Jimmy Lindberg

In Vitro Resistance Profile of the Hepatitis C Virus NS3/4A Protease Inhibitor TMC435

ABSTRACT TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC50s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to ∼2,000-fold for those with the D168V or D168I mutation, compared to the EC50 for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC50s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC50s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.

Induced-Fit Binding of the Macrocyclic Noncovalent Inhibitor TMC435 to its HCV NS3/NS4A Protease Target

The NS3 protein of hepatitis C virus (HCV), together with the NS4A peptide co-factor, comprises 685 residues and possesses domain-specific RNA helicase and serine protease activities. NS3/NS4A protease activity is essential to the HCV life cycle. Small-molecule inhibitors of NS3/NS4A protease have been widely explored and are typically grouped into two classes: linear peptidomimetics with a ketoamide functionality that reacts with the catalytic Ser to form a reversible enzyme–inhibitor adduct, and noncovalent peptidomimetics containing a macrocycle (e.g. Figure 1); macrocyclic ketoamide inhibitors have also been reported. Macrocycles, underrepresented in synthetic drugs, are helpful in improving the druglike character of molecules. TMC435 (1; Figure 1), a macrocyclic noncovalent inhibitor of NS3/NS4A protease with subnanomolar Ki values for genotype 1a and 1b NS3/ NS4A proteases, 11] was discovered by optimizing an earlier NS3/NS4A protease inhibitor, BILN-2061 (2 ; Figure 1). Key steps in the progression from 2 to 1 include reduction of macrocycle size, truncation of the P4 (P3 capping) group, conversion of the carboxylate “head group” to an acylsulfonamide, replacement of the P2 proline pyrrolidine with a cyclopentyl ring, and optimization of the substituted quinoline-thiazole ring system (Figure 1). 14–16] Despite exceeding three of four Lipinski criteria, 1 shows excellent pharmacokinetics in humans. We have determined the crystal structure of 1 bound to its NS3/NS4A protease target from the BK strain of genotype 1b HCV at a resolution of 2.4 (Figure 2; see Table S1 and Figure S1 in the Supporting Information). The threedimensional structure of the NS3 protease domain in complex with a truncated version of the NS4A cofactor was first reported in 1996, and that of an engineered single-chain NS3/NS4A protease–helicase construct in 1999. Currently there are multiple covalent NS3/NS4A protease–inhibitor complexes accessible at the PDB. This structure is the first noncovalent NS3/NS4A protease–inhibitor complex to be deposited at the PDB. Additionally, the new structure shows that the large P2 substituent of 1 induces an extended S2 subsite to accommodate this group; none of the previously available complex structures share this feature. We analyze the observed induced-fit binding of 1 to HCV NS3/NS4A protease, discuss key in vitro resistance mutations in the context of the complex, and disclose the new crystal structure for public analysis. The structure of the NS3/NS4A–1 complex shows the expected trypsin-like fold for the enzyme, with the inhibitor bound at the active site, spanning the S3–S1’ subsites (Figure 2; see Figure S1 in the Supporting Information). Unlike many other macrocyclic drugs that can be divided into functional (binding) and modulator (nonbinding) domains, essentially all of 1 is involved in binding to its target site (Figure 2). Two canonical substrate-like intermolecular hydrogen bonds are observed: the P1–P2 backbone amide N contacts Arg155:O, and the carbonyl O of the P2–P3 amide Figure 1. Macrocyclic (1, 2) and ketoamide (3) inhibitors of HCV NS3/ NS4A protease. Substrate positions from NS3/NS4A protease complex structures are indicated for 1 and 3.

Design, synthesis and SAR of potent statine-based BACE-1 inhibitors: exploration of P1 phenoxy and benzyloxy residues.

Several BACE-1 inhibitors with low nanomolar level activities, encompassing a statine-based core structure with phenyloxymethyl- and benzyloxymethyl residues in the P1 position, are presented. The novel P1 modification introduced to allow the facile exploration of the S1 binding pocket of BACE-1, delivered highly promising inhibitors.

Investigation of α-phenylnorstatine and α-benzylnorstatine as transition state isostere motifs in the search for new BACE-1 inhibitors.

Inhibition of the BACE-1 protease enzyme has over the recent decade developed into a promising drug strategy for Alzheimer therapy. In this report, more than 20 new BACE-1 protease inhibitors based on α-phenylnorstatine, α-benzylnorstatine, iso-serine, and β-alanine moieties have been prepared. The inhibitors were synthesized by applying Fmoc solid phase methodology and evaluated for their inhibitory properties. The most potent inhibitor, tert-alcohol containing (R)-12 (IC(50)=0.19μM) was co-crystallized in the active site of the BACE-1 protease, furnishing a novel binding mode in which the N-terminal amine makes a hydrogen bond to one of the catalytic aspartic acids.

Highly potent macrocyclic BACE-1 inhibitors incorporating a hydroxyethylamine core: Design, synthesis and X-ray crystal structures of enzyme inhibitor complexes

A series of P1-P3 linked macrocyclic BACE-1 inhibitors containing a hydroxyethylamine (HEA) isostere scaffold has been synthesized. All inhibitors comprise a toluene or N-phenylmethanesulfonamide P2 moiety. Excellent BACE-1 potencies, both in enzymatic and cell-based assays, were observed in this series of target compounds, with the best candidates displaying cell-based IC(50) values in the low nanomolar range. As an attempt to improve potency, a phenyl substituent aiming at the S3 subpocket was introduced in the macrocyclic ring. X-ray analyzes were performed on selected compounds, and enzyme-inhibitor interactions are discussed.

Design and synthesis of potent hydroxyethylamine (HEA) BACE-1 inhibitors carrying prime side 4,5,6,7-tetrahydrobenzazole and 4,5,6,7-tetrahydropyridinoazole templates.

A set of low molecular weight compounds containing a hydroxyethylamine (HEA) core structure with different prime side alkyl substituted 4,5,6,7-tetrahydrobenzazoles and one 4,5,6,7-tetrahydropyridinoazole was synthesized. Striking differences were observed on potencies in the BACE-1 enzymatic and cellular assays depending on the nature of the heteroatoms in the bicyclic ring, from the low active compound 4 to inhibitor 6, displaying BACE-1 IC(50) values of 44 nM (enzyme assay) and 65 nM (cell-based assay).

Backbone Assignment of the MALT1 Paracaspase by Solution NMR

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a unique paracaspase protein whose protease activity mediates oncogenic NF-κB signalling in activated B cell-like diffuse large B cell lymphomas (ABC-DLBCLs). ABC-DLBCLs are aggressive lymphomas with high resistance to current chemotherapies. Low survival rate among patients emphasizes the urgent need for alternative treatment options. The characterization of the MALT1 will be an essential tool for developing new target-directed drugs against MALT1 dependent disorders. As the first step in the atomic-level NMR studies of the system, here we report, the 15N/13C/1H backbone assignment of the apo form of the MALT1 paracaspase region together with the third immunoglobulin-like (Ig3) domain, 44 kDa, by high resolution NMR. In addition, the non-uniform sampling (NUS) based targeted acquisition procedure is evaluated as a mean of decreasing acquisition and analysis time for larger proteins.

Design and Synthesis of Novel Arylketo-containing P1-P3 Linked Macro- cyclic BACE-1 Inhibitors

A series of arylketo-containing P1-P3 linked macrocyclic BACE-1 inhibitors were designed, synthesized, and compared with compounds with a previously known and extensively studied corresponding P2 isophthalamide moiety with the aim to improve on permeability whilst retaining the enzyme- and cell-based activities. Several inhibitors displayed substantial increases in Caco-2 cell-based permeability compared to earlier synthesized inhibitors and notably also with retained activities, showing that this approach might yield BACE-1 inhibitors with improved properties.