Matheus Froeyen

Novel Inhibitors of Influenza Virus Fusion: Structure-Activity Relationship and Interaction with the Viral Hemagglutinin

ABSTRACT A new class of N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide inhibitors of influenza virus hemagglutinin (HA)-mediated membrane fusion that has a narrow and defined structure-activity relationship was identified. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza virus A/H3N2, the lead compound, 4c, displayed a 50% effective concentration of 3 to 23 μM and an antiviral selectivity index of 10. No activity was observed for A/H1N1, A/H5N1, A/H7N2, and B viruses. The activity of 4c was reduced considerably when added 30 min or later postinfection, indicating that 4c inhibits an early step in virus replication. 4c and its congeners inhibited influenza A/H3N2 virus-induced erythrocyte hemolysis at low pH. 4c-resistant virus mutants, selected in MDCK cells, contained either a single D112N change in the HA2 subunit of the viral HA or a combination of three substitutions, i.e., R220S (in HA1) and E57K (in HA2) and an A-T substitution at position 43 or 96 of HA2. The mutants showed efficiency for receptor binding and replication similar to that of wild-type virus yet displayed an increased pH of erythrocyte hemolysis. In polykaryon assays with cells expressing single-mutant HA proteins, the E57K, A96T, and D112N mutations resulted in 4c resistance, and the HA proteins containing R220S, A96T, and D112N mutations displayed an increased fusion pH. Molecular modeling identified a binding cavity for 4c involving arginine-54 and glutamic acid-57 in the HA2 subunit. Our studies with the new fusion inhibitor 4c confirm the importance of this HA region in the development of influenza virus fusion inhibitors.

Carbohydrate-binding Agents Cause Deletions of Highly Conserved Glycosylation Sites in HIV GP120 A NEW THERAPEUTIC CONCEPT TO HIT THE ACHILLES HEEL OF HIV

Mannose-binding proteins derived from several plants (i.e. Hippeastrum hybrid and Galanthus nivalis agglutinin) or prokaryotes (i.e. cyanovirin-N) inhibit human immunodeficiency virus (HIV) replication and select for drug-resistant viruses that show profound deletion of N-glycosylation sites in the GP120 envelope (Balzarini, J., Van Laethem, K., Hatse, S., Vermeire, K., De Clercq, E., Peumans, W., Van Damme, E., Vandamme, A.-M., Bolmstedt, A., and Schols, D. (2004) J. Virol. 78, 10617-10627; Balzarini, J., Van Laethem, K., Hatse, S., Froeyen, M., Van Damme, E., Bolmstedt, A., Peumans, W., De Clercq, E., and Schols, D. (2005) Mol. Pharmacol. 67, 1556-1565). Here we demonstrated that the N-acetylglucosamine-binding protein from Urtica dioica (UDA) prevents HIV entry and eventually selects for viruses in which conserved N-glycosylation sites in GP120 were deleted. In contrast to the mannose-binding proteins, which have a 50-100-fold decreased antiviral activity against the UDA-exposed mutant viruses, UDA has decreased anti-HIV activity to a very limited extent, even against those mutant virus strains that lack at least 9 of 22 (∼40%) glycosylation sites in their GP120 envelope. Therefore, UDA represents the prototype of a new conceptual class of carbohydrate-binding agents with an unusually specific and targeted drug resistance profile. It forces HIV to escape drug pressure by deleting the indispensable glycans on its GP120, thereby obligatorily exposing previously hidden immunogenic epitopes on its envelope.

A Novel, Highly Selective Inhibitor of Pestivirus Replication That Targets the Viral RNA-Dependent RNA Polymerase

ABSTRACT 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 μM. Comparable reduction of viral RNA synthesis (EC50 = 0.12± 0.02 μM) and production of infectious virus (EC50 = 0.074 ± 0.003 μM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was ∼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.

Investigation of the DNA-dependent cyclohexenyl nucleic acid polymerization and the cyclohexenyl nucleic acid-dependent DNA polymerization

DNA polymerases from different evolutionary families [Vent (exo−) DNA polymerase from the B-family polymerases, Taq DNA polymerase from the A-family polymerases and HIV reverse transcriptase from the reverse transcriptase family] were examined for their ability to incorporate the sugar-modified cyclohexenyl nucleoside triphosphates. All enzymes were able to use the cyclohexenyl nucleotides as a substrate. Using Vent (exo−) DNA polymerase and HIV reverse transcriptase, we were even able to incorporate seven consecutive cyclohexenyl nucleotides. Using a cyclohexenyl nucleic acid (CeNA) template, all enzymes tested were also able to synthesize a short DNA fragment. Since the DNA-dependent CeNA polymerization and the CeNA-dependent DNA polymerization is possible to a limited extend, we suggest CeNA as an ideal candidate to use in directed evolution methods for the development of a polymerase capable of replicating CeNA.

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

ABSTRACT 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 μM, 5 ± 1 μM, and 2.3 ± 0.3 μM, 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 Å 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.

Synthesis and Biological Evaluation of Bicyclic Nucleosides as Inhibitors of M. tuberculosis Thymidylate Kinase

Herein we describe the synthesis and conformational analysis of a series of bicyclic thymidine derivatives and their evaluation as inhibitors of thymidine monophosphate kinase from Mycobacterium tuberculosis (TMPKmt), based on previously discovered bicyclic sugar nucleosides. With a Ki value of 2.3 μm, 1‐[3‐aminomethyl‐3,5‐dideoxy‐2‐O,6‐N‐(thiocarbonyl)‐β‐D‐ribofuranosyl]thymine emerged as the most potent TMPK inhibitor of this series. Moreover, this promising compound displays inhibitory potency against Mycobacteria cultures with an IC99 value of 100 μg mL−1, thus promoting TMPKmt for the first time as a validated target for further inhibitory design. Attempts to rationalise the observed structure–activity relationship (SAR) involving molecular modelling and conformational analysis are described.

