RESUMEN
A human in vitro resorption assay has been developed using osteoclastoma-derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the alphavbeta3 integrin, and inhibitors of cathepsin K and the osteoclast ATPase. The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human alphavbeta3-mediated cell adhesion assay for the vitronectin receptor antagonists (r2 = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H+-ATPase inhibitors (r2 = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r2 = 0.80). Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue. These cells can be stored long-term in liquid nitrogen and upon thawing maintain their bone-resorbing phenotype. The cryopreserved cells can be cultured on bovine cortical bone for 24-48 h and resorption can be measured by either confocal microscopy or biochemical assays. The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds. In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C-telopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent assay. Resorption can be measured reproducibly using a 48-h incubation of osteoclasts on bone slices, or a 24-h incubation with bone particles. This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Óseas/química , Resorción Ósea , Tumor Óseo de Células Gigantes/química , Adenosina Trifosfatasas/metabolismo , Animales , Catepsina K , Catepsinas/metabolismo , Bovinos , Adhesión Celular , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Congelación , Humanos , Técnicas In Vitro , Fenotipo , Reproducibilidad de los ResultadosRESUMEN
We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Chz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.
Asunto(s)
Aldehídos/farmacología , Resorción Ósea , Catepsinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Oligopéptidos/farmacología , Animales , Artritis Experimental/metabolismo , Calcio/sangre , Catepsina K , Catepsinas/genética , Bovinos , Femenino , Humanos , Hormona Paratiroidea/farmacología , Paratiroidectomía , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/antagonistas & inhibidores , Tiroidectomía , Células Tumorales CultivadasRESUMEN
C7 mimetics, designed to lock three amino acid residues of a peptide chain into a gamma-turn conformation, were introduced sequentially between the P3 to P2' positions of a model HIV-1 protease substrate I (resulting in compounds II-IV) to probe its conformational requirements in binding to HIV-1 protease. Of these, compound IIIa with the C7 mimetic replacing Asn-Tyr-Pro, corresponding to the P2 through P1' positions of substrate, was found to be an inhibitor with a Ki of 147 microM. Reduction of the amide bond in the C7 mimetic of IIIa resulted in a novel constrained reduced-amide mimetic VIa with a Ki of 430 nM. This corresponds to over a 300-fold improvement in inhibitory activity over the original C7 mimetic. The inhibitory activity of mimetic VIa was in addition found to be 44-fold better than a similar linear reduced-amide containing inhibitor V. The synthesis of these mimetics are described.
Asunto(s)
Inhibidores de la Proteasa del VIH/síntesis química , Simpatomiméticos/síntesis química , Inhibidores de la Proteasa del VIH/metabolismo , Relación Estructura-Actividad , Simpatomiméticos/metabolismoRESUMEN
The rational design and synthesis of a highly potent inhibitor of HIV-1 protease have been accomplished. The inhibitor, SB 206343, is based on a model derived from the structure of the MVT-101/HIV-1 protease complex and contains a 4(5)-acylimidazole ring as an isosteric replacement for the P1'--P2' amide bond. It is a competitive inhibitor with an apparent inhibition constant of 0.6 nM at pH 6.0. The three-dimensional structure of SB 206343 bound in the active site of HIV-1 protease has been determined at 2.3 A resolution by X-ray diffraction techniques and refined to a crystallographic discrepancy factor, R (= sigma parallel Fo magnitude of/Fc parallel/sigma magnitude of), of 0.194. The inhibitor is held in the enzyme by a set of hydrophobic and polar interactions. N-3 of the imidazole ring participates in a novel hydrogen-bonding interaction with the bound water molecule, demonstrating the effectiveness of the imidazole ring as an isosteric replacement for the P1'--P2' amide bond in hydroxyethylene-based HIV-1 protease inhibitors. Also present are hydrogen-bonding interactions between N-1 of the imidazole ring and the carbonyl of Gly-127 as well as between the imidazole acyl carbonyl oxygen and the amide nitrogen of Asp-129, exemplifying the peptidomimetic nature of the 4(5)-acylimidazole isostere. All of these interactions are in qualitative agreement with those predicted by the model.
