Search

Antibody-drug conjugates (ADCs) have demonstrated a great therapeutic potential against cancer due to their target specificity and cytotoxicity. To exert a maximum therapeutic effect on cancerous cells, we have conjugated two different payloads to different amino acids, cysteines (cys) and lysines (lys), on trastuzumab, which is a humanised anti-HER2 monoclonal antibody. First, trastuzumab was conjugated with monomethyl auristatin E (MMAE), an antimitotic agent, through a cleavable linker (Val-Cit) to prepare ADC (Tmab-VcMMAE). Then, the ADC (Tmab-VcMMAE) was conjugated with a second antimitotic agent, Mertansine (DM1), via a non-cleavable linker Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) to form a dual conjugate (Tmab-VcMMAE-SMCC-DM1). Our results indicated that the dual-payload conjugate, Tmab-VcMMAE-SMCC-DM1, had a synergistic and superior cytotoxic effect compared to trastuzumab alone. Ultimately employing a dual conjugation approach has the potential to overcome treatment-resistance and tumour recurrences and could pave the way to employ other payloads to construct dual (or multiple) payload complexes. [ABSTRACT FROM AUTHOR]

A series of benzimidazolium salts having 2‐cyanobenzyl (2–6), 2‐(4‐nitrophenyl)ethyl (7–10), (1,3‐dioxoisoindolin‐2‐yl)butyl (11–17) or N‐methylphthalimide (18–22) substitution were synthesized and characterized by spectroscopic and analytical techniques. The in vitro cytotoxic activity of these salts was tested against human colon cancer (DLD‐1), human breast cancer (MDA‐MB‐231), and normal human noncancerous embryonic kidney (HEK‐293T) cell lines using the MTT assay method. The viability rates of the cells were determined by confocal microscopy. The majority of the newly synthesized compounds, with the exception of compounds 2, 3, 8, 11, 12, and 17, displayed high cytotoxic activity against cancerous cells. In fact, compounds 6 and 7 exhibited better results than the positive control drugs, cisplatin and busulfan, against the MDA‐MB‐231 cells. Therefore, compounds 6 and 7 could be considered as anticancer drug candidates for further development. [ABSTRACT FROM AUTHOR]

Benzimidazolium salts and their P yridine E nhanced P recatalyst P reparation S tabilization and I nitiation (PEPPSI) palladium N -heterocyclic carbene (Pd-NHC) based complexes have been synthesized and their structures characterized with a number of different instrumental techniques including NMR ( 1 H and 13 C), IR, EI-MS (for 2 ), X-ray (for 1 , 2 and 4 ) and elemental analysis. The cytotoxicity of all the compounds was tested using the human embryonic kidney (HEK-293T), human breast epithelial adenocarcinoma (MDA-MB-231), and human colon epithelial colorectal adenocarcinoma (DLD-1) cell lines. The benzimidazolium salts ( 2–5 ) had more cytotoxic activity against cancerous cells compared with the metal complexes ( 6–9 ), which curiously exhibited no activity against any of the cell lines. Based on the IC 50 values, compound 5 displayed the highest in vitro anticancer activity among compounds 2–9 . [ABSTRACT FROM AUTHOR]

Background/aim: Vaccines are often lyophilized in order to retain their stability and efficacy for a longer period of time. However, the same lyophilization process may also cause a major degradation of the vaccine, especially during early phases of manufacturing, leading to a loss of potency of the product. Many viral diseases, such as rabies, are acute and fatal unless the vaccine is administered prior to exposure or the onset of symptoms in the case of postexposure treatment. Materials and methods: We investigated the effect of lyophilization on the stability of the virus structure during rabies vaccine manufacturing using dynamic light scattering and transmission electron microscopy. Results: Our results indicate that some viruses lose their stability and efficacy in the course of lyophilization if the pH of the cell culture medium is controlled by solvated CO2 because the structure of the rabies virus is very sensitive to the solution pH: the virus either aggregates or its shape is deformed at low solution pH, whereas at high pH empty capsid shells are formed. Conclusion: Based on our findings, we developed a new formulation for the rabies vaccine that is stable in different buffers owing to the prevention of pH upshift upon lyophilization. [ABSTRACT FROM AUTHOR]

