Boping Liu

An adenosine nucleoside inhibitor of dengue virus

Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.

Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome.

Combination of α-glucosidase inhibitor and ribavirin for the treatment of dengue virus infection in vitro and in vivo

Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection.

Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleoside

ABSTRACT We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxy-methyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC50) and cytotoxic concentration (CC50) values of 0.7 μM and >100 μM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compounds antiviral activity in cell culture. Studies of rats showed that 14C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis.

Inhibition of Dengue Virus by an Ester Prodrug of an Adenosine Analog

ABSTRACT Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen that infects humans. Neither a vaccine nor an antiviral therapy is currently available for DENV. Here, we report an adenosine nucleoside prodrug that potently inhibits DENV replication both in cell culture and in a DENV mouse model. NITD449 (2′-C-acetylene-7-deaza-7-carbamoyladenosine) was initially identified as a parental compound that inhibits all four serotypes of DENV with low cytotoxicity. However, in vivo pharmacokinetic studies indicated that NITD449 had a low level of exposure in plasma when dosed orally. To increase the oral bioavailability, we covalently linked isobutyric acids to the 3′- and 5′-hydroxyl groups of ribose via ester linkage to NITD449, leading to the prodrug NITD203 (3′,5′-O-diisobutyryl-2′-C-acetylene-7-deaza-7-carbamoyl-adenosin). Pharmacokinetic analysis showed that upon oral dosing of the prodrug, NITD203 was readily converted to NITD449, resulting in improved exposure of the parental compound in plasma in both mouse and rat. In DENV-infected AG129 mice, oral dosing of the prodrug at 25 mg/kg of body weight reduced peak viremia by 30-fold. Antiviral spectrum analysis showed that NITD203 inhibited various flaviviruses (DENV, yellow fever virus, and West Nile virus) and hepatitis C virus but not Chikungunya virus (an alphavirus). Mode-of-action analysis, using a luciferase-reporting replicon, indicated that NITD203 inhibited DENV RNA synthesis. Although NITD203 exhibited potent in vitro and in vivo efficacies, the compound could not reach a satisfactory no-observable-adverse-effect level (NOAEL) in a 2-week in vivo toxicity study. Nevertheless, our results demonstrate that a prodrug approach using a nucleoside analog could potentially be developed for flavivirus antiviral therapy.

Exploring Aigialomycin D and Its Analogues as Protein Kinase Inhibitors for Cancer Targets

The natural product aigialomycin D (1) is a member of the resorcylic acid lactone (RAL) family possessing protein kinase inhibitory activities. This paper describes the synthesis of aigialomycin D and a series of its analogues and their activity for the inhibition of protein kinases related to cancer pathways. A preliminary study of these compounds in the inhibition of CDK2/cyclin A kinase has found that aigialomycin D and analogues 11 and 23 are moderate CDK2/cyclin A inhibitors with IC50 values of ca. 20 μM. Kinase profiling of aigialomycin D against a panel of kinases has led to the identification of MNK2 as a promising target (IC50 = 0.45 μM), and preliminary structure-activity relationship studies have been carried out to identify the essential functional groups for activity.

NMR structural characterization of the N-terminal active domain of the gyrase B subunit from Pseudomonas aeruginosa and its complex with an inhibitor.

The N‐terminal ATP binding domain of the DNA gyrase B subunit is a validated drug target for antibacterial drug discovery. Structural information for this domain (pGyrB) fromPseudomonas aeruginosa is still missing. In this study, the interaction between pGyrB and abis‐pyridylurea inhibitor was characterized using several biophysical methods. We further carried out structural analysis of pGyrB using NMR spectroscopy. The secondary structures of free and inhibitor bound pGyrB were obtained based on backbone chemical shift assignment. Chemical shift perturbation and NOE experiments demonstrated that the inhibitor binds to the ATP binding pocket. The results of this study will be helpful for drug development targetingP. aeruginosa.

Probing the binding mechanism of Mnk inhibitors by docking and molecular dynamics simulations.

Mitogen-activated protein kinases-interacting kinase 1 and 2 (Mnk1/2) activate the oncogene eukaryotic initiation factor 4E (eIF4E) by phosphorylation. High level of phosphorylated eIF4E is associated with various types of cancers. Inhibition of Mnk prevents eIF4E phosphorylation, making them potential therapeutic targets for cancer. Recently, we have designed and synthesized a series of novel imidazopyridine and imidazopyrazine derivatives that inhibit Mnk1/2 kinases with a potency in the nanomolar to micromolar range. In the current work we model the inhibition of Mnk kinase activity by these inhibitors using various computational approaches. Combining homology modeling, docking, molecular dynamics simulations, and free energy calculations, we find that all compounds bind similarly to the active sites of both kinases with their imidazopyridine and imidazopyrazine cores anchored to the hinge regions of the kinases through hydrogen bonds. In addition, hydrogen bond interactions between the inhibitors and the catalytic Lys78 (Mnk1), Lys113 (Mnk2) and Ser131 (Mnk1), Ser166 (Mnk2) appear to be important for the potency and stability of the bound conformations of the inhibitors. The computed binding free energies (ΔGPred) of these inhibitors are in accord with experimental bioactivity data (pIC50) with correlation coefficients (r(2)) of 0.70 and 0.68 for Mnk1 and Mnk2 respectively. van der Waals energies and entropic effects appear to dominate the binding free energy (ΔGPred) for each Mnk-inhibitor complex studied. The models suggest that the activities of these small molecule inhibitors arise from interactions with multiple residues in the active sites, particularly with the hydrophobic residues.

Escherichia coli topoisomerase IV E subunit and an inhibitor binding mode revealed by NMR spectroscopy

Bacterial topoisomerases are attractive antibacterial drug targets because of their importance in bacterial growth and low homology with other human topoisomerases. Structure-based drug design has been a proven approach of efficiently developing new antibiotics against these targets. Past studies have focused on developing lead compounds against the ATP binding pockets of both DNA gyrase and topoisomerase IV. A detailed understanding of the interactions between ligand and target in a solution state will provide valuable information for further developing drugs against topoisomerase IV targets. Here we describe a detailed characterization of a known potent inhibitor containing a 9H-pyrimido[4,5-b]indole scaffold against the N-terminal domain of the topoisomerase IV E subunit from Escherichia coli (eParE). Using a series of biophysical and biochemical experiments, it has been demonstrated that this inhibitor forms a tight complex with eParE. NMR studies revealed the exact protein residues responsible for inhibitor binding. Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the α4 and residues close to the loop of β1-α2 and residues in the loop of β3-β4 might improve the inhibitor potency.

Structure–Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells

Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.