Hepatitis C NS5B polymerase inhibitors: functional equivalents for the benzothiadiazine moiety

A series of quinoline derivatives was synthesized as potential bioisosteric replacements for the benzothiadiazine moiety of earlier Hepatitis C NS5B polymerase inhibitors. Several of these compounds exhibited potent activity in enzymatic and replicon assays.     

The UL97 protein kinase of human cytomegalovirus and homologues in other herpesviruses: impact on virus and host

The human herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6A (HHV-6A), HHV-6B, HHV-7 and HHV-8, establish persistent infections with possible recurrence during immunosuppression. HCMV replication is inhibited by the nucleoside analogue ganciclovir (GCV), the compound of choice for the treatment of HCMV diseases and preemptive treatment of infections. The viral UL97 protein (pUL97) which shares homologies with protein kinases and bacterial phosphotransferases is able to monophosphorylate GCV. Homologues of pUL97 are found in HSV (UL13), VZV (ORF47), EBV (BGLF4), HHV-6 (U69), HHV-8 (ORF36) as well as in murine CMV (M97) or rat CMV (R97). Several indolocarbazoles have been reported to be specific inhibitors of pUL97. The protein is important for efficient replication of the virus. Autophosphorylation of pUL97 was observed using different experimental systems. Most recently, it has been shown that pUL97 interacts with the DNA polymerase processivity factor pUL44. Indolocarbazole protein kinase inhibitors are promising lead compounds for the development of more specific inhibitors of HCMV.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of unsymmetrical 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives

Substituted 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives were investigated as inhibitors of genotype 1 HCV polymerase. Structure-activity relationship patterns for this class of compounds are discussed. It was found that the saturated alkane dialkyl units provided the most active analogs.     

Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.     

Hepatitis C NS5B polymerase inhibitors: 4,4-Dialkyl-1-hydroxy-3-oxo-3,4-dihydronaphthalene-3-yl benzothiadiazine derivatives

4,4-Dialkyl-1-hydroxy-3-oxo-3.4-dihydronaphthalene-3-yl benzothiadiazine derivatives were synthesized and evaluated as inhibitors of genotypes 1a and 1b HCV NS5B polymerase. A number of these compounds exhibited potent activity against genotypes 1a and 1b HCV polymerase in both enzymatic and cell culture activities. A representative compound also showed favorable pharmacokinetics in the rat.     

Naphthalene carboxamides as inhibitors of human cytomegalovirus DNA polymerase

ortho-Hydroxynaphthalene carboxamides have been identified as inhibitors of HCMV DNA polymerase. SAR investigations have demonstrated that both the amide and hydroxy functionalities are required for activity. Substitution on the naphthalene ring has led to inhibitors with submicromolar IC50s against HCMV polymerase. These compounds have been found to be >100-fold selective for inhibition of HCMV polymerase versus human alpha polymerase and display antiviral activity in a cell-based plaque reduction assay.     

Synthesis and anti-HCMV activity of 1-acyl-beta-lactams and 1-acylazetidines derived from phenylalanine

Different Phe-derived 1-acyl-beta-lactams, analogous to a series of 2-azetidinones acting as HCMV serine protease inhibitors, were synthesized. Some of these compounds were modest inhibitors of the HCMV replication. Interestingly, removal of the carbonyl group of the beta-lactam ring, most likely acting as the serine trap, resulted in an azetidine derivative with anti-HCMV activity comparable to that of the reference compound ganciclovir.     

Novel bicyclic furanopyrimidines with dual anti-VZV and -HCMV activity

Several novel bicyclic furanopyrimidine deoxy nucleosides have been designed, prepared and evaluated as anti-Varicella Zoster Virus agents. The compounds have long ether side chains. Uniquely amongst compounds of this family to date the present agents show dual anti- (VZV) and human cytomegalovirus (HCMV) activity. The lead compounds inhibit VZV at 10 nM and HCMV at 5 μM.     

Polyamine metabolism in MRC5 cells infected with different herpesviruses.

Both methyglyoxal bis(guanylhydrazone), an inhibitor of S-adenosyl-L-methionine decarboxylase (EC.4.1.1.50) and DL-α-methylornithine, an inhibitor of ornithine decarboxylase (EC.4.1.1.17), are shown to be potent inhibitors of the replication of human cytomegalovirus (HCMV) in MRC-5 cells. These compounds, both inhibitors of polyamine biosynthesis, do not affect the replication of either herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). This difference in antiviral effect is shown to be related to the stimulation of spermidine and spermine synthesis in host cells following HCMV infection and the inhibition of polyamine metabolism in HSV-1 or HSV-2-infected cells. Inhibition of HCMV replication by the inhibitors of polyamine biosynthesis is accompanied by a marked decrease in the formation of intranuclear, DNA-containing inclusions characteristic of HCMV infection. These results suggest significant differences in the mechanisms of replication of different herpesviruses.     