Understanding antibody aggregation is of great significance for the pharmaceutical industry. We studied the aggregation of five different therapeutic monoclonal antibodies (mAbs) with size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC), fluorescence spectroscopy, electron microscopy, and light scattering methods at various temperatures with the aim of gaining insight into the aggregation process and developing models of it. In particular, we find that the kinetics can be described by a second-order model and are non-Arrhenius. Thus, we develop a non-Arrhenius model to connect accelerated aggregation experiments at high temperature to long-term storage experiments at low temperature. We evaluate our model by predicting mAb aggregation and comparing it with long-term behavior. Our results suggest that the number of monomers and mAb conformations within aggregates vary with the size and age of the aggregates, and that only certain sizes of aggregates are populated in the solution. We also propose a kinetic model based on conformational changes of proteins and monomer peak loss kinetics from SEC-HPLC. This model could be employed for a detail analysis of mAb aggregation kinetics. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:2526-2542, 2011 [ABSTRACT FROM AUTHOR]

Intrinsic tryptophan (Trp) fluorescence is often used to determine conformational changes of proteins. The fluorescence of multi-Trp proteins is generally assumed to be additive. This assumption usually holds well if Trp residues are situated at long distances from each other in the absence of any excited state reactions involving these residues and therefore when energy transfer does not occur. Here, we experimentally demonstrate energy transfer among Trp residues and support it by a Master Equation kinetic model applied to a therapeutic monoclonal antibody (mAb). The mAbs are one of the most studied and important biologics for the pharmaceutical industry, and they contain many Trp residues in close proximity. Understanding mAb fluorescence is critical for interpreting fluorescence data and protein-structure relationships. We propose that Trp residues could be categorized into three types of emitters in the mAbs. Experimentally, we categorize them according to solvent accessibility based on dependence of their fluorescence lifetime on the external quencher concentration and their emission wavelength. Theoretically, we categorize with molecular dynamics simulations according to their solvent accessibility. This method of combinatorial mapping of fluorescence characteristics can be utilized to illuminate structural aspects as well as make comparisons of drug formulations for these pharmaceutical proteins. [ABSTRACT FROM AUTHOR]

Energy migration between tryptophan residues has been experimentally demonstrated in self-assembled peptide tapes. Each peptide contains 11 amino acids with a Trp at position 6. The peptide self-assembly is pH-sensitive and forms amphiphilic tapes, which further stack in ribbons (double tapes) and fibrils in water depending on the concentration. Fluorescence spectra, quenching, and anisotropy experiments showed that when the pH is lowered from 9 to 2, the peptide self-assembly buries the tryptophan in a hydrophobic and restricted environment in the interior of stable ribbons as expected on the basis of the peptide design. These fluorescence data support directly and for the first time the presence of such ribbons which are characterized by a highly packed and stable hydrophobic interior. In common with Trp in many proteins, fluorescence lifetimes are nonexponential, but the average lifetime is shorter at low pH, possibly due to quenching with neighboring Phe residues. Unexpectedly, time-resolved fluorescence anisotropy does not change significantly with self-assembly when in water. In highly viscous sucrose- water mixtures, the anisotropy decay at low pH was largely unchanged compared to that in water, whereas at high pH, the anisotropy decay increased significantly. We concluded that depolarization at low pH was not due to rotational diffusion but mainly due to energy migration between adjacent tryptophan residues. This was supported by a master equation kinetic model of Trp-Trp energy migration, which showed that the simulated and experimental results are in good agreement, although on average only three Trp residues were visited before emission. [ABSTRACT FROM AUTHOR]

Nanoparticle carriers offer the possibility of enhanced delivery of therapeutic payloads in tumor tissues due to tumor-selective accumulation through the enhanced permeability and retention effect (EPR). Gold nanoparticles (AuNP), in particular, possess highly appealing features for development as nanomedicines, such as biocompatibility, tunable optical properties and a remarkable ease of surface functionalization. Taking advantage of the latter, several strategies have been designed to increase treatment specificity of gold nanocarriers by attaching monoclonal antibodies on the surface, as a way to promote selective interactions with the targeted cells—an approach referred to as active-targeting. Here, we describe the synthesis of spherical gold nanoparticles surface-functionalized with an anti-HER2 antibody-drug conjugate (ADC) as an active targeting agent that carries a cytotoxic payload. In addition, we enhanced the intracellular delivery properties of the carrier by attaching a cell penetrating peptide to the active-targeted nanoparticles. We demonstrate that the antibody retains high receptor-affinity after the structural modifications performed for drug-conjugation and nanoparticle attachment. Furthermore, we show that antibody attachment increases cellular uptake in HER2 amplified cell lines selectively, and incorporation of the cell penetrating peptide leads to a further increase in cellular internalization. Nanoparticle-bound antibody-drug conjugates retain high antimitotic potency, which could contribute to a higher therapeutic index in high EPR tumors. [ABSTRACT FROM AUTHOR]

