RESUMO
Next-generation site-specific cysteine-based antibody-drug-conjugates (ADCs) broaden therapeutic index by precise drug-antibody attachments. However, manufacturing such ADCs for clinical validation requires complex full reduction and reoxidation processes, impacting product quality. To overcome this technical challenge, we developed a novel antibody manufacturing process through cysteine (Cys) metabolic engineering in Chinese hamster ovary cells implementing a unique cysteine-capping technology. This development enabled a direct conjugation of drugs after chemoselective-reduction with mild reductant tris(3-sulfonatophenyl)phosphine. This innovative platform produces clinical ADC products with superior quality through a simplified manufacturing process. This technology also has the potential to integrate Cys-based site-specific conjugation with other site-specific conjugation methodologies to develop multi-drug ADCs and exploit multi-mechanisms of action for effective cancer treatments.
Assuntos
Antineoplásicos , Imunoconjugados , Animais , Anticorpos , Antineoplásicos/uso terapêutico , Células CHO , Cricetinae , Cricetulus , Cisteína , Dissulfetos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Engenharia MetabólicaRESUMO
Prolyl 4-hydroxylases install a hydroxyl group in the 4R configuration on the gamma-carbon atom of certain (2S)-proline (Pro) residues in tropocollagen, elastin, and other proteins to form (2S,4R)-4-hydroxyproline (Hyp). The gauche effect arising from this prevalent post-translational modification enforces a C(gamma)-exo ring pucker and stabilizes the collagen triple helix. The Hyp diastereomer (2S,4S)-4-hydroxyproline (hyp) has not been observed in a protein, despite the ability of electronegative 4S substituents to enforce the more common C(gamma)-endo ring pucker of Pro. Here, we use density functional theory, spectroscopy, crystallography, and calorimetry to explore the consequences of hyp incorporation on protein stability using a collagen model system. We find that the 4S-hydroxylation of Pro to form hyp does indeed enforce a C(gamma)-endo ring pucker but a transannular hydrogen bond between the hydroxyl moiety and the carbonyl of hyp distorts the main-chain torsion angles that typically accompany a C(gamma)-endo ring pucker. This same transannular hydrogen bond enhances an n-->pi* interaction that stabilizes the trans conformation of the peptide bond preceding hyp, endowing hyp with the unusual combination of a C(gamma)-endo ring pucker and high trans/cis ratio. O-Methylation of hyp to form (2S,4S)-4-methoxyproline (mop) eliminates the transannular hydrogen bond and restores a prototypical C(gamma)-endo pucker. mop residues endow the collagen triple helix with much more conformational stability than do hyp residues. These findings highlight the critical importance of the configuration of the hydroxyl group installed on C(gamma) of proline residues.
Assuntos
Hidroxiprolina/química , Colágeno/química , Simulação por Computador , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Peptídeos/síntese química , Peptídeos/química , Proteínas/química , Estereoisomerismo , TermodinâmicaRESUMO
Using solid-state (23)Na NMR and quantum chemical calculations we have found that the Na(+) ion bound to a calix[4]arene-guanosine conjugate dimer resides slightly above the G-quartet plane and simultaneously coordinates to a water molecule in a square-pyramidal (penta-coordination) geometry.
RESUMO
Collagen is the most abundant protein in animals. Every third residue in a collagen strand is a glycine with phi, psi = -70 degrees, 175 degrees. A recent computational study suggested that replacing these glycine residues with D-alanine or D-serine would stabilize the collagen triple helix. This hypothesis is of substantial importance, as the glycine residues in collagen constitute nearly 10% of the amino acid residues in humans. To test this hypothesis, we synthesized a series of collagen mimic peptides that contain one or more D-alanine or D-serine residues replacing the canonical glycine residues. Circular dichroism spectroscopy and thermal denaturation experiments indicated clearly that the substitution of glycine with D-alanine or D-serine greatly disfavors the formation of a triple helix. Host-guest studies also revealed that replacing a single glycine residue with D-alanine is more destabilizing than is its replacement with L-alanine, a substitution that results from a common mutation in patients with collagen-related diseases. These data indicate that the glycine residues in collagen are not a surrogate for a D-amino acid and support the notion that the main-chain torsion angles of a glycine residue in the native structure (especially, phi > 0 degrees ) are critical determinants for its beneficial substitution with a D-amino acid in a protein.