Novel non-nucleoside human cytomegalovirus inhibitors based upon TSAO nucleoside derivatives: structure-activity relationships

TSAO derivatives are a unique group of potent and highly specific inhibitors of HIV-1 replication. We have recently reported 4'-ureido TSAO derivatives that are devoid of anti-HIV-1 activity, but inhibit human cytomegalovirus with an activity comparable to that of Ganciclovir. We herein report the synthesis and biological evaluation of novel 4'-ureido TSAO derivatives in order to evaluate the structural features required for anti-HCMV activity. Interestingly, these studies revealed that the compounds may inhibit HCMV at the DNA polymerase step via a non-nucleoside mechanism.     

Synthesis and characterization of novel quinazoline type inhibitors for mutant and wild-type EGFR and RICK kinases

The development of selective protein kinase inhibitors has become an important area of drug discovery for the treatment of different diseases. We report the synthesis and characterization of a series of novel quinazoline derivatives against three therapeutically important and pharmacologically related kinases: 1) epidermal growth factor receptor (EGFR; wild type and mutant) in the field of cancer, 2) receptor-interacting caspase-like apoptosis-regulatory kinase (RICK) in the field of inflammation, and 3) pUL97 of human cytomegalovirus (HCMV). For reference purpose we have synthesized the four clinically relevant quinazolines, including the lead compounds, which we previously identified for RICK and pUL97. A total of 52 quinazoline derivatives were synthesized and tested on the basis of these leads to specifically target the hydrophobic pocket of the ATP-binding site. Selected compounds were tested on wild-type and mutant forms of EGFR, RICK, and pUL97 kinases; their logP and logS values for assessing suitability as drugs were calculated and hit or lead compounds identified.     

Synthesis and Characterization of Novel Quinazoline Type Inhibitors for Mutant and Wild-Type EGFR and RICK Kinases

The development of selective protein kinase inhibitors has become an important area of drug discovery for the treatment of different diseases. We report the synthesis and characterization of a series of novel quinazoline derivatives against three therapeutically important and pharmacologically related kinases: 1) epidermal growth factor receptor (EGFR; wild type and mutant) in the field of cancer, 2) receptor-interacting caspase-like apoptosis-regulatory kinase (RICK) in the field of inflammation, and 3) pUL97 of human cytomegalovirus (HCMV). For reference purpose we have synthesized the four clinically relevant quinazolines, including the lead compounds, which we previously identified for RICK and pUL97. A total of 52 quinazoline derivatives were synthesized and tested on the basis of these leads to specifically target the hydrophobic pocket of the ATP-binding site. Selected compounds were tested on wild-type and mutant forms of EGFR, RICK, and pUL97 kinases; their logP and logS values for assessing suitability as drugs were calculated and hit or lead compounds identified.     

Imidazothiadiazine dioxides: synthesis and antiviral activity.

A new series of imidazothiadiazine dioxides, including the first acyclonucleosides derived from this heterocycle moiety, has been synthesized. A wide-spectrum antiviral screening was performed. Some of the N-1 benzyl imidazothiadiazines and the new acyclonucleosides showed interesting anti-CMV or anti-HIV activity. These structures could be considered as new lead compounds for antiviral drug research.     

Good oral absorption prediction on non-nucleoside benzothiadiazine dioxide human cytomegalovirus inhibitors using combined chromatographic and neuronal network techniques

The current drugs available against human cytomegalovirus (HCMV) suffer from a number of shortcomings such as toxic side effect, poor bioavailability and/or risk for emergence of drug-resistance virus strains. Due to these limitations, the development of new drugs against HCMV is of great interest. Taking into account the therapeutic potential of benzothiadiazines dioxides (BTD) derivatives, it is most important to know their oral bioavailability because all the current clinical drugs are poorly absorbed. In this work, the utility of CODES neural networks and biopartitioning micellar chromatography (BMC) in predicting pharmacokinetic properties has been used to estimate the oral absorption of BTD derivatives and their efficacy has been verified. The results indicate higher values for BTD derivatives than the currently licensed anti-viral agents.     

Tripeptidic BACE1 inhibitors devised by in-silico conformational structure-based design

Previously reported pentapeptidic BACE1 inhibitors, designed using a substrate-based approach, were used as lead compounds for the further design of non-peptidic BACE1 inhibitors. Although these peptidic and non-peptidic inhibitors, with a hydroxymethylcarbonyl isostere as a substrate transition-state mimic, exhibited potent BACE1 inhibitory activities, their molecular-sizes appeared a little too big (molecular weight of >600daltons) for developing practical anti-Alzheimer's disease drugs. To develop lower weight BACE1 inhibitors, a series of tripeptidic BACE1 inhibitors were devised using a design approach based on the conformation of a virtual inhibitor bound to the BACE1 active site, also called 'in-silico conformational structure-based design'. Although these tripeptidic BACE1 inhibitors contained some natural amino acid residues, they are expected to be useful as lead compounds for developing the next generation BACE1 inhibitors, due to their low molecular size and unique structural features compared with previously reported inhibitors.     