Accelerated aggregation studies were conducted around the melting temperature (T m ) to elucidate the kinetics of seeded BSA aggregation. Aggregation was tracked by SEC-HPLC and intrinsic fluorescence spectroscopy. Time evolution of monomer, dimer and soluble aggregate concentrations were globally analysed to reliably deduce mechanistic details pertinent to the process. Results showed that BSA aggregated irreversibly through both sequential monomer addition and aggregate–aggregate interactions. Sequential monomer addition proceeded only via non-native monomers, starting to occur only by 1–2 °C below the T m . Aggregate–aggregate interactions were the dominant mechanism below the T m due to an initial presence of small aggregates that acted as seeds. Aggregate–aggregate interactions were significant also above the T m , particularly at later stages of aggregation when sequential monomer addition seemed to cease, leading in some cases to insoluble aggregate formation. The adherence (or non-thereof) of the mechanisms to Arrhenius kinetics were discussed alongside possible implications of seeding for biopharmaceutical shelf-life and spectroscopic data interpretation, the latter of which was found to often be overlooked in BSA aggregation studies. [ABSTRACT FROM AUTHOR]

Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation. [ABSTRACT FROM AUTHOR]

Therapeutic proteins such as antibodies constitute the most rapidly growing class of pharmaceuticals for use in diverse clinical settings including cancer, chronic inflammatory diseases, kidney transplantation, cardiovascular medicine, and infectious diseases. Unfortunately, they tend to aggregate when stored under the concentrated conditions required in their usage. Aggregation leads to a decrease in antibody activity and could elicit an immunological response. Using full antibody atomistic molecular dynamics simulations, we identify the antibody regions prone to aggregation by using a technology that we developed called spatial aggregation propensity (SAP). SAP identifies the location and size of these aggregation prone regions, and allows us to perform target mutations of those regions to engineer antibodies for stability. We apply this method to therapeutic antibodies and demonstrate the significantly enhanced stability of our mutants compared with the wild type. The technology described here could be used to incorporate developability in a rational way during the screening of antibodies in the discovery phase for several diseases. [ABSTRACT FROM AUTHOR]

Recently, there has been an increased interest in iron–gold-based hybrid nanostructures, due to their combined outstanding optical and magnetic properties resulting from the usage of two separate metals. The synthesis of these nanoparticles involves thermal decomposition and modification of their surfaces using a variety of different methods, which are discussed in this review. In addition, different forms such as core–shell, dumbbell, flower, octahedral, star, rod, and Janus-shaped hybrids are discussed, and their unique properties are highlighted. Studies on combining optical response in the near-infrared window and magnetic properties of iron–gold-based hybrid nanoparticles as multifunctional nanoprobes for drug delivery, magnetic–photothermal heating as well as contrast agents during magnetic and optical imaging and magnetically-assisted optical biosensing to detect traces of targeted analytes inside the body has been reviewed. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are of high value in the diagnostic and treatment of many debilitating diseases such as cancers, auto-immune disorders and infections. Unfortunately, protein aggregation is one of the ongoing challenges, limiting the development and application of mAbs as therapeutic products by decreasing half-life, increasing immunogenicity and reducing activity. We engineered an aggregation-prone region of adalimumab, the top selling mAb product worldwide - with additional glycosylation sites to enhance its resistance to aggregation by steric hindrance as a next generation biologic. We found that the addition of N-glycans in the Fab domain significantly enhanced its conformational stability, with some variants increasing the melting temperature of the Fab domain by >6 °C. The mutations tested had minimal impact on antigen binding affinity, or affinity to Fcγ receptors responsible for effector function. Our findings highlight the significant utility of this rational engineering approach for enhancing the conformational stability of therapeutic mAbs and other next-generation antibody formats. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Triton X-100 (TX-100) is the most common surfactant used to split viruses during the production of influenza split-virus vaccines. It is a mild surfactant not known to denature the viral proteins; this property makes TX-100 useful for maintaining antigen conformational structure, and, as an added benefit, for partially stabilizing vaccine formulations against protein aggregation. Despite its benefits, TX-100 needs to be filtered out after virus splitting has been achieved, due to its toxicity in large quantities. Accordingly, residual TX-100 presence in vaccine formulations has implications for both formulation stability and safety, necessitating both accurate screening during processing to guide decision-making about filtration repeats and accurate quantitation in the final product. Accurate HPLC-based methods are used successfully for the latter but their use for routine screening during processing is far from ideal because they often require extensive sample preparation and are fairly slow, complicated and costly. Here, "deconstruction" of UV–Vis absorption spectra into components corresponding to different absorbing "species" is demonstrated as a novel and viable method for routine TX-100 screening in vaccine samples from different industrial processing steps. This method is fairly accurate and, more importantly, preparation-free, rapid, simple/user-friendly and comparatively inexpensive. It is evaluated in depth in terms of applicability conditions, limitations and potential for high-throughput adaptation as well as generalization to other complex biopharmaceutical formulations. [ABSTRACT FROM AUTHOR]

