Design, synthesis, and lead optimization of piperazinyl-pyrimidine analogues as potent small molecules targeting the viral capping machinery of Chikungunya virus.

The worldwide re-emerge of the Chikungunya virus (CHIKV), the high morbidity associated with it, and the lack of an available vaccine or antiviral treatment make the development of a potent CHIKV-inhibitor highly desirable. Therefore, an extensive lead optimization was performed based on the previously reported CHVB compound 1b and the reported synthesis route was optimized - improving the overall yield in remarkably shorter synthesis and work-up time. Hundred analogues were designed, synthesized, and investigated for their antiviral activity, physiochemistry, and toxicological profile. An extensive structure-activity relationship study (SAR) was performed, which focused mainly on the combination of scaffold changes and revealed the key chemical features for potent anti-CHIKV inhibition. Further, a thorough ADMET investigation of the compounds was carried out: the compounds were screened for their aqueous solubility, lipophilicity, their toxicity in CaCo-2 cells, and possible hERG channel interactions. Additionally, 55 analogues were assessed for their metabolic stability in human liver microsomes (HLMs), leading to a structure-metabolism relationship study (SMR). The compounds showed an excellent safety profile, favourable physicochemical characteristics, and the required metabolic stability. A cross-resistance study confirmed the viral capping machinery (nsP1) to be the viral target of these compounds. This study identified 31b and 34 as potent, safe, and stable lead compounds for further development as selective CHIKV inhibitors. Finally, the collected insight led to a successful scaffold hop (64b) for future antiviral research studies.

Novel Class of Chikungunya Virus Small Molecule Inhibitors That Targets the Viral Capping Machinery.

Despite the worldwide reemergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we aimed to identify the target of a novel class of CHIKV inhibitors, i.e., the CHVB series. CHVB compounds inhibit the in vitro replication of CHIKV isolates with 50% effective concentrations in the low-micromolar range. A CHVB-resistant variant (CHVBres) was selected that carried two mutations in the gene encoding nsP1 (responsible for viral RNA capping), one mutation in nsP2, and one mutation in nsP3. Reverse genetics studies demonstrated that both nsP1 mutations were necessary and sufficient to achieve ∼18-fold resistance, suggesting that CHVB targets viral mRNA capping. Interestingly, CHVBres was cross-resistant to the previously described CHIKV capping inhibitors from the MADTP series, suggesting they share a similar mechanism of action. In enzymatic assays, CHVB inhibited the methyltransferase and guanylyltransferase activities of alphavirus nsP1 proteins. To conclude, we identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity makes this chemical scaffold a potential candidate for CHIKV drug development.

Structure Elucidation of Coxsackievirus A16 in Complex with GPP3 Informs a Systematic Review of Highly Potent Capsid Binders to Enteroviruses.

The replication of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), which are the major cause of hand, foot and mouth disease (HFMD) in children, can be inhibited by the capsid binder GPP3. Here, we present the crystal structure of CVA16 in complex with GPP3, which clarifies the role of the key residues involved in interactions with the inhibitor. Based on this model, in silico docking was performed to investigate the interactions with the two next-generation capsid binders NLD and ALD, which we show to be potent inhibitors of a panel of enteroviruses with potentially interesting pharmacological properties. A meta-analysis was performed using the available structural information to obtain a deeper insight into those structural features required for capsid binders to interact effectively and also those that confer broad-spectrum anti-enterovirus activity.

More-powerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules.

Enterovirus 71 (HEV71) epidemics in children and infants result mainly in mild symptoms; however, especially in the Asia-Pacific region, infection can be fatal. At present, no therapies are available. We have used structural analysis of the complete virus to guide the design of HEV71 inhibitors. Analysis of complexes with four 3-(4-pyridyl)-2-imidazolidinone derivatives with varying anti-HEV71 activities pinpointed key structure-activity correlates. We then identified additional potentially beneficial substitutions, developed methods to reliably triage compounds by quantum mechanics-enhanced ligand docking and synthesized two candidates. Structural analysis and in vitro assays confirmed the predicted binding modes and their ability to block viral infection. One ligand (with IC50 of 25 pM) is an order of magnitude more potent than the best previously reported inhibitor and is also more soluble. Our approach may be useful in the design of effective drugs for enterovirus infections.

The main hepatitis B virus (HBV) mutants resistant to nucleoside analogs are susceptible in vitro to non-nucleoside inhibitors of HBV replication.