Asunto(s)
Amidas/síntesis química , Amidas/farmacología , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/farmacología , Imidazoles/síntesis química , Imidazoles/farmacología , Amidas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Proteasa del VIH/efectos de los fármacos , Proteasa del VIH/metabolismo , Imidazoles/química , Imidazoles/metabolismo , Modelos Moleculares , Estructura Molecular , Reproducibilidad de los Resultados , Estereoisomerismo , Relación Estructura-Actividad , Valina/análogos & derivados , Valina/síntesis química , Valina/química , Valina/farmacologíaRESUMEN
Peptidomimetic cathepsin K inhibitors have been designed using binding models which were based on the X-ray crystal structure of an amino acid-based, active site-spanning inhibitor complexed with cathepsin K. These inhibitors, which contain a benzyloxybenzoyl group in place of a Cbz-leucine moiety, maintained good inhibitory potency relative to the amino acid-based inhibitor, and the binding models were found to be very predictive of relative inhibitor potency. The binding mode of one of the inhibitors was confirmed by X-ray crystallography, and the crystallographically determined structure is in close qualitative agreement with the initial binding model. These results strengthen the validity of a strategy involving iterative cycles of structure-based design, inhibitor synthesis and evaluation, and crystallographic structure determination for the discovery of peptidomimetic inhibitors.
Asunto(s)
Benzoatos/síntesis química , Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/síntesis química , Diseño de Fármacos , Péptidos/química , Benzoatos/química , Benzoatos/metabolismo , Sitios de Unión , Catepsina K , Catepsinas/metabolismo , Cristalografía por Rayos X , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/metabolismo , Modelos Moleculares , Imitación Molecular , Relación Estructura-ActividadRESUMEN
Papain has been used as a surrogate enzyme in a drug design effort to obtain potent and selective inhibitors of cathepsin K, a new member of the papain superfamily of cysteine proteases that is selectively and highly expressed in osteoclasts and is implicated in bone resorption. Here we report the crystal structures of two papain-inhibitor complexes and the rational design of novel cathepsin K inhibitors. Unlike previously known crystal structures of papain-inhibitor complexes, our papain structures show ligand binding extending deep within the S'-subsites. The two inhibitor complexes, carbobenzyloxyleucinyl-leucinyl-leucinal and carbobenzyloxy-L-leucinyl-L-leucinyl methoxymethyl ketone, were refined to 2.2- and 2.5-A resolution with R-factors of 0.190 and 0. 217, respectively. The S'-subsite interactions with the inhibitors are dominated by an aromatic-aromatic stacking and an oxygen-aromatic ring edge interaction. The knowledge of S'-subsite interactions led to a design strategy for an inhibitor spanning both subsites and yielded a novel, symmetric inhibitor selective for cathepsin K. Simultaneous exploitation of both S- and S'-sites provides a general strategy for the design of cysteine protease inhibitors having high specificity to their target enzymes.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/química , Dipéptidos/química , Leupeptinas/química , Modelos Moleculares , Papaína/química , Sitios de Unión , Catepsina K , Cristalografía por Rayos X , Inhibidores de Cisteína Proteinasa/metabolismo , Dipéptidos/metabolismo , Diseño de Fármacos , Leupeptinas/metabolismo , Papaína/metabolismo , Estructura Terciaria de ProteínaRESUMEN
The synthesis, in vitro activities, and pharmacokinetics of a series of azepanone-based inhibitors of the cysteine protease cathepsin K (EC 3.4.22.38) are described. These compounds show improved configurational stability of the C-4 diastereomeric center relative to the previously published five- and six-membered ring ketone-based inhibitor series. Studies in this series have led to the identification of 20, a potent, selective inhibitor of human cathepsin K (K(i) = 0.16 nM) as well as 24, a potent inhibitor of both human (K(i) = 0.0048 nM) and rat (K(i,app) = 4.8 nM) cathepsin K. Small-molecule X-ray crystallographic analysis of 20 established the C-4 S stereochemistry as being critical for potent inhibition and that unbound 20 adopted the expected equatorial conformation for the C-4 substituent. Molecular modeling studies predicted the higher energy axial orientation at C-4 of 20 when bound within the active site of cathepsin K, a feature subsequently confirmed by X-ray crystallography. Pharmacokinetic studies in the rat show 20 to be 42% orally bioavailable. Comparison of the transport of the cyclic and acyclic analogues through CaCo-2 cells suggests that oral bioavailability of the acyclic derivatives is limited by a P-glycoprotein-mediated efflux mechanism. It is concluded that the introduction of a conformational constraint has served the dual purpose of increasing inhibitor potency by locking in a bioactive conformation as well as locking out available conformations which may serve as substrates for enzyme systems that limit oral bioavailability.
