RESUMEN
Targeting of cryptic binding sites represents an attractive but underexplored approach to modulating protein function with small molecules. Using the dimeric protease (Pr) from Kaposi's sarcoma-associated herpesvirus (KSHV) as a model system, we sought to dissect a putative allosteric network linking a cryptic site at the dimerization interface to enzyme function. Five cryogenic X-ray structures were solved of the monomeric protease with allosteric inhibitors bound to the dimer interface site. Distinct coordinated movements captured by the allosteric inhibitors were also revealed as alternative states in room-temperature X-ray data and comparative analyses of other dimeric herpesvirus proteases. A two-step mechanism was elucidated through detailed kinetic analyses and suggests an enzyme isomerization model of inhibition. Finally, a representative allosteric inhibitor from this class was shown to be efficacious in a cellular model of viral infectivity. These studies reveal a coordinated dynamic network of atomic communication linking cryptic binding site occupancy and allosteric inactivation of KHSV Pr that can be exploited to target other members of this clinically relevant family of enzymes.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/enzimología , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/farmacología , Cristalografía por Rayos X , Infecciones por Herpesviridae/tratamiento farmacológico , Herpesvirus Humano 8/química , Herpesvirus Humano 8/efectos de los fármacos , Humanos , Modelos Moleculares , Péptido Hidrolasas/química , Conformación Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacosRESUMEN
The Large-neutral Amino Acid Transporter 1 (LAT-1)--a sodium-independent exchanger of amino acids, thyroid hormones, and prescription drugs--is highly expressed in the blood-brain barrier and various types of cancer. LAT-1 plays an important role in cancer development as well as in mediating drug and nutrient delivery across the blood-brain barrier, making it a key drug target. Here, we identify four LAT-1 ligands, including one chemically novel substrate, by comparative modeling, virtual screening, and experimental validation. These results may rationalize the enhanced brain permeability of two drugs, including the anticancer agent acivicin. Finally, two of our hits inhibited proliferation of a cancer cell line by distinct mechanisms, providing useful chemical tools to characterize the role of LAT-1 in cancer metabolism.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Transportador de Aminoácidos Neutros Grandes 1/química , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Modelos Moleculares , Aminas/metabolismo , Análisis de Varianza , Línea Celular Tumoral , Cristalografía por Rayos X , Ácidos Ciclohexanocarboxílicos/metabolismo , Citometría de Flujo , Gabapentina , Glioblastoma/metabolismo , Células HEK293 , Humanos , Isoxazoles/farmacocinética , Leucina/metabolismo , Ligandos , Tritio , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds 2 and 3) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, ß, and γ). Inhibition data reveal that compound 2 has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) J. Biol. Chem. 266, 15591-15594] show DD2, compound 2, and compound 3 inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure-activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein-protein interactions.
Asunto(s)
Herpesvirus Humano 8/enzimología , Inhibidores de Proteasas/química , Serina Endopeptidasas/química , Regulación Alostérica , Humanos , Espectroscopía de Resonancia MagnéticaRESUMEN
Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.
Asunto(s)
Enfermedad de Chagas , Inhibidores de Topoisomerasa II , Triazoles , Trypanosoma , Tripanosomiasis Africana , Animales , Humanos , Ratones , Enfermedad de Chagas/tratamiento farmacológico , Microscopía por Crioelectrón , ADN-Topoisomerasas de Tipo II/metabolismo , Trypanosoma/efectos de los fármacos , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/uso terapéutico , Triazoles/química , Triazoles/farmacología , Triazoles/uso terapéutico , Tripanosomiasis Africana/tratamiento farmacológico , Evaluación Preclínica de MedicamentosRESUMEN
Human African Trypanosomiasis (HAT) is a vector-borne disease caused by kinetoplastid parasites of the Trypanosoma genus. The disease proceeds in two stages, with a hemolymphatic blood stage and a meningo-encephalic brain stage. In the latter stage, the parasite causes irreversible damage to the brain leading to sleep cycle disruption and is fatal if untreated. An orally bioavailable treatment is highly desirable. In this study, we present a brain-penetrant, parasite-selective 20S proteasome inhibitor that was rapidly optimized from an HTS singleton hit to drug candidate compound 7 that showed cure in a stage II mouse efficacy model. Here, we describe hit expansion and lead optimization campaign guided by cryo-electron microscopy and an in silico model to predict the brain-to-plasma partition coefficient Kp as an important parameter to prioritize compounds for synthesis. The model combined with in vitro and in vivo experiments allowed us to advance compounds with favorable unbound brain-to-plasma ratios (Kp,uu) to cure a CNS disease such as HAT.