Abstract The macrocycle para -sulfonatocalix[8]arene, sCX[8], was examined with 2 antibiotic drugs, ciprofloxacin (CIP) and isoniazid. The drugs were shown to form complexes with sCX[8] using proton nuclear magnetic resonance, thermogravimetric analysis, fluorescence spectroscopy, and molecular modeling. Both drugs form 1:1 hydrated (H 2 O: 13%-14% w/w) host-guest complexes, with sCX[8] binding around the pyridine ring of isoniazid, and around the piperazine and cyclopropane rings of CIP. From proton nuclear magnetic resonance, the binding constant of isoniazid to sCX[8] was 6.8 (±0.3) × 103 M−1. Addition of 2 equivalents of sCX[8] to CIP resulted in a 58% decrease in fluorescence, and time-resolved fluorescence anisotropy of CIP doubles with sCX[8]. Each drug binds into the cavity of the macrocycle, with binding stabilized via combinations of hydrogen bonding, electrostatic interactions, π-π stacking, and hydrophobic effects. The safety of sCX[8] was examined in vitro with human embryonic kidney 293 cells. The IC 50 of sCX[8] was 559 μM, which is a minimum of 5-fold higher than the concentration that would be used in the clinic. The in vitro effect of sCX[8] on the action of CIP was examined on a panel of bacterial lines. The results showed that sCX[8] has no inherent antibiotic activity and had no negative effect on the action of CIP. [ABSTRACT FROM AUTHOR]

We investigated the effect of an emerging biocompatible ionic liquid, choline dihydrogen phosphate (CDHP), on the stability of high-concentration formulations of Herceptin® (trastuzumab). Our results show that CDHP significantly suppresses unfolding and aggregation of trastuzumab, demonstrating great promise as an additive in the development of stable therapeutic antibody formulations. [ABSTRACT FROM AUTHOR]

It has been over four decades since the development of monoclonal antibodies (mAbs) using a hybridoma cell line was first reported. Since then more than thirty therapeutic antibodies have been marketed, mostly as oncology, autoimmune and inflammatory therapeutics. While antibodies are very efficient, their cost-effectiveness has always been discussed owing to their high costs, accumulating to more than one billion dollars from preclinical development through to market approval. Because of this, therapeutic antibodies are inaccessible to some patients in both developed and developing countries. The growing interest in biosimilar antibodies as affordable versions of therapeutic antibodies may provide alternative treatment options as well potentially decreasing costs. As certain markets begin to capitalize on this opportunity, regulatory authorities continue to refine the requirements for demonstrating quality, efficacy and safety of biosimilar compared to originator products. In addition to biosimilars, innovations in antibody engineering are providing the opportunity to design biobetter antibodies with improved properties to maximize efficacy. Enhancing effector function, antibody drug conjugates (ADC) or targeting multiple disease pathways via multi-specific antibodies are being explored. The manufacturing process of antibodies is also moving forward with advancements relating to host cell production and purification processes. Studies into the physical and chemical degradation pathways of antibodies are contributing to the design of more stable proteins guided by computational tools. Moreover, the delivery and pharmacokinetics of antibody-based therapeutics are improving as optimized formulations are pursued through the implementation of recent innovations in the field. [ABSTRACT FROM AUTHOR]