Asunto(s)
Azepinas/síntesis química , Catepsinas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Leucina/síntesis química , Administración Oral , Animales , Azepinas/química , Azepinas/farmacocinética , Azepinas/farmacología , Disponibilidad Biológica , Catepsina K , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Leucina/análogos & derivados , Leucina/química , Leucina/farmacocinética , Leucina/farmacología , Espectrometría de Masas , Modelos Moleculares , Estructura Molecular , Osteoclastos/efectos de los fármacos , Unión Proteica , Ratas , Estereoisomerismo , Relación Estructura-ActividadAsunto(s)
Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/síntesis química , Diaminas/síntesis química , Cetonas/síntesis química , Péptidos/química , Sitios de Unión , Catepsina K , Catepsinas/metabolismo , Cristalografía por Rayos X , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Diaminas/química , Diaminas/farmacología , Cetonas/química , Cetonas/farmacología , Modelos Moleculares , Conformación Molecular , Imitación Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Beef heart mitochondrial ATPase (F1) catalyzes the hydrolysis of the ATP analog adenyl-5-yl imidodiphosphate (AMP-PNP). The reaction products are inorganic phosphate and adenyl-5-yl phosphoramidate (AMP-PN) as determined by HPLC analysis. The hydrolysis occurs in both the presence and absence of added divalent metal ions and is stimulated by potassium. The kinetic properties of the hydrolytic reaction depend markedly on the identity of the added divalent metal. GMP-PNP and AMP-CPP are also hydrolyzed, while AMP-PCP is not. Adenyl-5-yl phosphoramidate is a potent effect of beef heart mitochondrial ATPase activity. Based on these data, a reinterpretation of work based on the assumption that AMP-PNP is not hydrolyzed is presented.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Adenilil Imidodifosfato/metabolismo , Antibacterianos , Mitocondrias Cardíacas/metabolismo , ATPasas de Translocación de Protón/metabolismo , Animales , Bovinos , Hidrólisis , Cinética , Miocardio/metabolismo , Péptidos/farmacología , Potasio/farmacologíaRESUMEN
The pH dependence of the peptidolytic reaction of recombinant human immunodeficiency virus type 1 protease has been examined over a pH range of 3-7 for four oligopeptide substrates and two competitive inhibitors. The pK values obtained from the pKis vs pH profiles for the unprotonated and protonated active-site aspartyl groups, Asp-25 and Asp-25', in the monoprotonated enzyme form were 3.1 and 5.2, respectively. Profiles of log V/K vs pH for all four substrates were "bell-shaped" in which the pK values for the unprotonated and protonated aspartyl residues were 3.4-3.7 and 5.5-6.5, respectively. Profiles of log V vs pH for these substrates were "wave-shaped" in which V was shifted to a constant lower value upon protonation of a residue of pK = 4.2-5.2. These results indicate that substrates bind only to a form of HIV-1 protease in which one of the two catalytic aspartyl residues is protonated. Solvent kinetic isotope effects were measured over a pH (D) range of 3-7 for two oligopeptide substrates, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 and Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2. The pH-independent value for DV/K was 1.0 for both substrates, and DV = 1.5-1.7 and 2.2-3.2 at low and high pH (D), respectively. The attentuation of both V and DV at low pH (D) is consistent with a change in rate-limiting step from a chemical one at high pH (D) to one in which a product release step or an enzyme isomerization step becomes partly rate-limiting at low pH (D). Proton inventory data is in accord with the concerted transfer of two protons in the transition state of a rate-limiting chemical step in which the enzyme-bound amide hydrate adduct collapses to form the carboxylic acid and amine products.
Asunto(s)
Proteasa del VIH/química , Secuencia de Aminoácidos , Sitios de Unión , Unión Competitiva , Catálisis , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Oligopéptidos/química , Isótopos de Oxígeno , Solventes , Especificidad por SustratoRESUMEN
This study examined the inhibition of azide as a probe of the magnesium regulation of beef heart mitochondrial ATPase (F1) catalysis. Azide elicited a slow hysteretic effect on both ATP and ITP hydrolysis of F1. This hysteretic effect was shown to be due to the consecutive binding of magnesium and azide, and to be independent of catalytic turnover. The azide binding site was also shown to be separate from the anion binding HCO3- site on F1. The results presented indicate that metal binding is important in the inhibition of the hydrolytic activity and regulation of F1. A model is presented which is consistent with the hysteretic inhibition of F1 by azide, in which there is a slow equilibration between free enzyme and the enzyme-magnesium-azide complex.