Assuntos
Aminoácidos/química , Colágeno/química , Glicina/química , Alanina/química , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Químicos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Serina/química , TermodinâmicaRESUMO
Cysteine-conjugated antibody-drug conjugates (ADCs) are manufactured using controlled partial reduction and conjugation chemistry with drug payloads that typically occur in intervals of 0, 2, 4, 6, and 8. Control of heterogeneity is of particular importance to the quality of ADC product because drug loading and distribution can affect the safety and efficacy of the ADC. Liquid chromatography ultra-violet (LC-UV)-based methods can be used to acquire the drug distribution profiles of cysteine-conjugated ADCs when analyzed using hydrophobic interaction chromatography (HIC). However, alternative analysis techniques are often required for structural identification when conjugated drugs do not possess discrete ultra-violet absorbance properties for precise assessment of the drug-to-antibody ratio (DAR). In this study, multidimensional chromatography was used as an efficient method for combining non-compatible techniques, such as HIC, with analysis by mass spectrometry (LC/LC/QTOF-MS) for rapid on-line structural elucidation of species observed in HIC distribution profiles of cysteine-conjugated ADCs. The methodology was tested using an IgG1 mAb modified by cysteine conjugation with a non-toxic drug mimic. Structural elucidation of peaks observed in the HIC analysis (1(st) dimension) were successfully identified based on their unique sub-unit masses via mass spectrometry techniques once dissociation occurred under denaturing reversed phase conditions (2(nd) dimension). Upon identification, the DAR values were determined to be 2.83, 4.44, and 5.97 for 3 drug load levels (low-, medium-, and high-loaded ADC batches), respectively, based on relative abundance from the LC-UV data. This work demonstrates that multidimensional chromatography coupled with MS, provides an efficient approach for on-line biotherapeutic characterization to ensure ADC product quality.
Assuntos
Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Cisteína/química , Imunoconjugados/química , Espectrometria de Massas/métodos , Humanos , Imunoconjugados/imunologia , Imunoglobulina G/química , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
This paper illustrates the use of a kinetically controlled exchange reaction to effect regioselective modification of a hydrogen-bonded assembly. Both the bound anion and cation can control the exchange of ligand into the different layers of a synthetic G-quadruplex.
RESUMO
Collagen is the most abundant protein in animals and the major component of connective tissues. Although collagen isolated from natural sources has long served as the basis for some biomaterials, natural collagen is difficult to modify and can engender pathogenic and immunological side effects. Collagen comprises a helix of three strands. Triple helices derived from synthetic peptides are much shorter (<10 nm) than natural collagen (approximately 300 nm), limiting their utility. Here, we describe the synthesis of short collagen fragments in which the three strands are held in a staggered array by disulfide bonds. Data from CD spectroscopy, dynamic light scattering, analytical ultracentrifugation, atomic force microscopy, and transmission electron microscopy indicate that these "sticky-ended" fragments self-assemble via intermolecular triple-helix formation. The resulting fibrils resemble natural collagen, and some are longer (>400 nm) than any known collagen. We anticipate that our self-assembly strategy can provide synthetic collagen-mimetic materials for a variety of applications.
Assuntos
Colágeno/química , Colágeno/metabolismo , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Colágeno/síntese química , Colágeno/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Estrutura Secundária de ProteínaRESUMO
This contribution describes the discovery and properties of a synthetic, low-molecular weight compound that transports Cl- across bilayer membranes. Such compounds have potential as therapeutics for cystic fibrosis and cancer. The H+/Cl- co-transport activities of acyclic tetrabutylamides 1-6 were compared by using a pH-stat assay with synthetic EYPC liposomes. The ion transport activity of the most active compound, trimer 3, was an order of magnitude greater than that of calix[4]arene tetrabutylamide C1 a macrocycle known to function as a synthetic ion channel. Trimer 3 has an unprecedented function for a synthetic compound, as it induces a stable potential in liposomes experiencing a transmembrane Cl-/SO42- gradient. Data from both pH-stat and 35Cl NMR experiments indicate that 3 co-transports H+/Cl-. Although 3 transports both Cl- and H+ the overall process is not electrically silent. Thus, trimer 3 induces a stable potential in LUVs due to a transmembrane anionic gradient. The ability of trimer 3 to transport Cl-, to maintain a transmembrane potential, along with its high activity at uM concentrations, its low molecular weight, and its simple preparation, make this compound a valuable lead in drug development for diseases caused by Cl- transport malfunction.
