RESUMO
The Biophorum Development Group (BPDG) is an industry-wide consortium enabling networking and sharing of best practices for the development of biopharmaceuticals. To gain a better understanding of current industry approaches for establishing biopharmaceutical drug product (DP) robustness, the BPDG-Formulation Point Share group conducted an intercompany collaboration exercise, which included a bench-marking survey and extensive group discussions around the scope, design, and execution of robustness studies. The results of this industry collaboration revealed several key common themes: (1) overall DP robustness is defined by both the formulation and the manufacturing process robustness; (2) robustness integrates the principles of quality by design (QbD); (3) DP robustness is an important factor in setting critical quality attribute control strategies and commercial specifications; (4) most companies employ robustness studies, along with prior knowledge, risk assessments, and statistics, to develop the DP design space; (5) studies are tailored to commercial development needs and the practices of each company. Three case studies further illustrate how a robustness study design for a biopharmaceutical DP balances experimental complexity, statistical power, scientific understanding, and risk assessment to provide the desired product and process knowledge. The BPDG-Formulation Point Share discusses identified industry challenges with regard to biopharmaceutical DP robustness and presents some recommendations for best practices.
Assuntos
Indústria Farmacêutica/métodos , Preparações Farmacêuticas/química , Biofarmácia/métodos , Química Farmacêutica/métodos , Ensaios Clínicos como Assunto , Desenho de Fármacos , Humanos , Colaboração Intersetorial , Medição de Risco , Tecnologia Farmacêutica/métodosRESUMO
Endocannabinoids such as 2-arachidonylglycerol (2-AG) are ligands for cannabinoid receptors that contribute to the transmission and modulation of pain signals. The antinociceptive effect of exogenous 2-AG suggests that inhibition of monoglyceride lipase (MGLL), the enzyme responsible for degrading 2-AG and arresting signaling, may be a target for pain modulation. Here we describe the characterization of MGLL ligands following a high-throughput screening campaign. Ligands were discovered using ThermoFluor, a label-free affinity-based screening tool that measures ligand binding via modulation of protein thermal stability. A kinetic fluorescent assay using the substrate 4-methylcoumarin butyrate was used to counterscreen confirmed HTS positives. A comparison of results from binding and inhibition assays allowed elucidation of compound mechanism of action. We demonstrate the limit of each technology and the benefits of using orthogonal assay techniques in profiling compounds.
Assuntos
Domínio Catalítico/efeitos dos fármacos , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Monoacilglicerol Lipases/antagonistas & inibidores , Ácidos Araquidônicos/química , Endocanabinoides , Inibidores Enzimáticos/química , Glicerídeos/química , Ensaios de Triagem em Larga Escala , Humanos , Hidrólise , Concentração Inibidora 50 , Cinética , Monoacilglicerol Lipases/química , Monoacilglicerol Lipases/metabolismo , Ligação Proteica , Solubilidade , Especificidade por SubstratoRESUMO
Thermodynamic principles of cooperativity and allostery have long been used as a starting point to begin understanding the interplay between ligand binding events. Understanding the nature of allosteric effects requires an experimental technique that can be used to quantify ligand binding energies and simultaneously give experimental insights into the conformational dynamics at play upon ligand binding. CD spectroscopy provides macroscopic information about the relative secondary and tertiary structures present in a protein. Here, we use this spectroscopic technique with thermal shift assays wherein ligand binding constants can be quantified based on their stabilizing effect against thermally induced protein denaturation. Binding constants for two ligands are used to determine a pairwise coupling free energy which defines the shared energy that favors or opposes binding of the second ligand binding event in an allosteric system. In CD-based thermal shift assays, temperature is the driving force for protein unfolding and can also influence protein conformational dynamics present in the unbound protein or ligand-bound proteins. Dihydrofolate reductase (DHFR) and glutamate dehydrogenase (GDH) are proposed as example test systems. NADP and methotrexate bind DHFR with positive cooperativity. Mammalian GDH exhibits negative cooperativity with respect to binding of NAD and NADPH coenzyme molecules, activation by ADP, and inhibition by GTP.