Asunto(s)
Azidas/farmacología , Mitocondrias Cardíacas/enzimología , ATPasas de Translocación de Protón/antagonistas & inhibidores , Animales , Azidas/antagonistas & inhibidores , Sitios de Unión/efectos de los fármacos , Unión Competitiva , Catálisis , Bovinos , Cinética , Magnesio/farmacología , Matemática , Modelos QuímicosRESUMEN
Several divalent metal ions were used as kinetic probes of the beef heart mitochondrial adenosinetriphosphatase (F1) under a variety of conditions, and the relationship between the properties of the catalytic metal ion and the catalytic activity of the enzyme was examined. Vmax for ATP hydrolysis was largest when metal ions characterized by intermediate values of acidity of coordinated water molecules (pKa) and metal-nucleotide stability constants (Kstab) were present. As temperature increased, the peak of Vmax vs. pKa (or Kstab) shifted to lower initial values of pKa or Kstab. The solvent deuterium isotope effect on Vmax (DV) was normal and largest when the metal ion present during F1-catalyzed ATP hydrolysis was most acidic and the metal nucleotide stability constant was large. When an active site tyrosine on F1 was nitrated, Vmax was most affected when the metal ion present was least acidic and the metal nucleotide stability constant was small. The isotope effect on V/K (DV/K) was normal, small, and apparently independent of the metal ion present. ADP inhibition of F1-catalyzed ATP hydrolysis is competitive, and the Ki is independent of the metal ion present. The degree of Pi inhibition of F1 is dependent on the metal ion present. The inhibition by Pi is competitive at low temperature and becomes noncompetitive as temperature increases. These and previous results support a mechanism whereby a water molecule coordinated to the metal ion of an enzyme-bound gamma-monodentate metal-ATP complex is deprotonated to begin a series of events whereby a beta,gamma-bidentate metal-ATP complex is produced. Upon hydrolysis, the bond between the metal ion and the beta-phosphate of ADP in the Pi-metal-ADP complex is broken before products (ADP and metal-Pi) are released.
Asunto(s)
Mitocondrias Cardíacas/enzimología , ATPasas de Translocación de Protón/metabolismo , Adenosina Difosfato/farmacología , Animales , Cationes Bivalentes , Bovinos , Cinética , ATPasas de Translocación de Protón/aislamiento & purificación , TermodinámicaRESUMEN
Purified HIV-1 protease hydrolyzes H-Ser-Gln-Asn-Leu-Phe(NO2)-Leu-Asp-Gly-NH2 (Peptide 1) and acetyl-Arg-Lys-Ile-Leu-Phe(NO2)-Leu-Asp-Gly-NH2 (Peptide 2) between the (p-nitro)phenylalanyl and leucyl residues. The cleavage of Peptides 1 and 2 resulted in a decrease in uv absorbance at 310 nm. The HIV-1 protease-catalyzed peptidolysis of Peptides 1 and 2 was characterized by a linear time course at substrate turnover of less than 20%. The solubilities of these substrates at pH 5.0 were sufficient to provide initial rate measurements over a concentration range of 0.05-0.5 mM. Steady-state kinetic data and inhibition constants using both spectrophotometric and high performance liquid chromatography (HPLC) analysis of the peptidolysis of these substrates resulted in comparable values.
Asunto(s)
Endopeptidasas/análisis , Productos del Gen pol/análisis , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Proteasa del VIH , VIH-1/enzimología , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Oligopéptidos , Proteínas Recombinantes , Espectrofotometría/métodosRESUMEN
Simian immunodeficiency virus protease (SIV-PR) was produced in Escherichia coli with a recombinant expression system in which the mature enzyme autoprocessed from a precursor form. Recombinant SIV and HIV-1 (human immunodeficiency virus, type 1) proteases were purified from bacterial cell lysates by use of sequential steps of ammonium sulfate precipitation and size-exclusion and ion-exchange chromatography. The amino acid composition, amino-terminal sequence, and molecular weight (monomer) of the recombinant SIV-PR were in accord with that of the 99 amino acid polypeptide predicted from the SIVMac-PR nucleotide sequence. The active form of SIV-PR was shown to be dimeric by gel filtration chromatography. Inhibition by pepstatin A, time-dependent inactivation by 1,2-epoxy-3-(4-nitrophenoxy)propane, and pH rate profiles using oligopeptide substrates demonstrated that SIV-PR behaves as an aspartic protease. Recombinant HIV-1 Pr55gag precursor was processed in vitro by SIV-PR and HIV-1 PR with indistinguishable proteolytic patterns upon NaDodSO4-polyacrylamide gel electrophoresis. Oligopeptide substrates for HIV-1 PR were found to be suitable substrates for recombinant SIV-PR with the exception of a peptide containing the site identified for p66/p51 cleavage (Phe*Tyr) within HIV-1 reverse transcriptase (RT). Several synthetic peptide analogue inhibitors of HIV-1 PR were also potent inhibitors of SIV-PR, indicating that SIV infection in macaques and rhesus monkeys should be useful models for the preclinical evaluation of acquired immunodeficiency syndrome (AIDS) therapeutics targeted towards the virally encoded HIV-1 protease.