Cross-linking of natural and synthetic polymers is widely explored to achieve the desired material properties (mechanical strength, drug loading capacity, swelling and erosion rates). However, the potential of polymers produced by crosslinking poly (methyl vinyl ether-co-maleic acid) (PMVE/MA) and pectin (PE) in pharmaceutics is mainly unexplored so far. We have investigated the effect of various esterification conditions and pectin content on the physicochemical properties. Materials have been characterized by fourier transform infrared, differential scanning calorimetry and scanning electron microscopy. In addition, swelling and bioadhesive features of PMVE/MA-PE hydrogel systems were investigated. A band shift for the carbonyl group from 1706 to 1776 cm −1 , and glass transition ( Tg ) increased from 55.4 ± 0.9 °C to 119.5 ± 0.3 °C confirmed the formation of esterification reaction within the cross-linked films. Cross-linked PMVE/MA:PE films with a ratio of 5 demonstrated a superior mass increase when compared to 2.5, 3.125, 3.75, 6.25, and 7.5 ratios of the same hydrogel film. Formulations containing PMVE/MA and pectin with a ratio of 3.75 showed superior bioadhesive features. For the first time, we engineered three-dimensional printing based swell-able microneedle arrays made out of cross-linked PMVE/MA-PE. Microneedle arrays height and aspect ratio were ranged from 702.5 ± 11.9 μm to 726 ± 23.3 μm and 3.12 ± 0.20 to 3.29 ± 0.21, respectively. Cross-linked PMVE/MA-PE Microneedle arrays (10-2, 24 h) indicated the least height loss, 22.33 ± 4.15%, during axial compression test; whilst, transverse failure of cross-linked PMVE/MA-PE Microneedle arrays was varied from 0.15 ± 0.05 to 0.25 ± 0.04 N/needle. In conclusion, we obtained a novel cross-linked polymer system with promising features of drug delivery and bio-analytical applications. [ABSTRACT FROM AUTHOR]

The extensive presence of large (high molecular weight) protein aggregates in biopharmaceutical formulations is a concern for formulation stability and possibly safety. Tests to screen large aggregate content in such bioformulations are therefore needed for rapid and reliable quality control in industrial settings. Herein, non-commercial seasonal influenza split-virus vaccine samples, produced using various strains and extracted from selected industrial processing steps, were used as model complex bioformulations. Orthogonal characterization through transmission electron microscopy, UV–Vis absorption spectroscopy, fluorescence emission spectroscopy, high-performance liquid chromatography and single-radial immunodiffusion revealed that large, amorphous protein aggregates are formed after virus splitting and their presence is linked mainly, albeit not only, to surfactant (Triton X-100) content in a sample. Importantly, the presence of large virus aggregates in purified whole virus samples and large protein aggregates in vaccine samples was found to correlate with broadening/shouldering in Nile Red fluorescence spectra. Accordingly, decomposition of Nile Red spectra into components allowed the development of a novel, rapid, reliable and user-friendly test with high-throughput potential for screening large aggregate content in influenza split-virus vaccines. The test can be adapted for screening other complex biopharmaceutical formulations, provided relevant controls are done for informed decomposition of fluorescence spectra into their components. [ABSTRACT FROM AUTHOR]

Five new palladium complexes were efficiently synthesized from the reaction of benzimidazolium salts, potassium carbonate (K 2 CO 3 ) and palladium chloride (PdCl 2 ) in pyridine (for 3 – 5 ) or 3-chloropyridine (for 6 and 7 ). The synthesized complexes were characterized and tested in Suzuki-Miyaura cross-coupling reaction as catalysts. In the presence of catalysts 3 – 7 , biaryl products were obtained in moderate yields when phenylboronic acid was used as boronic acid derivative. However, the coupling of thianaphthene-2-boronic acid with 1-chloro-4-nitrobenzene generated low yields although a longer period of time was used in comparison to the coupling of phenylboronic acid with aryl chlorides. [ABSTRACT FROM AUTHOR]

