties.
9-(2-Phosphonomethoxyethyl)-2,6-diamino-[8-3 H]purine (4), 9-(2-phosphonomethoxyethyl)-[8-3 H]guanine (6) and (R)-9-(2-phosphonomethoxypropyl)-[8-3 H]adenine (11) with specific activities of 10.9, 7.9 and 16 Ci/mmol, respectively, were prepared by a catalytic dehalogenation of the corresponding 8-bromo derivatives 1, 2 and 9. The rate of the exchange of the tritium label on C-8 of the purine ring in title compounds with the hydrogen of water under physiological pH at 20°C was studied using 3 H NMR. The loss of 3 H-label attained 7% in [8-3 H]tenofovir (11), 10% in [8-3 H]PMEDAP (4) and 12% in [8-3 H]PMEG (6) after the period of 3 weeks. Storage at a temperature of -196°C in liquid nitrogen ensured a better than 97% radiochemical purity of the prepared labeled compounds even after a six-month period.
Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF) are key virulence factors with adenylate cyclase (AC) activity that substantially contribute to the pathogenesis of whooping cough and anthrax, respectively. There is an urgent need to develop potent and selective inhibitors of bacterial ACs with prospects for development of potential antibacterial therapeutics and to study their molecular interactions with the target enzymes. Novel fluorescent 5-chloroanthraniloyl-substituted acyclic nucleoside phosphonates (Cl-ANT-ANPs) were designed and synthesized in the form of their diphosphates (Cl-ANT-ANPpp) as competitive ACT and EF inhibitors with submicromolar potency (IC50 values 11 – 622 nM). Fluorescence experiments indicated that Cl-ANT-ANPpp analogues bind to the ACT active site and docking studies suggested that the Cl-ANT group interacts with Phe306 and Leu60. Interestingly, the increase of direct fluorescence with Cl-ANT-ANPpp having an ester linker was strictly CaM-dependent, whereas Cl-ANT-ANPpp analogues with an amide linker, upon binding to ACT, increased the fluorescence even in the absence of CaM. Such a dependence of binding on structural modification could be exploited in the future design of potent inhibitors of bacterial ACs. Furthermore, one Cl-ANT-ANP in the form of a bisamidate prodrug was able to inhibit B. pertussis ACT activity in macrophage cells with IC50 = 12 µM.
Chronic
hepatitis B (CHB) remains a major public health problem
worldwide, with limited treatment options, but inducing an antiviral
response by innate immunity activation may provide a therapeutic alternative.
We assessed the cytokine-mediated anti-hepatitis B virus (HBV) potential
for stimulating the cyclic GMP–AMP synthase–stimulator
of interferon genes (STING) pathway using STING agonists in primary
human hepatocytes (PHH) and nonparenchymal liver cells (NPCs). The
natural STING agonist, 2′,3′-cyclic GMP–AMP,
the synthetic analogue 3′,3′-c-di(2′F,2′dAMP),
and its bis(pivaloyloxymethyl) prodrug had strong indirect cytokine-mediated
anti-HBV effects in PHH regardless of HBV genotype. Furthermore, STING
agonists induced anti-HBV cytokine secretion in vitro, in both human and mouse NPCs, and triggered hepatic T cell activation.
Cytokine secretion and lymphocyte activation were equally stimulated
in NPCs isolated from control and HBV-persistent mice. Therefore,
STING agonists modulate immune activation regardless of HBV persistence,
paving the way toward a CHB therapy.
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