Assuntos
Termodinâmica , Regulação Alostérica/fisiologia , Dicroísmo Circular , Glutamato Desidrogenase/metabolismo , Ligantes , Ligação Proteica , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Measuring the strength of binding of low molecular weight ligands to a target protein is a significant challenge to fragment-based drug discovery that must be solved. Thermal shift assays are uniquely suited for this purpose, due to the thermodynamic effects of a ligand on protein thermal stability. We show here how to implement a thermal shift assay, describing the basic features and analysis of the protein unfolding data. We then describe in detail the effects of a ligand on the observed stability of the protein to produce a shift in stability. The anatomy of ligand-induced thermal shift data is discussed in detail. We describe the unique aspects of concentration-response curves, the effect of protein unfolding energetics, and the stoichiometry of the interaction. We outline a typical assay development strategy for optimizing dye type and concentration, protein concentration, and buffer conditions. Guidelines are presented to demonstrate the limits of detection for weak-binding ligands, as applied to sulfonamide-based inhibitors of carbonic anhydrase II and applied to nucleotide binding to the death-associated protein kinase 1 catalytic domain.
Assuntos
Descoberta de Drogas/métodos , Naftalenossulfonato de Anilina/química , Proteínas Reguladoras de Apoptose/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/química , Inibidores da Anidrase Carbônica/química , Proteínas Quinases Associadas com Morte Celular , Corantes Fluorescentes/química , Ligantes , Peso Molecular , Nucleotídeos , Ligação Proteica , Desnaturação Proteica , Estabilidade Proteica , Proteínas/química , Sulfonamidas/farmacologia , TermodinâmicaRESUMO
A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.
Assuntos
Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ácidos Araquidônicos/química , Ácidos Araquidônicos/metabolismo , Moduladores de Receptores de Canabinoides/química , Moduladores de Receptores de Canabinoides/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Endocanabinoides , Glicerídeos/química , Glicerídeos/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica , Eletricidade EstáticaRESUMO
The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.
Assuntos
Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/química , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Soluções Tampão , Fluorescência , Concentração de Íons de Hidrogênio , Metais/farmacologia , Concentração Osmolar , Estrutura Quaternária de Proteína/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/isolamento & purificação , Receptor de Fator Estimulador de Colônias de Macrófagos/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/normas , Sais/farmacologia , Soluções/farmacologia , TermodinâmicaRESUMO
Calmodulin is a central mediator of calcium-dependent signal transduction pathways and regulates the activity of a large number of diverse targets. Calcium-dependent interactions of calmodulin with regulated proteins are of generally high affinity but of quite variable thermodynamic origins. Here we investigate the influence of the binding of the calmodulin-binding domain of calmodulin kinase I on the fast internal dynamics of calcium-saturated calmodulin. NMR relaxation was used to probe motion on the backbone (viewed through the backbone amide NH group) and the side chains (viewed through methyl groups). The distribution of the amplitudes of side chain dynamics is trimodal. The microscopic details of side chain motion are compared with those of a thermodynamically and structurally similar complex of calmodulin with the calmodulin-binding domain of the smooth muscle myosin light chain kinase. While there are no significant differences in backbone dynamics and no net change in methyl-bearing side chain dynamics, a large redistribution of the amplitude of methyl dynamics is observed between the two complexes. The variation in dynamics was largely localized to the heterogeneously dynamic target-binding interface, suggesting that differential dynamics of the binding surface plays a functional role in the high-affinity binding interactions of calmodulin. These results begin to reveal a fundamental role for residual protein entropy in molecular recognition by calmodulin.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Entropia , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Galinhas , Ligação de Hidrogênio , Modelos Moleculares , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , TitulometriaRESUMO
ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.
Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Animais , Varredura Diferencial de Calorimetria , Anidrase Carbônica I/química , Anidrase Carbônica I/metabolismo , Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/química , Bovinos , Estabilidade Enzimática , Humanos , Técnicas In Vitro , Cinética , Desnaturação Proteica , Espectrometria de Fluorescência , TermodinâmicaRESUMO
The temperature dependence of the fast internal dynamics of calcium-saturated calmodulin in complex with a peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light chain kinase is examined using 15N and 2H NMR relaxation methods. NMR relaxation studies of the complex were carried out at 13 temperatures that span 288-346 K. The dynamics of the backbone and over four dozen methyl-bearing side chains, distributed throughout the calmodulin molecule, were probed. The side chains show a much more variable and often considerably larger response to temperature than the backbone. A significant variation in the temperature dependence of the amplitude of motion of individual side chains is seen. The amplitude of motion of some side chains is essentially temperature-independent while many show a simple roughly linear temperature dependence. In a few cases, angular order increases with temperature, which is interpreted as arising from interactions with neighboring residues. In addition, a number of side chains display a nonlinear temperature dependence. The significance of these and other results is illuminated by several simple interpretative models. Importantly, analysis of these models indicates that changes in generalized order parameters can be robustly related to corresponding changes in residual entropy. A simple cluster model that incorporates features of cooperative or conditional motion reproduces many of the unusual features of the experimentally observed temperature dependence and illustrates that side chain interactions result in a dynamically changing environment that significantly influences the motion of internal side chains. This model also suggests that the intrinsic entropy of interacting clusters of side chains is only modestly reduced from that of independent side chain motion. Finally, estimates of protein heat capacity support the view that the major contribution to the heat capacity of protein solutions largely arises from local bond vibrations and solvent interactions and not from torsional oscillations of side chains.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Músculo Liso/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Fragmentos de Peptídeos/metabolismo , Temperatura , Animais , Sítios de Ligação , Calmodulina/química , Galinhas , Entropia , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Quinase de Cadeia Leve de Miosina/química , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Termodinâmica , Água/metabolismoRESUMO
Calmodulin (CaM) is an intracellular calcium-binding protein essential for many pathways in eukaryotic signal transduction. Although a structure of Ca(2+)-saturated Paramecium CaM at 1.0 A resolution (1EXR.pdb) provides the highest level of detail about side-chain orientations in CaM, information about an end state alone cannot explain driving forces for the transitions that occur during Ca(2+)-induced conformational switching and why the two domains of CaM are saturated sequentially rather than simultaneously. Recent studies focus attention on the contributions of interdomain linker residues. Electron paramagnetic resonance showed that Ca(2+)-induced structural stabilization of residues 76-81 modulates domain coupling [Qin and Squier (2001) Biophys. J. 81, 2908-2918]. Studies of N-domain fragments of Paramecium CaM showed that residues 76-80 increased thermostability of the N-domain but lowered the Ca(2+) affinity of sites I and II [Sorensen et al. (2002) Biochemistry 41, 15-20]. To probe domain coupling during Ca(2+) binding, we have used (1)H-(15)N HSQC NMR to monitor more than 40 residues in Paramecium CaM. The titrations demonstrated that residues Glu78 to Glu84 (in the linker and cap of helix E) underwent sequential phases of conformational change. Initially, they changed in volume (slow exchange) as sites III and IV titrated, and subsequently, they changed in frequency (fast exchange) as sites I and II titrated. These studies provide evidence for Ca(2+)-dependent communication between the domains, demonstrating that spatially distant residues respond to Ca(2+) binding at sites I and II in the N-domain of CaM.
Assuntos
Cálcio/química , Calmodulina/química , Paramecium/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Cátions Bivalentes/química , Dados de Sequência Molecular , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Prótons , Ratos , TitulometriaRESUMO
The peptide antibiotic ramoplanin is highly effective against several drug-resistant gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by binding to Lipid intermediates I and II at a location different than the N-acyl-D-Ala-D-Ala dipeptide site targeted by vancomycin. Lipid I/II capture physically occludes these substrates from proper utilization by the late-stage PG biosynthesis enzymes MurG and the transglycosylases. Key structural features of ramoplanin responsible for antibiotic activity and PG molecular recognition have been discovered by antibiotic semisynthetic modification in conjunction with NMR analyses. These results help define a minimalist ramoplanin pharmacophore and introduce the possibility of generating ramoplanin-derived peptide or peptidomimetic antibiotics for use against VRE, MRSA, and related pathogens.