This study reports the synthesis, characterisation and antimicrobial activity of five novel silverN-heterocyclic carbene (Ag–NHC) complexes obtained byN-propylphthalimide and N-methyldioxane substituted benzimidazolium salts with silver oxide. The reactions were performed at room temperature for 24 h in the absence of light. The obtained complexes were identified and characterised by1H and13C NMR, FT-IR and elemental analysis techniques. The minimum inhibitory concentration (MIC) of the complexes was determined forE. coli, P. aeruginosa, E. faecalis, S. aureus, C. tropicalisandC. albicans in vitrothrough agar and broth dilution. The results indicated that these complexes exhibit antimicrobial activity. In particular, complex3presented the significant broad spectrum antimicrobial activity. [ABSTRACT FROM PUBLISHER]

Pillar[ n ]arenes are a new family of nanocapsules that have shown application in a number of areas, but because of their poor water solubility their biomedical applications are limited. Recently, a method of synthesizing water-soluble pillar[ n ]arenes was developed. In this study, carboxylated pillar[ n ]arenes (WP[ n ], n = 6 or 7) have been examined for their ability to form host-guest complexes with compounds relevant to drug delivery and biodiagnostic applications. Both pillar[ n ]arenes form host-guest complexes with memantine, chlorhexidine hydrochloride, and proflavine by 1 H nuclear magnetic resonance and modeling. Binding is stabilized by hydrophobic effects within the cavities, and hydrogen bonding and electrostatic interactions at the portals. Encapsulation within WP[6] results in the complete and efficient quenching of proflavine fluorescence, giving rise to “on” and “off” states that have potential in biodiagnostics. The toxicity of the pillar[ n ]arenes was examined using in vitro growth assays with the OVCAR-3 and HEK293 cell lines. The pillar[ n ]arenes are relatively nontoxic to cells except at high doses and after prolonged continuous exposure. Overall, the results show that there could be a potentially large range of medical applications for carboxylated pillar[ n ]arene nanocapsules. [ABSTRACT FROM AUTHOR]

New benzimidazolium salts ( 2 , 3 ) and their silver ( 4 , 5 ) and palladium ( 6 , 7 ) N -heterocyclic carbene (Ag- and Pd-NHC) complexes were designed, synthesized and structurally characterized by NMR ( 1 H and 13 C), IR, HRMS and UV–Vis spectroscopic methods. All the synthesized compounds were tested in human colon cancer (DLD-1), breast cancer (MDA-MB-231) and embryonic kidney (HEK 293T) (non-cancerous) cell lines for in vitro cytotoxicity. Compound 4 specifically showed low IC 50 values for these cell lines (IC 50 values 12.41 ± 2.89, 11.98 ± 2.52 and 4.21 ± 1.74 μmol/L in DLD-1, MDA-MB-231 and HEK 293T cell lines, respectively). The Pd-NHC complexes ( 6 , 7 ) did not exhibit cytotoxic activity (IC 50 values >200 in DLD-1, MDA-MB-231, HEK 293T cell lines). Therefore, Ag-NHC complexes can be further investigated as potential anticancer agents. [ABSTRACT FROM AUTHOR]

Abstract: Therapeutic antibodies of many different IgG subclasses (IgG1, IgG2 and IgG4) are used in the treatment of various cancers, rheumatoid arthritis and other inflammatory and infectious diseases. These antibodies are stored for long durations under high concentrations as required in the disease treatment. Unfortunately, these antibodies aggregate under these storage conditions, leading to a decrease in antibody activity and raising concerns about causing an immunological response. Thus, there is a tremendous need to identify the aggregation-prone regions in different classes of antibodies. We use the SAP (spatial-aggregation-propensity) technology based on molecular simulations to determine the aggregation-prone motifs in the constant regions of IgG1 classes of antibodies. Mutations engineered on these aggregation-prone motif regions led to antibodies of enhanced stability. Fourteen aggregation-prone motifs are identified, with each motif containing one to seven residues. While some of these motifs contain residues that are neighbors in primary sequence, others contain residues that are far apart in primary sequence but are close together in the tertiary structure. Comparison of the IgG1 sequence with those of other subclasses (IgG2, IgG3 and IgG4) showed that these aggregation-prone motifs are largely preserved among all IgG subclasses. Other broader classes of antibodies (IgA1, IgD, IgE and IgM), however, differed in these motif regions. The aggregation-prone motifs identified were therefore common to all IgG subclasses, but differ from those of non-IgG classes. Moreover, since the motifs identified are in the constant regions, they are applicable for all antibodies within the IgG class irrespective of the variable region. Thus, the motif regions identified could be modified on all IgGs to yield antibodies of enhanced stability. [Copyright &y& Elsevier]