Assuntos
Acetamidas/química , Cloretos/química , Bicamadas Lipídicas/química , Acetamidas/metabolismo , Acetatos/química , Cloretos/metabolismo , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana , PrótonsRESUMO
Nucleic acid quadruplexes are composed of guanine quartets stabilized by specific metal ions. X-ray diffraction can provide high-resolution information on the structure and metal binding properties of quadruplexes, but only if they can be crystallized. NMR can provide detailed information on the solution structure of such quadruplexes but little quantitative data concerning the metal binding site. Here we apply extended X-ray absorption fine structure (EXAFS) measurements to characterize the metal ion binding site, in frozen solution, of the unimolecular quadruplex formed by the thrombin binding aptamer, d(G(2)T(2)G(2)TGTG(2)T(2)G(2)) (TBA), in the presence of Pb(2+) ions. The Pb L(III) -edge X-ray absorption spectrum of this metal-DNA complex is very similar to that we obtain for a Pb(2+)-stabilized quartet system of known structure constructed from a modified guanine nucleoside (G1). The Fourier transforms of the Pb(2+) complexes with both TBA and G1 show a first-shell interaction at about 2.6 A, and a weaker, broader shell at 3.5-4.0 A. Quantitative analysis of the EXAFS data reveals the following: (i) very close agreement between interatomic distances at the metal coordination site for the Pb(2+)-G1 complex determined by EXAFS and by X-ray crystallography; (ii) similarly close agreement between interatomic distances measured by EXAFS for the Pb(2+)-G1 and Pb(2+)-TBA complexes. These results provide strong evidence for binding of the Pb(2+) ion in the region between the two quartets in the Pb(2+)-TBA complex, coordinated to the eight surrounding guanine O6 atoms. The specific binding of Pb(2+) to DNA examined here may be relevant to the genotoxic effects of this environmentally important heavy metal. Furthermore, these results demonstrate the utility of EXAFS as a method for quantitative characterization of specific metal binding sites in nucleic acids in solution.
Assuntos
DNA/química , Sítios de Ligação , Dicroísmo Circular , Cristalografia por Raios X , DNA/metabolismo , Guanosina/metabolismo , Chumbo/metabolismoRESUMO
The ion transport activity of calix[4]arene tetrabutylamide 1,3-alt 2 was studied in liposomes, planar lipid bilayers, and HEK-293 cells. These experiments, when considered together with (1)H NMR and X-ray crystallography data, indicate that calix[4]arene tetrabutylamide 2 (1) forms ion channels in bilayer membranes, (2) mediates ion transport across cell membranes at positive holding potential, (3) alters the pH inside liposomes experiencing a Cl(-) gradient, and (4) shows a significant Cl(-)/SO(4)(2)(-) transport selectivity. An analogue, calix[4]arene tetramethylamide 1, self-assembles in the presence of HCl to generate solid-state structures with chloride-filled and water-filled channels. Structureminus signactivity studies indicate that the hydrophobicity, amide substitution, and macrocyclic framework of the calixarene are essential for HCl binding and transport. Calix[4]arene tetrabutylamide 2 is a rare example of an anion-dependent, synthetic ion channel.
Assuntos
Calixarenos , Ácido Clorídrico/química , Canais Iônicos/química , Bicamadas Lipídicas/química , Fenóis/química , Amidas/química , Amidas/metabolismo , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Cloretos/química , Cloretos/metabolismo , Humanos , Ácido Clorídrico/metabolismo , Ligação de Hidrogênio , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Lipossomos , Potenciais da Membrana , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Técnicas de Patch-Clamp , Fenóis/metabolismo , Sulfatos/química , Sulfatos/metabolismoRESUMO
In this paper, we report on the formation and properties of a water-stabilized dimer comprising calix[4]arene-guanosine conjugate cG 2. The 1,3-alternate calixarene cG 2 was poorly soluble in dry CDCl(3) and gave an ill-resolved NMR spectrum, consistent with its nonspecific aggregation. The compound was much more soluble in water-saturated CDCl(3). Two sets of well-resolved (1)H NMR signals for the guanosine residues in cG 2, present in a 1:1 ratio, indicated that the compound's D(2) symmetry had been broken and provided the first hint that cG 2 dimerizes in water-saturated CDCl(3). The resulting dimer, (cG 2)(2).(H(2)O)(n)(), has a unique property: it extracts alkali halide salts from water into organic solution. This dimer is a rare example of a self-assembled ion pair receptor. The identity of the (cG 2)(2).NaCl.(H(2)O)(n)() dimer was confirmed by comparing its self-diffusion coefficient in CDCl(3), determined by pulsed-field gradient NMR, with that of control compound cA 3, an adenosine analogue. The dimer's stoichiometry was also confirmed by quantitative measurement of the cation and anion using ion chromatrography. Two-dimensional NMR and ion-induced NMR shifts indicated that the cation binding site is formed by an intermolecular G-quartet and the anion binding site is provided by the 5'-amide NH groups. Once bound, the salt increases the dimer's thermal stability. Both (1)H NMR and ion chromatography measurements indicated that the cG 2 dimer has a modest selectivity for extracting K(+) over Na(+) and Br(-) over Cl(-). The anion's identity also influences the association process: NaCl gives a soluble, discrete dimer whereas addition of NaBPh(4) to (cG 2)(2).(H(2)O)(n)() leads to extensive aggregation and precipitation. This study suggests a new direction for ion pair receptors, namely, the use of molecular self-assembly. The study also highlights water's ability to stabilize a functional noncovalent assembly.