Iminodiacetic-phosphoramidates as metabolic prototypes for diversifying nucleic acid polymerization in vivo

Previous studies in our laboratory proved that certain functional groups are able to mimic the pyrophosphate moiety and act as leaving groups in the enzymatic polymerization of deoxyribonucleic acids by HIV-1 reverse transcriptase. When the potential leaving group possesses two carboxylic acid moieties linked to the nucleoside via a phosphoramidate bond, it is efficiently recognized by this error-prone enzyme, resulting in nucleotide incorporation into DNA. Here, we present a new efficient alternative leaving group, iminodiacetic acid, which displays enhanced kinetics and an enhanced elongation capacity compared to previous results obtained with amino acid deoxyadenosine phosphoramidates. Iminodiacetic acid phosphoramidate of deoxyadenosine monophosphate (IDA-dAMP) is processed by HIV-1 RT as a substrate for single nucleotide incorporation and displays a typical Michaelis–Menten kinetic profile. This novel substrate also proved to be successful in primer strand elongation of a seven-base template overhang. Modelling of this new substrate in the active site of the enzyme revealed that the interactions formed between the triphosphate moiety, magnesium ions and enzymes residues could be different from those of the natural triphosphate substrate and is likely to involve additional amino acid residues. Preliminary testing for a potential metabolic accessibility lets us to envision its possible use in an orthogonal system for nucleic acid synthesis that would not influence or be influenced by genetic information from the outside.

A novel benzonitrile analogue inhibits rhinovirus replication

OBJECTIVES To study the characteristics and the mode of action of the anti-rhinovirus compound 4-[1-hydroxy-2-(4,5-dimethoxy-2-nitrophenyl)ethyl]benzonitrile (LPCRW_0005). METHODS The antiviral activity of LPCRW_0005 was evaluated in a cytopathic effect reduction assay against a panel of human rhinovirus (HRV) strains. To unravel its precise molecular mechanism of action, a time-of-drug-addition study, resistance selection and thermostability assays were performed. The crystal structure of the HRV14/LPCRW_0005 complex was elucidated as well. RESULTS LPCRW_0005 proved to be a selective inhibitor of the replication of HRV14 (EC(50) of 2 ± 1 μM). Time-of-drug-addition studies revealed that LPCRW_0005 interferes with the earliest stages of virus replication. Phenotypic drug-resistant virus variants were obtained (≥30-fold decrease in susceptibility to the inhibitory effect of LPCRW_0005), which carried either an A150T or A150V amino acid substitution in the VP1 capsid protein. The link between the mutant genotype and drug-resistant phenotype was confirmed by reverse genetics. Cross-resistance studies and thermostability assays revealed that LPCRW_0005 has a similar mechanism of action to the capsid binder pleconaril. Elucidation of the crystal structure of the HRV14/LPCRW_0005 complex revealed the existence of multiple hydrophobic and polar interactions between the VP1 pocket and LPCRW_0005. CONCLUSIONS LPCRW_0005 is a novel inhibitor of HRV14 replication that acts as a capsid binder. The compound has a chemical structure that is markedly smaller than that of other capsid binders. Structural studies show that LPCRW_0005, in contrast to pleconaril, leaves the toe end of the pocket in VP1 empty. This suggests that extended analogues of LPCRW_0005 that fill the full cavity could be more potent inhibitors of rhinovirus replication.

Insight into ligand selectivity in HCV NS5B polymerase: molecular dynamics simulations, free energy decomposition and docking

Modeling studies were performed on HCV NS5B polymerase in an effort to design new inhibitors. The binding models of five different scaffold inhibitors were investigated and compared by using molecular dynamics simulations, free energy calculation and decomposition. Our results show Tyr448 plays the most critical role in the binding of most inhibitors. In addition, favorable contributions of residues Pro197, Arg200, Cys366, Met414 and Tyr448 in a deep hydrophobic pocket prove to be important for the selectivity of inhibitors. Furthermore, an optimized docking protocol was presented based on cross-docking the five inhibitors in the palm binding site of this enzyme using the Autodock program. This protocol was used later to virtually screen NCI and Maybridge diversity set libraries. The binding site was profiled via the statistics and analysis of the hydrogen bond networks formed between the receptor and the top-ranked diversity set compounds. Based on our detailed binding site analysis two useful rules were proposed to guide the selection of promising hits.

Highly potent and selective inhibition of bovine viral diarrhea virus replication by γ-carboline derivatives

Several novel γ-carboline derivatives were identified as selective inhibitors of bovine viral diarrhea virus (BVDV) replication in cell cultures. Among them, 3,4,5-trimethyl-γ-carboline (SK3M4M5M) was the most active against BVDV (Nose strain) in MDBK cells, with a 50% effective concentration of 0.017±0.005μM and a selectivity index of 435. The compound inhibited viral RNA synthesis in a dose-dependent fashion. In a time of drug-addition experiment during a single viral replication cycle, SK3M4M5M lost its antiviral activity when first added at 8h or later after infection, which coincides with the onset of viral RNA synthesis. When selected γ-carboline derivatives, including SK3M4M5M, were examined for their inhibitory effect on the mutant strains resistant to some classes of nonnucleoside BVDV RNA-dependent RNA polymerase inhibitors, all of which target the top of the finger domain of the polymerase, the strains displayed cross-resistance to the γ-carboline derivatives. These results indicate that the γ-carboline derivatives may possibly target a hot spot of the RNA-dependent RNA polymerase. Although SK3M4M5M was highly active against BVDV, the compound proved inactive against hepatitis C virus (HCV) in HCV RNA replicon cells.