RESUMEN
Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/farmacología , Resonancia Magnética Nuclear Biomolecular , Bibliotecas de Moléculas Pequeñas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Modelos Moleculares , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
The type I interferon alpha family consists of small proteins that have clinically important anti-infective and anti-tumor activity. Interferon alpha-2b (Intron A) combination therapy with ribavirin is the current standard of care for the treatment of chronic hepatitis C virus infection. A drawback to the therapy however, is the short serum half-life and rapid clearance of the interferon alpha protein. Schering-Plough has developed a semi-synthetic form of Intron A by attaching a 12-kDa mono-methoxy polyethylene glycol to the protein (PEG Intron) which fulfills the requirements of a long-acting interferon alpha protein while providing significant clinical benefits. A detailed physicochemical and biological characterization of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Though pegylation appeared to decrease the specific activity of the interferon alpha-2b protein, the potency of PEG Intron, independent of protein concentration, was comparable to the Intron A standard at both the molecular and cellular level. Importantly, PEG Intron has demonstrated an enhanced pharmacokinetic profile in both animal and human studies. Recently, PEG Intron in combination with ribavirin has been shown to be very effective in reducing hepatitis C viral load and maintaining effective sustained viral suppression in patients. Because of the improved clinical benefits, it is anticipated that the PEG Intron plus ribavirin combination therapy will become the new standard of care for the treatment of chronic hepatitis C.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Interferón-alfa , Interferón-alfa/química , Interferón-alfa/farmacología , Polietilenglicoles , Animales , Antivirales/uso terapéutico , Dicroismo Circular , Quimioterapia Combinada , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Isomerismo , Proteínas Recombinantes , Ribavirina/uso terapéuticoRESUMEN
Derivatization of protein-based therapeutics with polyethylene glycol (pegylation) can often improve pharmacokinetic and pharmacodynamic properties of the proteins and thereby, improve efficacy and minimize dosing frequency. This review will provide an overview of pegylation technology and pegylated protein-based drugs being used or investigated clinically. The novel therapeutic, PEG Intron(R), formed by attaching a 12-kDa mono-methoxy polyethylene glycol (PEG) to the interferon alpha-2b protein, will be discussed in detail in terms of its structure, biological activities, pharmacokinetic properties, and clinical efficacy for the treatment of chronic hepatitis C. Detailed physicochemical and biological characterization studies of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Pegylation of Intron A at pH 6.5 results in a mixture of > or = 95% mono-pegylated isoforms with the predominant species (approximately 50%) derivatized to the His(34) residue with the remaining positional isomers pegylated at various lysines, the N-terminal cysteine, as well as serine, tyrosine, and another histidine residue. The anti-viral activity for each pegylated isomer showed that the highest specific activity (37%) was associated with the His(34)-pegylated isomer. Though pegylation decreases the specific activity of the interferon alpha-2b protein in vitro, the potency of PEG Intron was comparable to the Intron A standard at both the molecular and cellular level. The substituted IFN had an enhanced pharmacokinetic profile in both animal and human studies, and, when combined with ribavirin, was very effective in reducing hepatitis C viral load and maintaining sustained viral suppression in patients.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Interferón-alfa/química , Interferón-alfa/uso terapéutico , Polietilenglicoles/química , Polietilenglicoles/uso terapéutico , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Fenómenos Químicos , Química Física , Citocinas/química , Citocinas/uso terapéutico , Humanos , Concentración de Iones de Hidrógeno , Interferón alfa-2 , Interferón-alfa/farmacocinética , Interferón-alfa/farmacología , Datos de Secuencia Molecular , Polietilenglicoles/farmacocinética , Polietilenglicoles/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Relación Estructura-ActividadRESUMEN
NMR-based screening of a customized fragment library identified 16 small-molecule hits that bind weakly (K(D) approximately 100 microM to 10 mM) to substrate binding sites of the NS4A-bound NS3 protease of the hepatitis C virus (HCV). Analogues for five classes of NMR hits were evaluated by a combination of NMR and biochemical data yielding SAR and, in most cases, optimized hits with improved potencies (K(D) approximately K(I) approximately 40 microM to 1 mM). NMR chemical shift perturbation data were used to establish the binding location and orientation of the active site directed scaffolds in these five analogue series. Two of these scaffolds, which bind the enzyme at the proximal S1-S3 and S2' substrate binding sites, were linked together producing competitive inhibitors of the HCV NS3 protease with potencies in the micromolar range. This example illustrates that the low molecular weight scaffolds discovered from structure-based NMR screening can be optimized with focused structure-guided chemistry to produce potent nonpeptidic small-molecule inhibitors of the HCV NS3 protease.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Inhibidores Enzimáticos/química , Hepacivirus/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Anilidas/química , Derivados del Benceno/química , Sitios de Unión , Bases de Datos Factuales , Inhibidores Enzimáticos/síntesis química , Indoles/química , Péptidos y Proteínas de Señalización Intracelular , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Pyridine carboxamide-based inhibitors of the hepatitis C virus (HCV) NS5B polymerase were diversified and optimized to a variety of topologically related scaffolds. In particular, the 2-methyl nicotinic acid scaffold was developed into inhibitors with improved biochemical (IC50-GT1b = 0.014 µM) and cell-based HCV replicon potency (EC50-GT1b = 0.7 µM). Biophysical and biochemical characterization identified this novel series of compounds as palm site binders to HCV polymerase.
