Medicinal chemistry

Design and synthesis of anti-viral agents, structure-activity relationships

One of our main interests is the design and synthesis of anti-viral agents that inhibit the function of certain viral proteins and the exploration of their structure-activity relationships. Current targets are hepatitis B (HBV), influenza A and hepatitis C (HCV) viruses.

Inhibition of hepatitis B virus replication by N-hydroxyisoquinolinediones and related polyoxygenated heterocycles
Hepatitis B virus (HBV) chronically infects >250 million people and kills nearly a million annually, and current antivirals cannot clear the infection or adequately suppress disease. The virus replicates by reverse transcription, and the dominant antiviral drugs are nucleos(t)ide analogs that target the viral reverse transcriptase. We are developing antivirals targeting the other essential viral enzymatic activity, the ribonuclease H (RNaseH).
HBV RNaseH inhibitors with nanomolar efficacies against viral replication in culture have been identified in the α-hydroxytropolone, N-hydroxyisoquinolinedione, N-hydroxypyridinedions, and N-hydroxynapthyridinone chemotypes. We are a Lab that focuses on RNaseH inhibitors, current synthetic approaches, and challenges to optimizing the inhibitors into leads for clinical assessment.
Novel flutimide analogues targeting the influenza virus PA endonuclease
Influenza is a highly contagious infectious disease that affects millions of people every year. In the twentieth century, influenza caused more fatalities in Europe than any other infectious disease. Current vaccines against influenza virus require annual updating due to continuous variation of the viral antigens. Thus, anti-influenza virus drugs are vital as a first line of defense. At present, two classes of antivirals are available: the neuraminidase inhibitors oseltamivir and zanamivir, and the M2 proton channel blockers amantadine and rimantadine. For both drug classes, viral resistance is an emerging concern and, hence, novel antiviral agents with an alternative mode of action and favorable resistance profile are urgently needed. A promising new target is the influenza virus PA endonuclease, which performs the "cap-snatching" reaction during viral mRNA synthesis, and is essential for virus replication. The crystal structure of the N-terminal part of PA (PA-Nter) containing the catalytic endonuclease domain, was recently revealed, enabling structure-based development of PA inhibitors. Our group synthesized and evaluated several analogues of the natural compound Flutimide (a fungus-derived influenza virus endonuclease inhibitor with a 2,6-diketo-Δ3-piperazine motif). Using an enzymatic PA endonuclease activity assay, we show that these compounds block the enzymatic activity of PA-Nter. Theoretical calculations are also undertaken for providing a structural rationale for the interaction between the synthesized analogues and the viral protein and a number of molecular interactions contributing to binding affinity and specificity were elucidated. Theoretical results are supported by biochemical analyses of the enzymatic activity inhibition. Overall, our data reveal exciting strategies for the design and optimization of novel influenza virus inhibitors that target the viral PA endonucleaser.
New synthetic aminoadamantane compounds
We are also interested in new aminoadamantane compounds with potent anti-influenza virus A activity, bearing high selectivity indices (see list of publications). The reference compounds in this series, amantadine and rimantadine are current anti-influenza virus A drugs. Their protonated form blocks the influenza A M2 ion channel protein activity via interactions of the drug with the transmembrane domain of the tetrameric M2 protein, that is, the 25 residue M2TM peptide tetramer (M2TM sequence of Udorn H3N2 strain is: SSDPLVVAASIIGILHLILWILDRL).
Scaffold Hybridization Strategy towards potent hydroxamate-based inhibitors of Flaviviridae viruses and Trypanosoma species
Infections with Flaviviridae viruses, such as hepatitis C virus (HCV) and dengue virus (DENV) pose global health threats. Infected individuals are at risk of developing chronic liver failure or haemorrhagic fever respectively, often with a fatal outcome if left untreated. Diseases caused by tropical parasites of the Trypanosoma species, T. brucei and T. cruzi, constitute significant socioeconomic burden in sub-Saharan Africa and continental Latin America, yet drug development is under-funded. Anti-HCV chemotherapy is associated with severe side effects and high cost, while Dengue has no clinically approved therapy and antiparasitic drugs are outdated and difficult to administer. Moreover, drug resistance is an emerging concern. Consequently, the need for new revolutionary chemotherapies is urgent. By utilizing a molecular framework combination approach, we combined two distinct chemical entities with proven antiviral and trypanocidal activity into a novel hybrid scaffold attached by an acetohydroxamic acid group (CH2CONHOH), aiming at derivatives with dual activity. The novel spiro-carbocyclic substituted hydantoin analogues were rationally designed, synthesized and evaluated for their potency against three HCV genotypes (1b, 3a, 4a), DENV and two Trypanosoma species (T. brucei, T. cruzi). They exhibited significant EC50 values and remarkable selectivity indices. Several modifications were undertaken to further explore the structure activity relationships (SARs) and confirm the pivotal role of the acetohydroxamic acid metal binding group.
Design and Synthesis of Antiparasitic Agents, Structure-Activity Relationships

Human African trypanosomiasis (HAT), is caused by tsetse fly transmitted parasites of the Trypanosoma brucei species complex. Over 50 million people in sub-Saharan Africa are at risk. In 2009, there were 30,000 cases, although during epidemics, this level can increase >10-fold. Chagas disease (or American trypanosomiasis), is caused by Trypanosoma cruzi and affects 8-10 million people in Latin America, resulting in >15,000 deaths per year. Therapy for trypanosomal infections is unsatisfactory due to toxic side effects, the development of resistance, and in many cases, the need for parenteral administration. With no immediate prospect of vaccines, there is a great need to develop new antitrypanosome agents with an acceptable efficacy and safety profile.