Asunto(s)
Endopeptidasas/aislamiento & purificación , Proteasa del VIH/aislamiento & purificación , VIH-1/enzimología , Proteínas de los Retroviridae/aislamiento & purificación , Virus de la Inmunodeficiencia de los Simios/enzimología , Secuencia de Aminoácidos , Animales , Endopeptidasas/química , Endopeptidasas/clasificación , Proteasa del VIH/clasificación , Proteasa del VIH/genética , Inhibidores de la Proteasa del VIH , VIH-1/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/farmacología , Conformación Proteica , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas de los Retroviridae/antagonistas & inhibidores , Proteínas de los Retroviridae/clasificación , Proteínas de los Retroviridae/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Especificidad por SustratoRESUMEN
Using binding models which were based on the X-ray crystal structure of an amino acid-based active site-spanning inhibitor complexed with cathepsin K, Cbz-leucine mimics have been developed, leading ultimately to the design of a potent cathepsin K inhibitor free of amino acid components. These mimics, which consist of alpha-substituted biphenylacetyl groups in place of Cbz-leucine moieties, effectively mimic all aspects of the Cbz-leucine moieties which are important for inhibitor binding. The predicted directions of binding for the inhibitors were confirmed by mass spectral analysis of their complexes with cathepsin K, which gave results consistent with acylation of the enzyme and loss of the acylhydrazine portion of the inhibitor which binds on the S' side of the active site. The binding models were found to be very predictive of relative inhibitor potency as well as direction of inhibitor binding. These results strengthen the validity of a strategy involving iterative cycles of structure-based design and inhibitor synthesis and evaluation for the discovery of non-peptide inhibitors.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Diseño de Fármacos , Catepsina K , Cinética , Modelos MolecularesRESUMEN
Potential advantages of C2-symmetric inhibitors designed for the symmetric HIV-1 protease include high selectivity, potency, stability, and bioavailability. Pseudo-C2-symmetric monools and C2-symmetric diols, containing central hydroxymethylene and (R,R)-dihydroxyethylene moieties flanked by a variety of hydrophobic P1/P1' side chains, were studied as HIV-1 protease inhibitors. The monools and diols were synthesized in 8-10 steps from D-(+)-arabitol and D-(+)-mannitol, respectively. Monools with ethyl or isobutyl P1/P1' side chains were weak inhibitors of recombinant HIV-1 protease (Ki > 10 microM), while benzyl P1/P1' side chains afforded a moderately potent inhibitor (apparent Ki = 230 nM). Diols were 100-10,000x more potent than analogous monools, and a wider range of P1/P1' side chains led to potent inhibition. Both classes of compounds exhibited lower apparent Ki values under high-salt conditions. Surprisingly, monool and diol HIV-1 protease inhibitors were potent inhibitors of porcine pepsin, a prototypical asymmetric monomeric aspartic protease. These results were evaluated in the context of the pseudosymmetric structure of monomeric aspartic proteases and their evolutionary kinship with the retroviral proteases. The X-ray crystal structure of HIV-1 protease complexed with a symmetric diol was determined at 2.6 A. Contrary to expectations, the diol binds the protease asymmetrically and exhibits 2-fold disorder in the electron density map. Molecular dynamics simulations were conducted beginning with asymmetric and symmetric HIV-1 protease/inhibitor model complexes. A more stable trajectory resulted from the asymmetric complex, in agreement with the observed asymmetric binding mode. A simple four-point model was used to argue more generally that van der Waals and electrostatic force fields can commonly lead to an asymmetric association between symmetric molecules.