The conformation of a bisindolylmaleimide may be controlled by the size of a macrocyclic ring in which ills constrained. A range of techniques were used to demonstrate that the tether controls both the ratio of the two limiting conformers (syn and anti) in solution and the extent of conjugation between the maleimide and indole rings. Screening the conformationally diverse bisindolylmaleimides against a panel of protein kinases allowed their ATP binding sites to be compared using a chemical approach which, like sequence alignment, does not require detailed structural information. This approach lead to the conclusion that several AGC group protein kinases (including PKCα, PKCβ, MSK1, p70 S6K, PDK-1, and MAPKAP-K1α) may be best inhibited by bisindolylmaleimides which adopt a compressed approximately C2-symmetric anti conformation; in constrast, GSK3β may be best inhibited by bisindolylmaleimides whose ground state is a distorted syn conformation. It is concluded that PDK-1, whose structure has been determined by X-ray crystallography, and its mutants, may serve as particularly useful surrogates for the study of PKC inhibitors. [ABSTRACT FROM AUTHOR]

Examines the femtosecond electron transfer reactions between polynucleotides and DNA. Concept of nonadiabatic electron transfer; Influence of pyrimidine on the charge transfer in DNA; Use of Monte Carlo model for DNA charge exchange.

Protein aggregation constitutes a recurring complication in the manufacture and clinical use of therapeutic monoclonal antibodies (mAb) and mAb derivatives. Antibody aggregates can reduce production yield, cause immunogenic reactions, decrease the shelf-life of the pharmaceutical product and impair the capacity of the antibody monomer to bind to its cognate antigen. A common strategy to tackle protein aggregation involves the identification of surface-exposed aggregation-prone regions (APR) for replacement through protein engineering. It was shown that the insertion of N-glycosylation sequons on amino acids proximal to an aggregation-prone region can increase the physical stability of the protein by shielding the APR, thus preventing self-association of antibody monomers. We recently implemented this approach in the Fab region of full-size adalimumab and demonstrated that the thermodynamic stability of the Fab domain increases upon N-glycosite addition. Previous experimental data reported for this technique have lacked appropriate confirmation of glycan occupancy and structural characterization of the ensuing glycan profile. Herein, we mutated previously identified candidate positions on the Fab domain of Trastuzumab and employed tandem mass spectrometry to confirm attachment and obtain a detailed N-glycosylation profile of the mutants. The Trastuzumab glycomutants displayed a glycan profile with significantly higher structural heterogeneity compared to the HEK Trastuzumab antibody, which contains a single N-glycosylation site per heavy chain located in the CH2 domain of the Fc region. These findings suggest that Fab N-glycosites have higher accessibility to enzymes responsible for glycan maturation. Further, we have studied effects on additional glycosylation on protein stability via accelerated studies by following protein folding and aggregation propensities and observed that additional glycosylation indeed enhances physical stability and prevent protein aggregation. Our findings shed light into mAb glycobiology and potential implications in the application of this technique for the development of "biobetter" antibodies. [ABSTRACT FROM AUTHOR]

Therapeutic antibody technology heavily dominates the biologics market and continues to present as a significant industrial interest in developing novel and improved antibody treatment strategies. Many noteworthy advancements in the last decades have propelled the success of antibody development; however, there are still opportunities for improvement. In considering such interest to develop antibody therapies, this review summarizes the array of challenges and considerations faced in the design, manufacture, and formulation of therapeutic antibodies, such as stability, bioavailability and immunological engagement. We discuss the advancement of technologies that address these challenges, highlighting key antibody engineered formats that have been adapted. Furthermore, we examine the implication of novel formulation technologies such as nanocarrier delivery systems for the potential to formulate for pulmonary delivery. Finally, we comprehensively discuss developments in computational approaches for the strategic design of antibodies with modulated functions. [ABSTRACT FROM AUTHOR]