Long-term treatment of chronic hepatitis B with nucleos(t)ide analogs can lead to the emergence of HBV resistant mutants of the polymerase gene. The development of drugs with a different mode of action is warranted to prevent antiviral drug resistance. Only a few non-nucleosidic molecules belonging to the family of phenylpropenamides (AT-61 & AT-130) and heteroaryldihydropyrimidines (BAY41-4109) can prevent RNA encapsidation or destabilize nucleocapsids, respectively. The sensitivity of the main nucleos(t)ide analog- resistant mutants to these inhibitors was evaluated in vitro. HepG2 stable cell lines permanently expressing wild type (WT) HBV or the main HBV mutants resistant to lamivudine and/or adefovir (rtL180M+rtM204V, rtV173L+rtL180M+rtM204V, rtM204I, rtL180M+rtM204I, rtN236T, rtA181V, rtA181V+rtN236T, rtA181T, rtA181T+rtN236T) were treated with AT-61, AT-130 or BAY-41 4109. Analysis of intracellular encapsidated viral DNA showed that all mutants were almost as sensitive to these molecules as WT HBV; indeed, the fold-resistance ranged between 0.7 and 2.3. Furthermore, the effect of a combination of either AT-61 or AT-130 with BAY41-4109, and the combination of these compounds with tenofovir was studied on wild type HBV as well as on a lamivudine and an adefovir-resistant mutant (rtL180M+M204V and rtN236T, respectively). These combinations of compounds resulted in inhibition of viral replication but showed slight antagonistic effects on the three HBV species. Based on this in vitro study, BAY-41 4109, AT-61 and AT-130 molecules that interfere with capsid morphogenesis are active against the main lamivudine- and adefovir-resistant mutants. These results suggest that targeting nucleocapsid functions may represent an interesting approach to the development of novel HBV inhibitors to prevent and combat drug resistance.

Towards the design of combination therapy for the treatment of enterovirus infections.

We report here on a comparative study of the activity of 10 enterovirus inhibitors against poliovirus 1, enterovirus 71 and human rhinovirus 14. Three of the selected molecules (Pleconaril, BTA-798 and V-073) are in clinical development. The in vitro antiviral activity of pairwise combinations of inhibitors indicated that most combinations resulted in an additive to slightly synergistic antiviral activity. However, the combination of ribavirin with a nucleoside polymerase inhibitor resulted in a pronounced antagonistic effect.

A pyrazolotriazolopyrimidinamine inhibitor of bovine viral diarrhea virus replication that targets the viral RNA-dependent RNA polymerase.

[7-[3-(1,3-Benzodioxol-5-yl)propyl]-2-(2-furyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] (LZ37) was identified as a selective inhibitor of in vitro bovine viral diarrhea virus (BVDV) replication. The EC(50) values for inhibition of BVDV-induced cytopathic effect (CPE) formation, viral RNA synthesis and production of infectious virus were 4.3+/-0.7microM, 12.9+/-1microM and 5.8+/-0.6microM, respectively. LZ37 proved inactive against the hepatitis C virus and the flavivirus yellow fever. LZ37 inhibits BVDV replication at a time point that coincides with the onset of intracellular viral RNA synthesis. Drug-resistant mutants carried the F224Y mutation in the viral RNA-dependent RNA polymerase (RdRp). LZ37 showed cross-resistance with the imidazopyrrolopyridine AG110 [which selects for the E291G drug resistance mutation] as well as with the imidazopyridine BPIP [which selects for the F224S drug-resistant mutation]. LZ37 did not inhibit the in vitro activity of purified recombinant BVDV RdRp. Molecular modelling revealed that F224 is located near the tip of the finger domain of the RdRp. Docking of LZ37 in the crystal structure of the BVDV RdRp revealed several potential contacts including: (i) hydrophobic contacts of LZ37 with A221, A222, G223, F224 and A392; (ii) a stacking interaction between F224 side chain and the ring system of LZ37 and (iii) a hydrogen bond between the amino function of LZ37 and the O backbone atom of A392. It is concluded that LZ37 interacts with the same binding site as BPIP or VP32947 at the top of the finger domain of the polymerase that is a "hot spot" for inhibition of pestivirus replication.

Proof of concept for the reduction of classical swine fever infection in pigs by a novel viral polymerase inhibitor.

5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is a representative of a class of imidazopyridines with potent in vitro antiviral activity against pestiviruses including classical swine fever virus (CSFV). This study analysed whether the lead compound, BPIP, was able to reduce virus replication in infected piglets. The compound, administered in feed, was readily bioavailable and was well tolerated. Eight specific-pathogen-free pigs received a daily dose of 75 mg kg(-1) (mixed in feed) for a period of 15 consecutive days, starting 1 day before infection with the CSFV field isolate Wingene. BPIP-treated pigs developed a short, transient viraemia (one animal remained negative) and leukopenia (three animals did not develop leukopenia). Virus titres at peak viraemia (7 days post-infection) were markedly lower (approximately 1000-fold) than in untreated animals (P=0.00005) and the viral genome load in blood was also significantly lower (P

N-benzoxazol-2-yl-N'-1-(isoquinolin-3-yl-ethylidene)-hydrazine, a novel compound with antitumor activity, induces radicals and dissipation of mitochondrial membrane potential.