Asunto(s)
Quinolinas , Trypanosoma , Tripanosomiasis Africana , Animales , Microscopía por Crioelectrón , Modelos Animales de Enfermedad , Humanos , Ratones , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Quinolinas/farmacología , Quinolinas/uso terapéutico , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitologíaRESUMEN
Artificial intelligence (AI) is becoming established in drug discovery. For example, many in the industry are applying machine learning approaches to target discovery or to optimize compound synthesis. While our organization is certainly applying these sorts of approaches, we propose an additional approach: using AI to augment human intelligence. We have been working on a series of recommendation systems that take advantage of our existing laboratory processes, both wet and computational, in order to provide inspiration to our chemists, suggest next steps in their work, and automate existing workflows. We will describe five such systems in various stages of deployment within the Novartis Institutes for BioMedical Research. While each of these systems addresses different stages of the discovery pipeline, all of them share three common features: a trigger that initiates the recommendation, an analysis that leverages our existing systems with AI, and the delivery of a recommendation. The goal of all of these systems is to inspire and accelerate the drug discovery process.
Asunto(s)
Inteligencia Artificial , Química Farmacéutica/métodos , Descubrimiento de Drogas/métodos , Investigación Farmacéutica/métodos , Química Farmacéutica/organización & administración , Bases de Datos de Compuestos Químicos , Correo Electrónico , Humanos , Investigación Farmacéutica/organización & administración , Investigadores/psicología , Encuestas y CuestionariosRESUMEN
LL-37 is a broad-spectrum human antimicrobial peptide in the cathelicidin family. Potency assays in the form of minimal inhibitory concentration and vesicle leakage indicate that the single-tryptophan mutants, F6W and F17W, are as effective at killing bacteria and disrupting membranes as the native, tryptophan-free LL-37 peptide. Steady-state fluorescence and UV resonance Raman spectroscopy of F6W and F17W reveal molecular details of these tryptophan residues. The local environment polarity, hydrogen bond strength of the indole N-H moiety, and rotational freedom decrease for both F6W and F17W in the presence of carbonate ions relative to in pure distilled water; these results are consistent with burial of the hydrophobic region of alpha-helical LL-37 in oligomeric cores induced in the presence of carbonate ions. Differences in the spectroscopic properties of the carbonate-induced alpha-helical forms of F6W and F17W reflect the presence of a local lysine residue near F6W that makes the microenvironment of F6W more polar than that of F17W. In the presence of lipid vesicles, the mutants undergo additional loss of environment polarity, hydrogen bond strength, and rotational freedom. Quenching experiments utilizing brominated lipids reveal that the tryptophan residues in both mutants are essentially equidistant from the bilayer center and that bromines closer to the bilayer center, in the 9,10 positions, quench fluorescence more efficiently than those closer to the headgroups (6,7 positions). These results support carpeting or toroidal pore mechanisms of membrane disruption by LL-37 and demonstrate that the combination of tryptophan mutants and sensitive spectroscopic tools may provide important molecular clues about antimicrobial action.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Mutación Puntual , Péptidos Catiónicos Antimicrobianos/química , Bromo/química , Bromo/metabolismo , Humanos , Enlace de Hidrógeno , Lisina/química , Lisina/genética , Lisina/metabolismo , Lípidos de la Membrana/química , Lípidos de la Membrana/genética , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Mutación Puntual/genética , Mutación Puntual/fisiología , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Espectrometría Raman , Triptófano/química , Triptófano/genética , Triptófano/metabolismo , CatelicidinasRESUMEN
Refolding curves of the integral membrane protein outer membrane protein A (OmpA) were measured to determine the conformational stabilities of this model system for membrane protein folding. Wild-type OmpA exhibits a free energy of unfolding (DeltaG degrees H2O) of 10.5 kcal/mol. Mutants, containing a single tryptophan residue at the native positions 7, 15, 57, 102, or 143, are less stable than wild-type OmpA, with DeltaG degrees H2O values of 6.7, 4.8, 2.4, 4.7, and 2.8 kcal/mol, respectively. The trend observed here is discussed in terms of noncovalent interactions, including aromatic interactions and hydrogen bonding. The effect of the soluble tail on the conformational stability of the transmembrane domain of OmpA was also investigated via truncated single-Trp mutants; DeltaG degrees H2O values for four of the five truncated mutants are greater by >2.7 kcal/mol relative to the full-length versions, suggesting that the absence of the soluble domain may destabilize the unfolded transmembrane domain. Finally, dynamic light scattering experiments were performed to measure the effects of urea and protein on vesicle size and stability. Urea concentrations greater than 1 M cause an increase in vesicle size, and these diameters are unaltered in the presence of protein. These dynamic light scattering results complement the fluorescence studies and illustrate the important effects of vesicle size on protein conformational stability.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Pliegue de Proteína , Triptófano/metabolismo , Liposomas Unilamelares/química , Proteínas de la Membrana Bacteriana Externa/genética , Modelos Moleculares , Mutación , Conformación Proteica , Renaturación de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Dispersión de Radiación , Solubilidad , Espectrometría de Fluorescencia , Termodinámica , Liposomas Unilamelares/metabolismo , Urea/farmacologíaRESUMEN
Protein folding is an exploding area of research in biophysics and physical chemistry. Here, we describe the integration of several techniques, including absorption spectroscopy, fluorescence spectroscopy, and Förster resonance energy transfer (FRET) measurements, to probe important topics in protein folding. Cytochrome c is used as a model protein; comparison of conformational stabilities ( ΔGH2O∘) measured via two chemical denaturants, urea and guanidinium hydrochloride, illustrate important concepts in protein folding and intermolecular interactions. In addition, the determination of intraprotein distances based upon the FRET pair Trp-59 and the heme group for unfolded states of cytochrome c highlights the evolution of the protein structure under unfolding conditions. Analysis and discussion of these results provide opportunities to gain in-depth understanding of models for protein folding while enhancing students' skills with optical techniques. Collectively, the combination of optical spectroscopy, rigorous quantitative analysis, and a focus on biophysics illustrates the significance of fundamental research at the growing intersection of chemistry, biology, and physics.