Assuntos
Antibacterianos/química , Depsipeptídeos , Peptídeos Cíclicos/química , Antibacterianos/farmacologia , Dimerização , Desenho de Fármacos , Estabilidade de Medicamentos , Glicosilação , Conformação Molecular , Ornitina , Peptídeos Cíclicos/farmacologia , Peptidoglicano/biossíntese , Peptidoglicano/efeitos dos fármacos , Peptidoglicano/metabolismo , Relação Estrutura-AtividadeRESUMO
The peptide antibiotic ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by interrupting late-stage membrane-associated glycosyltransferase reactions catalyzed by the transglycosylase and MurG enzymes. The mechanism of ramoplanin involves sequestration of lipid-anchored PG biosynthesis intermediates, physically occluding these substrates from proper utilization by these enzymes. In this report, we describe the first molecular-level details of the interaction of ramoplanin with PG biosynthesis intermediates. NMR analysis in conjunction with chemical dissection of the PG monomer revealed that the ramoplanin octapeptide D-Hpg-D-Orn-D-alloThr-Hpg-D-Hpg-alloThr-Phe-D-Orn recognizes MurNAc-Ala-gamma-D-Glu pyrophosphate, the minimum component of PG capable of high-affinity complexation and fibril formation. Ramoplanin therefore recognizes a PG binding locus different from the N-acyl-D-Ala-D-Ala moiety targeted by vancomycin. Because ramoplanin is structurally less complex than glycopeptide antibiotics such as vancomycin, peptidomimetic chemotherapeutics derived from this recognition sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, and related pathogens.
Assuntos
Antibacterianos/química , Bacillus subtilis , Depsipeptídeos , Peptídeos Cíclicos , Peptidoglicano/química , Sequência de Aminoácidos , Glicopeptídeos/química , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Peptidoglicano/biossíntese , Peptidoglicano/ultraestrutura , Conformação ProteicaRESUMO
Ca(2+)-saturated calmodulin (CaM) directly associates with and activates CaM-dependent protein kinase I (CaMKI) through interactions with a short sequence in its regulatory domain. Using heteronuclear NMR (13)C-(15)N-(1)H correlation experiments, the backbone assignments were determined for CaM bound to a peptide (CaMKIp) corresponding to the CaM-binding sequence of CaMKI. A comparison of chemical shifts for free CaM with those of the CaM. CaMKIp complex indicate large differences throughout the CaM sequence. Using NMR techniques optimized for large proteins, backbone resonance assignments were also determined for CaM bound to the intact CaMKI enzyme. NMR spectra of CaM bound to either the CaMKI enzyme or peptide are virtually identical, indicating that calmodulin is structurally indistinguishable when complexed to the intact kinase or the peptide CaM-binding domain. Chemical shifts of CaM bound to a peptide (smMLCKp) corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase are also compared with the CaM. CaMKI complexes. Chemical shifts can differentiate one complex from another, as well as bound versus free states of CaM. In this context, the observed similarity between CaM. CaMKI enzyme and peptide complexes is striking, indicating that the peptide is an excellent mimetic for interaction of calmodulin with the CaMKI enzyme.
Assuntos
Calmodulina/química , Calmodulina/metabolismo , Peptídeos/química , Animais , Calmodulina/farmacologia , Núcleo Celular/metabolismo , Galinhas , DNA/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismoRESUMO
Amide hydrogen exchange has been used to examine the structural dynamics and energetics of the interaction of a peptide corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase (smMLCKp) with calcium-saturated calmodulin. Heteronuclear NMR (15)N-(1)H correlation spectroscopy was used to quantify amide proton exchange rates of the uniformly (15)N-labeled domain bound to calmodulin. A key feature of a proposed model for molecular recognition by calmodulin [Ehrhardt et al. (1995) Biochemistry 34, 2731-2738] is tested by examination of the dependence of amide hydrogen exchange on applied hydrostatic pressure. Hydrogen exchange rates and corresponding protection factors (1/K(op)) for individual amide protons of the bound smMLCKp domain span 5 orders of magnitude at ambient pressure. Individual protection factors decrease significantly in a linear fashion with increasing hydrostatic pressure. A common pressure dependence is revealed by a constant large negative volume change across the residues comprising the core of the bound helical domain. The pattern of protection factors and their response to hydrostatic pressure is consistent with a structural reorganization that results in the concerted disruption of ion pairs between calmodulin and the bound domain. These observations reinforce a model for the molecular recognition pathway where formation of the initial encounter complex is followed by helix-coil transitions in the bound state and subsequent concerted formation of the extensive ion pair network defining the intermolecular contact surface between CaM and the target domain in the final, compact complex structure.