Christian Lerner

A Real-World Perspective on Molecular Design.

We present a series of small molecule drug discovery case studies where computational methods were prospectively employed to impact Roche research projects, with the aim of highlighting those methods that provide real added value. Our brief accounts encompass a broad range of methods and techniques applied to a variety of enzymes and receptors. Most of these are based on judicious application of knowledge about molecular conformations and interactions: filling of lipophilic pockets to gain affinity or selectivity, addition of polar substituents, scaffold hopping, transfer of SAR, conformation analysis, and molecular overlays. A case study of sequence-driven focused screening is presented to illustrate how appropriate preprocessing of information enables effective exploitation of prior knowledge. We conclude that qualitative statements enabling chemists to focus on promising regions of chemical space are often more impactful than quantitative prediction.

Molecular recognition at the active site of catechol-o-methyltransferase: energetically favorable replacement of a water molecule imported by a bisubstrate inhibitor.

Biologically active catechols, such as l-DOPA and the neurotransmitter dopamine, are inactivated by methylation. This reaction is catalyzed by the enzyme catechol-O-methyltransferase (COMT) in the presence of S-adenosylmethionine (SAM) and Mg ions. Small nitrocatechol-based inhibitors of COMT find application in the treatment of Parkinson disease by blocking unwanted methylation of the administered l-DOPA, thereby enhancing dopamine levels in the brain. 3] Recent studies have pointed towards additional therapeutic applications of COMT inhibition in other disorders of the central nervous system, such as schizophrenia and depression. We have developed a series of potent bisubstrate inhibitors for COMT which are competitive for both the catechol and the SAM binding sites. Based on the X-ray crystal structure of ligand 1 (IC50 = 9 nm) [7a] in a ternary complex with COMT and a Mg ion (PDB code: 1JR4), we started a detailed exploration of the molecular recognition properties of the entire active site of the enzyme. Importantly, we found that potentially hepatotoxic nitro groups, which are mandatory in catechol-based monosubstrate inhibitors, are not required for high-affinity bisubstrate inhibition. We substituted the nitro group in position 5 of 1 with appropriate lipophilic residues, such as the 4-fluorophenyl ring in 2 (IC50 = 31 nm), and found that the high, competitive inhibitory potency was maintained. Computer modeling studies suggested that the newly introduced lipophilic residue occupies a hydrophobic cleft near the surface of the enzyme. This initial proposal is validated here experimentally by X-ray crystallography. The crystal structure of 1 in a ternary complex with COMT and a Mg ion shows that the adenine moiety forms two hydrogen bonds, a moderately strong and a weak one (d(N···O): 3.0 and 3.4 , respectively), to a water molecule

Molecular Recognition at the Active Site of Catechol-O-methyltransferase (COMT): Adenine Replacements in Bisubstrate Inhibitors

L-Dopa, the standard therapeutic for Parkinsons disease, is inactivated by the enzyme catechol-O-methyltransferase (COMT). COMT catalyzes the transfer of an activated methyl group from S-adenosylmethionine (SAM) to its catechol substrates, such as L-dopa, in the presence of magnesium ions. The molecular recognition properties of the SAM-binding site of COMT have been investigated only sparsely. Here, we explore this site by structural alterations of the adenine moiety of bisubstrate inhibitors. The molecular recognition of adenine is of special interest due to the great abundance and importance of this nucleobase in biological systems. Novel bisubstrate inhibitors with adenine replacements were developed by structure-based design and synthesized using a nucleosidation protocol introduced by Vorbrüggen and co-workers. Key interactions of the adenine moiety with COMT were measured with a radiochemical assay. Several bisubstrate inhibitors, most notably the adenine replacements thiopyridine, purine, N-methyladenine, and 6-methylpurine, displayed nanomolar IC(50) values (median inhibitory concentration) for COMT down to 6 nM. A series of six cocrystal structures of the bisubstrate inhibitors in ternary complexes with COMT and Mg(2+) confirm our predicted binding mode of the adenine replacements. The cocrystal structure of an inhibitor bearing no nucleobase can be regarded as an intermediate along the reaction coordinate of bisubstrate inhibitor binding to COMT. Our studies show that solvation varies with the type of adenine replacement, whereas among the adenine derivatives, the nitrogen atom at position 1 is essential for high affinity, while the exocyclic amino group is most efficiently substituted by a methyl group.

Catechol-O-methyltransferase in complex with substituted 3'-deoxyribose bisubstrate inhibitors

The biological activity of catechol neurotransmitters such as dopamine in the synapse is modulated by transporters and enzymes. Catechol-O-methyltransferase (COMT; EC 2.1.1.6) inactivates neurotransmitters by catalyzing the transfer of a methyl group from S-adenosylmethionine to catechols in the presence of Mg²⁺. This pathway also inactivates L-DOPA, the standard therapeutic for Parkinsons disease. Depletion of catechol neurotransmitters in the prefrontal cortex has been linked to schizophrenia. The inhibition of COMT therefore promises improvements in the treatment of these diseases. The concept of bisubstrate inhibitors for COMT has been described previously. Here, ribose-modified bisubstrate inhibitors were studied. Three high-resolution crystal structures of COMT in complex with novel ribose-modified bisubstrate inhibitors confirmed the predicted binding mode but displayed subtle alterations at the ribose-binding site. The high affinity of the inhibitors can be convincingly rationalized from the structures, which document the possibility of removing and/or replacing the ribose 3-hydroxyl group and provide a framework for further inhibitor design.

A Novel Endonuclease Inhibitor Exhibits Broad-Spectrum Anti-Influenza Virus Activity In Vitro

ABSTRACT Antiviral drugs are important in preventing and controlling influenza, particularly when vaccines are ineffective or unavailable. A single class of antiviral drugs, the neuraminidase inhibitors (NAIs), is recommended for treating influenza. The limited therapeutic options and the potential risk of antiviral resistance are driving the search for additional small-molecule inhibitors that act on influenza virus proteins. The acid polymerase (PA) of influenza viruses is a promising target for new antivirals because of its essential role in initiating virus transcription. Here, we characterized a novel compound, RO-7, identified as a putative PA endonuclease inhibitor. RO-7 was effective when added before the cessation of genome replication, reduced polymerase activity in cell-free systems, and decreased relative amounts of viral mRNA and genomic RNA during influenza virus infection. RO-7 specifically inhibited the ability of the PA endonuclease domain to cleave a nucleic acid substrate. RO-7 also inhibited influenza A viruses (seasonal and 2009 pandemic H1N1 and seasonal H3N2) and B viruses (Yamagata and Victoria lineages), zoonotic viruses (H5N1, H7N9, and H9N2), and NAI-resistant variants in plaque reduction, yield reduction, and cell viability assays in Madin-Darby canine kidney (MDCK) cells with nanomolar to submicromolar 50% effective concentrations (EC50s), low toxicity, and favorable selective indices. RO-7 also inhibited influenza virus replication in primary normal human bronchial epithelial cells. Overall, RO-7 exhibits broad-spectrum activity against influenza A and B viruses in multiple in vitro assays, supporting its further characterization and development as a potential antiviral agent for treating influenza.