RESUMEN
Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces.
Asunto(s)
Herpesvirus Humano 8/enzimología , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Unión Proteica/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
All members of the human herpesvirus protease (HHV Pr) family are active as weakly associating dimers but inactive as monomers. A small-molecule allosteric inhibitor of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low micromolar affinity. A 2.0-Å-resolution X-ray crystal structure of a C-terminal truncated KSHV Pr-inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 Å from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease via a similar mechanism. As all HHV Prs are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics that allosterically regulate enzymatic activity by disrupting protein-protein interactions.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Herpesvirus Humano 8/enzimología , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Sitio Alostérico , Sitios de Unión , Dominio Catalítico , Dicroismo Circular , Cristalografía por Rayos X , Dimerización , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis , Unión Proteica , Conformación Proteica , Serina Endopeptidasas/genéticaRESUMEN
Ultraviolet resonance Raman (UVRR) spectra of tryptophan compounds in various solvents and a model peptide are presented and reveal systematic changes that reflect solvent polarity, hydrogen bond strength, and cation-pi interaction. The commonly utilized UVRR spectral marker for environment polarity that has been based on off-resonance Raman data, the tryptophan Fermi doublet ratio I1360/I1340, exhibits different values in on- and off-resonance Raman spectra as well as for different tryptophan derivatives. Specifically, the UVRR Fermi doublet ratio for indole ranges from 0.3 in polar solvents to 0.8 in nonpolar solvents, whereas the respective values reported here and previously for off-resonance Raman spectra are 0.5-1.3. UVRR Fermi doublet ratios for the more biologically relevant molecule, N-acetyl tryptophan ethyl ester (NATEE), are in a smaller range of 1.1 (polar solvent) to 1.7 (nonpolar solvent) and correlate to the solvent polarity/polarization parameters pi* and ETN. As has been reported previously, several UVRR modes are also sensitive to the hydrogen bond strength of the indole N-H moiety. Here, we report a new unambiguous marker for H-bonding: the ratio of the W10 (approximately 1237 cm-1) intensity to that of the W9 (approximately 1254 cm-1) mode (RW10). This ratio is 0.7 for NATEE in the absence of hydrogen bond acceptors and increases to 3.1 in the presence of strong hydrogen bond acceptors, with a value of 2.3 in water. The W8 and W17 modes shift more than +10 and approximately -5 cm-1 upon increase in hydrogen bond strength; this range for W17 is smaller than that reported previously and reflects a more realistic range for proteins and peptides in solution. Finally, our data provide evidence for change in the W18 and W16 relative intensity in the presence of cation-pi interactions. These UVRR markers are utilized to interpret spectra of model membrane-bound systems tryptophan octyl ester and the peptide toxin melittin. These spectra reveal the importance of intra- and intermolecular hydrogen bonding and cation-pi interactions that likely influence the partitioning of membrane-associated biomolecules to lipid bilayers or self-associated soluble oligomers. The UVRR analysis presented here modifies and augments prior reports and provides an unambiguous set of spectral makers that can be applied to elucidate the molecular microenvironment and structure of a wide range of complex systems, including anchoring tryptophan residues in membrane proteins and peptides.
Asunto(s)
Proteínas de la Membrana/química , Solventes/química , Espectrometría Raman , Triptófano/química , Enlace de Hidrógeno , Meliteno/química , Espectrometría de Fluorescencia , Triptófano/análogos & derivadosRESUMEN
The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of <200-nm bilayer vesicles composed of anionic and neutral lipids as well as cholesterol. Vesicle disruption, or peptide potency, was monitored with a sensitive fluorescence leakage assay. Detailed molecular information on peptide-membrane interactions and peptide structure was further gained through vibrational spectroscopy combined with circular dichroism. Finally, steady-state fluorescence experiments yielded insight into the local environment of native or engineered tryptophan residues in melittin and human cathelicidin embedded in bilayer vesicles. Collectively, our results provide clues to the functional structures of the engineered and toxic peptides and may impact the design of synthetic antibiotic peptides that can be used against the growing number of antibiotic-resistant pathogens.