RESUMEN
A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit (3), with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores (10 and 23) were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180 degrees in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates, as revealed by 3. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Espectroscopía de Resonancia Magnética , Bibliotecas de Moléculas Pequeñas/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Guanidinas/síntesis química , Guanidinas/química , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Estudios de Validación como AsuntoRESUMEN
Fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design were used to identify novel inhibitors for BACE-1. A rapid optimization of an initial NMR hit was achieved by a combination of NMR and a functional assay, resulting in the identification of an isothiourea hit with a K(d) of 15 microM for BACE-1. NMR data and the crystal structure revealed that this hit makes H-bond interactions with the two catalytic aspartates, occupies the nonprime side region of the active site of BACE-1, and extends toward the S3 subpocket (S3sp). A focused NMR-based search for heterocyclic isothiourea isosteres resulted in several distinct classes of BACE-1 active site directed compounds with improved chemical stability and physicochemical properties. The strategy for optimization of the 2-aminopyridine lead series to potent inhibitors of BACE-1 was demonstrated. The structure-based design of a cyclic acylguanidine lead series and its optimization into nanomolar BACE-1 inhibitors are the subject of the companion paper
Asunto(s)
Aminopiridinas/química , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Espectroscopía de Resonancia Magnética , Bibliotecas de Moléculas Pequeñas/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-ActividadRESUMEN
Accumulation of the cytotoxic 40- to 42-residue beta-amyloid peptide represents the primary pathological process in Alzheimer's disease (AD). BACE1 (beta-site APP cleaving enzyme 1) is responsible for the initial required step in the neuronal amyloidogenic processing of beta-amyloid precursor protein and is a major drug target for the therapeutic intervention of AD. In the present study, BACE1 is initially synthesized as an immature precursor protein containing part of the pre domain and the entire pro domain, and undergoes autocatalytic conversion to yield the well-folded mature BACE1 enzyme. To understand the mechanism of the conversion and the role of the pro domain, we monitored the autocatalytic conversion of BACE1 by heteronuclear NMR spectroscopy and used chemical shift perturbations as a probe to study the structural changes accompanying the autocatalytic conversion. NMR data revealed local conformational changes from a partially disordered to a well-folded conformation associated with the conversion. The conformational changes are largely concentrated in the NH(2)-terminal lobe. Conversely, the active site conformations are conserved during the autocatalytic conversion. The precursor and mature BACE1 proteins were further characterized for their ability to interact with a substrate-based transition state BACE1 peptide inhibitor. The precursor BACE1 rapidly adopted the bound conformation in the presence of the inhibitor, which is identical to the bound conformation of the mature protein. The interaction of the inhibitor with both the precursor BACE1 and the fully processed BACE1 is in slow exchange on the NMR time scale, indicating a tight binding interaction. Overall, the NMR data demonstrated that the pro domain does not hinder inhibitor binding and may assist in the proper folding of the protein. The fully processed BACE1 represents a high quality well-folded protein which is highly stable over a long period of time, and is suitable for evaluation of inhibitor binding by NMR for drug intervention.
Asunto(s)
Endopeptidasas/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Catálisis , Endopeptidasas/química , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
The reported competition STD NMR method combines saturation transfer difference (STD) NMR with competition binding experiments to allow the detection of high-affinity ligands that undergo slow chemical exchange on the NMR time-scale. With this technique, the presence of a competing high-affinity ligand in the compound mixture can be detected by the disappearance or reduction of the STD signals of a low-affinity indicator ligand. This is demonstrated on a BACE1 (beta-site amyloid precursor protein cleaving enzyme 1) protein-inhibitor system. This method can also be used to derive an approximate value, or a lower limit, for the dissociation constant of the potential ligand based on the reduction of the signal intensity of the STD indicator, which is illustrated on an HSA (human serum albumin) model system. This leads to important applications of the competition STD NMR method for lead discovery: it can be used (i) for compound library screening against a broad range of drug targets to identify both high- and low-affinity ligands and (ii) to rank order analogs rapidly and derive structure-activity relationships, which are used to optimize these NMR hits into viable drug leads.