Based on our previous results on structure activity relationship (SAR) of trypanocidal agents we synthesize derivatives with a variety of structural modification at different positions of our lead compound, in order to determine how structural features affect the trypanocidal activity of compounds and improve their ADME, toxicological and pharmacokinetic properties (see list of publications).

Figure: Vector and parasite.



Figure 1: The HBV RNaseH. (A) The RNaseH is the C-terminal domain of the multifunctional HBV polymerase protein. The RNaseH can be expressed as a functional recombinant protein with N-terminal maltose-binding protein (MBP) and C-terminal hexahistidine (H6) tags. TP, terminal protein domain that primes DNA synthesis; Sp, spacer domain; RT, reverse transcriptase domain; RNaseH, RNase H domain. The relative locations of the carboxylic amino acids (D and E) that presumably coordinate the catalytic Mg2+ ions are shown for the recombinant RNaseH. (B) HBV replication cycle. Newly synthesized genomes can be secreted as mature virions or converted via "recycling" to the nuclear cccDNA. DNA is in blue and RNA is in red. The stage at which RNaseH inhibitors act is indicated. Panel B reprinted with permission from Tavis, J. E., Cheng, X., Hu, Y., Totten, M., Cao, F., Michailidis, E., Aurora, R., Meyers, M. J., Jacobsen, E. J., Parniak, M. A., and Sarafianos, S. G. (2013) The hepatitis B virus ribonuclease H is sensitive to inhibitors of the human immunodeficiency virus ribonuclease H and integrase enzymes. PLoS Pathog. 9 (1), e1003125. Copyright 2013 Tavis, Cheng, Hu, Totten, Cao, Michailidis, Aurora, Meyers, Jacobsen, Parniak, Sarafianos.

Figure 2

Figure 3: Influenza A virus.

Figure 4: Theoretical binding mode of inhibitors to the active site of PA endonuclease. The protein is depicted as a ribbon colored according to residue position (red: N-terminal, purple: C-terminal). Residues involved in inhibitor binding are depicted in a stick representation. The two manganese ions coordinated in the active site are depicted as blue opaque spheres. The three red spheres define hydration sites of high energy as determined by Szmap. B) Binding of the 5-fluoro analogue demonstrates its enhanced capabilities of accommodating stacking and hydrophobic interactions with respect to DPBA. Interestingly, the inhibitor is oriented in a manner which positions the 5-substituent in a N-terminal cavity offering possibilities for additional stabilizing interactions. The 5-susbtituent is also close to high-energy hydrations sites, thus permitting their partial displacement which would possibly lead to affinity improvement.

Figure 5: Structural elements of AM2 and BM2 important for channel function. (A) and (B) show the C-terminal tryptophan gates of the AM2 (PDB code: 2RLF)(Schnell and Chou 2008) and BM2 (PDB code: 2KIX)(Wang, Pielak et al. 2009) channels, respectively. (C) and (D) show the amino acid sidechains important for proton relay, selection, and gating in the TM domains of AM2 and BM2, respectively. (E) and (F) show the N-terminal constriction of the AM2 and BM2 channel, respectively.

Figure 6: A) The predicted binding mode of the lead compound 16 (ball and stick model with orange carbons) in the active site of HCV NS5B polymerase, shown in a ribbon representation. The ligand is oriented appropriately to achieve efficient chelation of the metal ions (shown as plum color spheres) by the acetohydroxamic acid group inside the viral enzyme pocket. The inhibitor is further stabilized by a hydrogen bond formed between the hydantoin ring carbonyl and R158. Additional hydrophobic and cation-π interactions between the sidechains of R222 and K51 with the cycloalkyl and aromatic fragments of the ligand, respectively, offer further stabilization to the complex. B) A two-dimensional representation of the key interactions anchoring the inhibitor in the viral protein active site. The two positions of the lead, where modifications were introduced for deriving SAR of the new compounds, are also depicted.

Figure 7: Inhibitory effect of compounds on HCV RNA levels and protein expression in a subgenomic HCV Con1 replicon assay. A) Quantification of (+) strand HCV RNA by RT-qPCR in Huh7.5-4a replicon cells treated with serial dilutions of compounds 17a, 18 or the solvent DMSO. Values from compound-treated cells are expressed as percentage of those obtained with cells that received the solvent (control). mRNA levels of the housekeeping gene YWHAZ were used for normalization. B) Indirect immunofluorescence for NS5A (left panels) in Huh7.5-4a replicon cells, treated with serial dilutions of compound 18 or the solvent DMSO. Nuclei were stained with propidium iodide (PI; middle panels). Merged images are shown at the right. Bar, 50 μm.