Asunto(s)
Dipéptidos/farmacología , Glicoles/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/efectos de los fármacos , VIH-1/enzimología , Alcoholes/química , Alcoholes/metabolismo , Dipéptidos/síntesis química , Dipéptidos/química , Dipéptidos/metabolismo , Glicoles/síntesis química , Glicoles/metabolismo , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Pepsina A/antagonistas & inhibidores , Conformación Proteica , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Relación Estructura-ActividadRESUMEN
The structure-based design and synthesis of lactam-constrained azapeptide inhibitors of human cathepsin K are described. Enhanced stability to proteolytic cleavage over acyclic analogues is discussed.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/farmacología , Osteoclastos/enzimología , Pirrolidinonas/síntesis química , Aminas/química , Catepsina K , Catepsinas/metabolismo , Cristalografía por Rayos X , Inhibidores de Cisteína Proteinasa/metabolismo , Diseño de Fármacos , Humanos , Modelos Moleculares , Péptidos/química , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacología , Relación Estructura-ActividadRESUMEN
To more rapidly prepare members of the 1,3-bis(acylamino)-2-butanone class of cysteine protease inhibitors, a solid-phase synthesis was developed. 1-Azido-3-amino-2,2-dimethoxybutane (4), which has the two amino groups differentiated and the ketone protected as a a ketal, served as a surrogate for the 1,3-diamino-2-butanone core. Amine (4) was coupled to the BAL-resin-linked carboxylic acids derived from alpha-amino acid esters. Evaluation of a small combinatorial array by measuring inhibition constants (Ki,appS) against cathepsins K, L, and B provided some structure-activity relationship trends with respect to selectivity and potency. Novel, potent inhibitors of cathepsins K and L were identified.
Asunto(s)
Butanonas/síntesis química , Catepsinas/antagonistas & inhibidores , Técnicas Químicas Combinatorias/métodos , Inhibidores de Cisteína Proteinasa/síntesis química , Endopeptidasas , Butanonas/química , Butanonas/farmacología , Catepsina K , Catepsina L , Cisteína Endopeptidasas , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Diseño de Fármacos , Cinética , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
The human immunodeficiency virus type 1 (HIV-1) protease is a potential target of acquired immune deficiency syndrome (AIDS) therapy. A highly potent, perfectly symmetrical phosphinate inhibitor of this enzyme, SB204144, has been synthesized. It is a competitive inhibitor of HIV-1 protease, with an apparent inhibition constant of 2.8 nM at pH 6.0. The three-dimensional structure of SB204144 bound to the enzyme has been determined at 2.3-A resolution by X-ray diffraction techniques and refined to a crystallographic discrepancy factor, R (= sigma parallel F(o) magnitude to - Fc parallel/sigma magnitude of F(o)), of 0.178. The inhibitor is held in the enzyme active site by a set of hydrophobic and hydrophilic interactions, including an interaction between Arg8 and the center of the terminal benzene rings of the inhibitor. The phosphinate establishes a novel interaction with the two catalytic aspartates; each oxygen of the central phosphinic acid moiety interacts with a single oxygen of one aspartic acid, establishing a very short (2.2-2.4 A) oxygen-oxygen contact. As with the structures of penicillopepsin bound to phosphinate and phosphonate inhibitors [Fraser, M. E., Strynadka, N. C., Bartlett, P. A., Hanson, J. E., & James, M. N. (1992) Biochemistry 31, 5201-14], we interpret this short distance and the stereochemical environment of each pair of oxygens in terms of a hydrogen bond that has a symmetric single-well potential energy curve with the proton located midway between the two atoms.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Inhibidores de la Proteasa del VIH/síntesis química , VIH-1/enzimología , Compuestos Organofosforados/síntesis química , Ácidos Fosfínicos , Valina/análogos & derivados , Secuencia de Aminoácidos , Sitios de Unión , Catálisis/efectos de los fármacos , Cristalización , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/metabolismo , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Alcoholes del Azúcar/química , Valina/síntesis química , Valina/química , Valina/metabolismo , Difracción de Rayos XRESUMEN
The design, synthesis, enzymologic, and protein mass spectrometric characterization of benzodioxocin-3-one and N-acyl-3-amino-3-buten-2-one inhibitors of the cysteine protease cathepsin K are described. The benzodioxocin-3-one ring system is chemically unstable giving rise to a mixture of N-acyl-3-amino-3-buten-2-one and hemiketals. This mixture of N-acyl-3-amino-3-buten-2-one and hemiketals potently inhibits recombinant, human cathepsin K (IC50 = 36 nM) by a time-independent, irreversible mechanism. Formation of a covalent adduct between cathepsin K and inhibitor has been confirmed by mass spectrometry.