Role of a mutation in human cytomegalovirus gene UL104 in resistance to benzimidazole ribonucleosides

The benzimidazole D-ribonucleosides TCRB and BDCRB are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Two HCMV strains resistant to these compounds were selected and had resistance mutations in genes UL89 and UL56. Proteins encoded by these two genes are the two subunits of the HCMV "terminase" and are necessary for cleavage and packaging of viral genomic DNA, a process inhibited by TCRB and BDCRB. We now report that both strains also have a previously unidentified mutation in UL104, the HCMV portal protein. This mutation, which results in L21F substitution, was introduced into the genome of wild-type HCMV by utilizing a recently cloned genome of HCMV as a bacterial artificial chromosome. The virus with this mutation alone was not resistant to BDCRB, suggesting that this site is not involved in binding benzimidazole nucleosides. As in previous proposals for mutations in UL104 of murine cytomegalovirus and HCMV strains resistant to BAY 38-4766, we hypothesize that this mutation could compensate for conformational changes in mutant UL89 and UL56 proteins, since the HCMV terminase is likely to interact with the portal protein during cleavage and packaging of genomic DNA.     

Mechanistic study of HCV polymerase inhibitors at individual steps of the polymerization reaction

Little is known about the mechanism of HCV polymerase-catalyzed nucleotide incorporation and the individual steps employed by this enzyme during a catalytic cycle. In this paper, we applied various biochemical tools and examined the mechanism of polymerase catalysis. We found that formation of a productive RNA-enzyme complex is the slowest step followed by RNA dissociation and initiation of primer strand synthesis. Various groups have reported several classes of small molecule inhibitors of hepatitis C virus NS5B polymerase; however, the mechanism of inhibition for many of these inhibitors is not clear. We undertook a series of detailed mechanistic studies to characterize the mechanisms of inhibition for these HCV polymerase inhibitors. We found that the diketoacid derivatives competitively bind to the elongation NTP pocket in the active site and inhibit both the initiation and elongation steps of polymerization. While both benzimidazoles and benzothiadiazines are noncompetitive with respect to the active site elongation NTP pocket, benzothiadiazine compounds competitively bind to the initiation pocket in the active site and inhibit only the initiation step of de novo RNA polymerization. The benzimidazoles bind to the thumb allosteric pocket and inhibit the conformational changes during RNA synthesis. We also observed a cross interaction between the thumb allosteric pocket and the initiation pocket using inhibitor-inhibitor cross competition studies. This information will be very important in designing combination therapies using two small molecule drugs to treat hepatitis C virus.     

Identification of inhibitors for a virally encoded protein kinase by 2 different screening systems: in vitro kinase assay and in-cell activity assay

The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates. About 20% of all compounds tested inhibited pUL97 kinase activity by more than 50% at a concentration of 10 microM. These hits belonged to various structural classes. To elucidate their potential to inhibit pUL97 in a cellular context, all compounds of the library were also tested in a cell-based activity assay. For this reason, a HEK293 cell line was established that ectopically expressed pUL97. When these cells were incubated with ganciclovir (GCV), pUL97 phosphorylated GCV to its monophosphate, which subsequently became phosphorylated to cytotoxic metabolites by cellular enzymes. Thereby, pUL97 converted cells into a GCV-sensitive phenotype. Inhibition of the pUL97 kinase activity resulted in protection of the cells against the cytotoxic effects of GCV. In total, 199 compounds of the library were cellular active at nontoxic concentrations, and 93 of them inhibited pUL97 in the in vitro kinase assay. Among these, promising inhibitors of HCMV replication were identified. The 2-fold screening system described here should facilitate the development of pUL97 inhibitors into potent drug candidates.     

Synthesis of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine-5-carboxamides with broad-spectrum human herpesvirus polymerase inhibition

A versatile synthesis of 4-oxo-4,7-dihydrofuro[2,3-b]pyridine-5-carboxylate esters has been developed which has lead to the identification of a new series of non-nucleoside inhibitors of human herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.     

Recent advances in drug discovery of benzothiadiazine and related analogs as HCV NS5B polymerase inhibitors

Hepatitis C virus (HCV) is a major health burden, with an estimated 170 million chronically infected individuals worldwide, and a leading cause of liver transplantation. Patients are at increased risk of developing liver cirrhosis, hepatocellular carcinoma and even liver failure. In the past two decades, several approaches have been adopted to inhibit non-structural viral proteins. The RNA-dependent RNA polymerase (NS5B) of HCV is one of the attractive validated targets for development of new drugs to block HCV infection. In this review, we report the recent progress made towards identifying and developing benzothiadiazines as HCV NS5B polymerase inhibitors. The substituted benzothiadiazine class was identified by HTS in 2002 as an NS5B inhibitor. Further optimization and modification of the core has improved the potency and pharmacokinetic properties of substituted benzothiadiazines. Research on palm site-binding benzothiadiazine analogs and related derivatives and analogs is discussed in this article.