The novel compound N-benzoxazol-2-yl-N'-1-(isoquinolin-3-yl-ethylidene)-hydrazine (EPH136) has been shown to exhibit antitumor activity in vitro and in vivo. A COMPARE analysis showed that the patterns of cellular effects of EPH136 are not related to any of 175 standard antitumor agents with a known mechanism of action. In order to help identify the mechanism of action we employed a bioinformatics approach called partial least squares modelling in latent variables in which the expression levels of approximately 8,000 genes in each of 56 untreated NCI panel cell lines were correlated with the respective IC(50) values of each cell line following treatment with EPH136. The 60 genes found to be most important for the antiproliferative effect of EPH136 are involved in nucleoside, nucleotide, nucleic acid binding and metabolism, developmental processes, protein modification and metabolism. In addition, using a DNA microarray we measured the expression of approximately 5,000 known genes following treatment of HT-29 colon carcinoma cells with a two-fold IC(50) concentration of EPH136. The genes that were up-regulated more than two-fold compared to untreated controls belong to the same classes as found by the bioinformatic approach. Many of these proteins are regulated by oxidation/reduction and so we concluded that formation of radicals may be involved in the mechanism of action. We show here that EPH136 leads to generation of oxygen radicals, swelling of mitochondria and dissipation of the mitochondrial membrane potential. The antiproliferative activity of EPH136 was prevented by the radical scavenger N-acetylcysteine. Cells with elevated glutathione exhibited resistance to EPH136. In summary, the mechanism of the novel experimental anticancer drug EPH136 is generation of radicals and dissipation of the mitochondrial membrane potential.

Comparative in vitro anti-hepatitis C virus activities of a selected series of polymerase, protease, and helicase inhibitors.

We report here a comparative study of the anti-hepatitis C virus (HCV) activities of selected (i) nucleoside polymerase, (ii) nonnucleoside polymerase, (iii) alpha,gamma-diketo acid polymerase, (iv) NS3 protease, and (v) helicase inhibitors, as well as (vi) cyclophilin binding molecules and (vii) alpha 2b interferon in four different HCV genotype 1b replicon systems.

The imidazopyrrolopyridine analogue AG110 is a novel, highly selective inhibitor of pestiviruses that targets the viral RNA-dependent RNA polymerase at a hot spot for inhibition of viral replication.

Ethyl 2-methylimidazo[1,2-a]pyrrolo[2,3-c]pyridin-8-carboxylate (AG110) was identified as a potent inhibitor of pestivirus replication. The 50% effective concentration values for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect, viral RNA synthesis, and production of infectious virus were 1.2 +/- 0.5 microM, 5 +/- 1 microM, and 2.3 +/- 0.3 microM, respectively. AG110 proved inactive against the hepatitis C virus and a flavivirus. AG110 inhibits BVDV replication at a time point that coincides with the onset of intracellular viral RNA synthesis. Drug-resistant mutants carry the E291G mutation in the viral RNA-dependent RNA polymerase (RdRp). AG110-resistant virus is cross-resistant to the cyclic urea compound 1453 which also selects for the E291G drug resistance mutation. Moreover, BVDV that carries the F224S mutation (because of resistance to the imidazopyridine 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine [BPIP]and VP32947) is also resistant to AG110. AG110 did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). Molecular modeling revealed that E291 is located in a small cavity near the tip of the finger domain of the RdRp about 7 A away from F224. Docking of AG110 in the crystal structure of the BVDV RdRp revealed several potential contacts including with Y257. The E291G mutation might enable the free rotation of Y257, which might in turn destabilize the backbone of the loop formed by residues 223 to 226, rendering more mobility to F224 and, hence, reducing the affinity for BPIP and VP32947. It is concluded that a single drug-binding pocket exists within the finger domain region of the BVDV RdRp that consists of two separate but potentially overlapping binding sites rather than two distinct drug-binding pockets.

Antiviral 2,5-disubstituted imidazo[4,5-c]pyridines: further optimization of anti-hepatitis C virus activity.

Substituted 5-benzyl-2-phenyl-5H-imidazo[4,5-c]pyridines represent a novel class of compounds with activity against pestiviruses and the hepatitis C virus (HCV). Several series of analogues with modifications of the substituents in positions 2 and 5 were prepared. These efforts resulted in the discovery of several compounds with potent antiviral activity of which 2-(2,3-difluorophenyl)-5-[4-(trifluoromethyl)benzyl]-5H-imidazo[4,5-c]pyridine (46) was most potent against HCV (EC(50) of 0.10 microM and a selectivity index of 1080).

Antiviral 2,5-disubstituted imidazo[4,5-c]pyridines: from anti-pestivirus to anti-hepatitis C virus activity.

A novel class of inhibitors of the hepatitis C virus [substituted 2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridines] is described. Introduction of a fluorine in position 2 of the 2-phenyl substituent of the lead anti-pestivirus compound 1 (5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine) resulted in an analogue with selective activity against HCV in the subgenomic replicon system.

Substituted 5-benzyl-2-phenyl-5H-imidazo[4,5-c]pyridines: a new class of pestivirus inhibitors.

A novel class of inhibitors of pestiviruses (5-substituted 2-phenyl-5H-imidazo[4,5-c]pyridines) is described. Modification of the substituent in position 5 resulted in analogues with high activity (EC(50)<100nM) and selectivity (SI>1000) against the pestivirus BVDV (bovine viral diarrhea virus).

A novel, highly selective inhibitor of pestivirus replication that targets the viral RNA-dependent RNA polymerase.

We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 +/- 0.01 microM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 +/- 0.02 microM) and production of infectious virus (EC50= 0.074 +/- 0.003 microM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was approximately 2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed.