ABSTRACT
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 (15)N/(13)C/(1)H 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.
Subject(s)
Caspases/chemistry , Neoplasm Proteins/chemistry , Humans , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
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.
ABSTRACT
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).
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Azoles/chemical synthesis , Benzoxazoles/chemical synthesis , Drug Design , Enzyme Inhibitors/chemical synthesis , Ethylamines/chemical synthesis , Pyridines/chemical synthesis , Animals , Azoles/chemistry , Azoles/pharmacology , Benzoxazoles/chemistry , Benzoxazoles/pharmacology , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Ethylamines/chemistry , Ethylamines/pharmacology , Humans , Inhibitory Concentration 50 , Male , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Structure , Pyridines/chemistry , Pyridines/pharmacologyABSTRACT
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.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/chemical synthesis , Ethylamines/chemistry , Amyloid Precursor Protein Secretases/metabolism , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Ethylamines/chemical synthesis , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
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 ß-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.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Phenylbutyrates/pharmacology , Protease Inhibitors/pharmacology , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Mass Spectrometry , Phenylbutyrates/chemistry , Protease Inhibitors/chemistryABSTRACT
Highly potent BACE-1 protease inhibitors have been developed from an inhibitors containing a hydroxyethylene (HE) core displaying aryloxymethyl or benzyloxymethyl P1 side chain and a methoxy P1' side chain. The target molecules were synthesized in good overall yields from chiral carbohydrate starting materials. The inhibitors show high BACE-1 potency and good selectivity against cathepsin D, where the most potent inhibitor furnishes BACE-1 K(i) << 1 nM and displays >1000-fold selectivity over cathepsin D.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Ethylenes/chemical synthesis , Amyloid Precursor Protein Secretases/chemistry , Aspartic Acid Endopeptidases/chemistry , Cathepsin D/antagonists & inhibitors , Crystallography, X-Ray , Drug Design , Ethylenes/chemistry , Ethylenes/pharmacology , Humans , Hydrogen Bonding , Models, Molecular , Stereoisomerism , Structure-Activity RelationshipABSTRACT
In a preceding study we have described the development of a new hydroxyethylene (HE) core motif displaying P1 aryloxymethyl and P1' methoxy substituents delivering potent BACE-1 inhibitors. In a continuation of this work we have now explored the SAR of the S1' pocket by introducing a set of P1' alkoxy groups and evaluated them as BACE-1 inhibitors. Previously the P1 and P1' positions of the classical HE template have been relatively little explored due to the complexity of the chemical routes involved in modifications at these positions. However, the chemistries developed for the current HE template renders substituents in both the P1 and P1' positions readily available for SAR exploration. The BACE-1 inhibitors prepared displayed K(i) values in the range of 1-20 nM, where the most potent compounds featured small P1' groups. The cathepsin D selectivity which was high for the smallest P1' substituents (P1'=ethoxy, fold selectively >1500) dropped for larger groups (P1'=benzyloxy, fold selectivity of 3). We have also confirmed the importance of both the hydroxyl group and its stereochemistry preference for this HE transition state isostere by preparing both the deoxygenated analogue and by inverting the configuration of the hydroxyl group to the R-configuration, which as expected resulted in large activity drops. Finally substituting the hydroxyl group by an amino group having the same configuration (S), which previously have been described to deliver potent BACE-1 inhibitors with advantageous properties, surprisingly resulted in a large drop in the inhibitory activity.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Ethylenes/chemistry , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Ultraviolet , Structure-Activity RelationshipABSTRACT
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 (EC(50)s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to approximately 2,000-fold for those with the D168V or D168I mutation, compared to the EC(50) 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 EC(50)s), 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 EC(50)s). 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.
Subject(s)
Hepacivirus/drug effects , Hepatitis C/drug therapy , Heterocyclic Compounds, 3-Ring/pharmacology , Protease Inhibitors/pharmacology , Sulfonamides/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/pharmacology , Cell Line , Drug Resistance, Viral/drug effects , Drug Resistance, Viral/genetics , Drug Synergism , Genotype , Hepacivirus/enzymology , Hepacivirus/genetics , Hepatitis C/virology , Humans , In Vitro Techniques , Interferon-alpha/pharmacology , Mutagenesis , Simeprevir , Viral Nonstructural Proteins/genetics , Virus Replication/drug effectsSubject(s)
Carrier Proteins/antagonists & inhibitors , Heterocyclic Compounds, 3-Ring/chemistry , Macrocyclic Compounds/chemistry , Protease Inhibitors/chemistry , Sulfonamides/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Binding Sites , Carrier Proteins/metabolism , Crystallography, X-Ray , Drug Design , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Intracellular Signaling Peptides and Proteins , Protease Inhibitors/pharmacology , Protein Binding , Simeprevir , Sulfonamides/pharmacology , Viral Nonstructural Proteins/metabolism , Viral Proteins/metabolismABSTRACT
We herein describe the design and synthesis of a series of BACE-1 inhibitors incorporating a P1-substituted hydroxylethylene transition state isostere. The synthetic route starting from commercially available carbohydrates yielded a pivotal lactone intermediate with excellent stereochemical control which subsequently could be diversified at the P1-position. The final inhibitors were optimized using three different amines to provide the residues in the P2'-P3' position and three different acids affording the residues in the P2-P3 position. In addition we report on the stereochemical preference of the P1'-methyl substituent in the synthesized inhibitors. All inhibitors were evaluated in an in vitro BACE-1 assay where the most potent inhibitor, 34-(R), exhibited a BACE-1 IC(50) value of 3.1 nM.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Ethylenes/chemistry , Cell Line , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Molecular Structure , Stereoisomerism , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Amino Acids/chemistry , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Drug Design , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Crystallography, X-Ray , Humans , Models, Molecular , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Protease Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
New and potent inhibitors of the malarial aspartic proteases plasmepsin (Plm) I and II, from the deadliest malaria parasite Plasmodium falciparum, have been synthesized utilizing Suzuki coupling reactions on previously synthesized bromobenzyloxy-substituted statine-like inhibitors. The enzyme inhibition activity has been improved up to eight times by identifying P1 substituents that effectively bind to the continuous S1-S3 crevice of Plasmepsin I and II. By replacement of the bromo atom in the P1 p-bromobenzyloxy-substituted inhibitors with different aryl substituents, several inhibitors exhibiting K(i) values in the low nanomolar range for both Plm I and II have been identified. Some of these inhibitors are also effective in attenuating parasite growth in red blood cells, with the best inhibitors, compounds 2 and 4, displaying 70% and 83% inhibition, respectively, at a concentration of 5 microM. The design was partially guided by the X-ray crystal structure disclosed herein of the previously synthesized inhibitor 1 in complex with plasmepsin II.
Subject(s)
Antiprotozoal Agents/chemical synthesis , Antiprotozoal Agents/pharmacology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/chemistry , Plasmodium falciparum/enzymology , Animals , Antiprotozoal Agents/chemistry , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Plasmodium falciparum/drug effects , Protozoan Proteins , Structure-Activity RelationshipABSTRACT
Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This activity is universally required for fatty acid biosynthesis as well as for the production of a number of small molecules, pH homeostasis, and other functions. At least three different carbonic anhydrase families are known to exist, of which the alpha-class found in humans has been studied in most detail. In the present work, we describe the structures of two of the three beta-class carbonic anhydrases that have been identified in Mycobacterium tuberculosis, i.e. Rv1284 and Rv3588c. Both structures were solved by molecular replacement and then refined to resolutions of 2.0 and 1.75 A, respectively. The active site of Rv1284 is small and almost completely shielded from solvent, whereas that of Rv3588c is larger and quite open to solution. Differences in coordination of the active site metal are also observed. In Rv3588c, an aspartic acid side chain displaces a water molecule and coordinates directly to the zinc ion, thereby closing the zinc coordination sphere and breaking the salt link to a nearby arginine that is a feature of Rv1284. The two carbonic anhydrases thus exhibit both of the metal coordination geometries that have previously been observed for structures in this family. Activity studies demonstrate that Rv3588c is a completely functional carbonic anhydrase. The apparent lack of activity of Rv1284 in the present assay system is likely exacerbated by the observed depletion of zinc in the preparation.
Subject(s)
Carbonic Anhydrases/chemistry , Carbonic Anhydrases/physiology , Mycobacterium tuberculosis/enzymology , Amino Acid Sequence , Aspartic Acid/chemistry , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Dimerization , Hydrogen-Ion Concentration , Ions , Ligands , Metals/chemistry , Models, Molecular , Molecular Sequence Data , Multigene Family , Protein Conformation , Protein Structure, Tertiary , Salts/pharmacology , Serine/chemistry , Solvents/chemistry , Structure-Activity Relationship , Time Factors , Zinc/chemistryABSTRACT
HIV-1 protease is a pivotal enzyme in the later stages of the viral life cycle which is responsible for the processing and maturation of the virus particle into an infectious virion. As such, HIV-1 protease has become an important target for the treatment of AIDS, and efficient drugs have been developed. However, negative side effects and fast emerging resistance to the current drugs have necessitated the development of novel chemical entities in order to exploit different pharmacokinetic properties as well as new interaction patterns. We have used X-ray crystallography to decipher the structure-activity relationship of fluoro-substitution as a strategy to improve the antiviral activity and the protease inhibition of C2-symmetric diol-based inhibitors. In total we present six protease-inhibitor complexes at 1.8-2.3 A resolution, which have been structurally characterized with respect to their antiviral and inhibitory activities, in order to evaluate the effects of different fluoro-substitutions. These C2-symmetric inhibitors comprise mono- and difluoro-substituted benzyloxy side groups in P1/P1' and indanoleamine side groups in P2/P2'. The ortho- and meta-fluorinated P1/P1'-benzyloxy side groups proved to have the most cytopathogenic effects compared with the nonsubstituted analog and related C2-symmetric diol-based inhibitors. The different fluoro-substitutions are well accommodated in the protease S1/S1' subsites, as observed by an increase in favorable Van der Waals contacts and surface area buried by the inhibitors. These data will be used in the development of potent inhibitors with different pharmacokinetic profiles towards resistant protease mutants.
Subject(s)
Benzene Derivatives/chemistry , Benzene Derivatives/pharmacology , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/pharmacology , HIV-1/enzymology , Hydrocarbons, Fluorinated/chemistry , Hydrocarbons, Fluorinated/pharmacology , Amino Acids/chemistry , Amino Acids/metabolism , Benzene Derivatives/metabolism , Binding Sites , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , HIV Protease/chemistry , HIV Protease/genetics , HIV Protease/metabolism , HIV Protease Inhibitors/metabolism , Humans , Hydrocarbons, Fluorinated/metabolism , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Binding , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , Structure-Activity RelationshipABSTRACT
A series of HIV-1 protease inhibitors having new tetrahydrofuran P2/P2' groups have been synthesised and tested for protease inhibition and antiviral activity. Six novel 4-aminotetrahydrofuran derivatives were prepared starting from commercially available isopropylidene-alpha-D-xylofuranose yielding six symmetrical and six unsymmetrical inhibitors. Promising sub nanomolar HIV-1 protease inhibitory activities were obtained. The X-ray crystal structure of the most potent inhibitor (23, K(i) 0.25 nM) co-crystallised with HIV-1 protease is discussed and the binding compared with inhibitors 1a and 1b.
Subject(s)
Furans/chemistry , HIV Protease Inhibitors/chemical synthesis , HIV Protease Inhibitors/pharmacology , HIV Protease/chemistry , HIV-1/enzymology , Crystallography, X-Ray , Drug Design , HIV Protease/drug effects , HIV Protease Inhibitors/chemistry , Indicators and Reagents , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular ConformationABSTRACT
The K103N substitution is a frequently observed HIV-1 RT mutation in patients who do not respond to combination-therapy. The drugs Efavirenz, MSC194 and PNU142721 belong to the recent generation of NNRTIs characterized by an improved resistance profile to the most common single point mutations within HIV-1 RT, including the K103N mutation. In the present study we present structural observations from Efavirenz in complex with wild-type protein and the K103N mutant and PNU142721 and MSC194 in complex with the K103N mutant. The structures unanimously indicate that the K103N substitution induces only minor positional adjustments of the three inhibitors and the residues lining the binding pocket. Thus, compared to the corresponding wild-type structures, these inhibitors bind to the mutant in a conservative mode rather than through major rearrangements. The structures implicate that the reduced inhibitory efficacy should be attributed to the changes in the chemical environment in the vicinity of the substituted N103 residue. This is supported by changes in hydrophobic and electrostatic interactions to the inhibitors between wild-type and K103N mutant complexes. These potent inhibitors accommodate to the K103N mutation by forming new interactions to the N103 side chain. Our results are consistent with the proposal by Hsiou et al. [Hsiou, Y., Ding, J., Das, K., Clark, A.D. Jr, Boyer, P.L., Lewi, P., Janssen, P.A., Kleim, J.P., Rosner, M., Hughes, S.H. & Arnold, E. (2001) J. Mol. Biol. 309, 437-445] that inhibitors with good activity against the K103N mutant would be expected to have favorable interactions with the mutant asparagines side chain, thereby compensating for resistance caused by stabilization of the mutant enzyme due to a hydrogen-bond network involving the N103 and Y188 side chains.