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The viscosity of highly concentrated therapeutic monoclonal antibody (mAb) formulations at concentrations ≥ 100 mg/mL can significantly affect the stability, processing, and drug product development for subcutaneous delivery. An early identification of a viscosity prone mAb during candidate selection stages are often beneficial for downstream processes. Higher order structure of mAbs may often dictate their viscosity behavior at high concentration. Thus it is beneficial to gauge or rank-order their viscosity behavior using noninvasive structural fingerprinting methods and to potentially screen for suitable viscosity lowering excipients.In this study, Dynamic Light Scattering (DLS) and 2D NMR based methyl fingerprinting were used to correlate viscosity behavior of a set of Pfizer mAbs. The viscosities of mAbs were determined. Respective Fab and Fc domains were generated for studies.Methyl fingerprinting of intact mAbs allows for differentiation of viscosity prone mAbs from well behaved ones even at 30-40 mg/ml, where bulk viscosity of the solutions are near identical. For viscosity prone mAbs, peak broadening and or distinct chemical shift changes were noted in intact and fragment fingerprints, unlike the well-behaved mAbs, indicative of protein protein interactions (PPI).Fab-Fab or Fab-Fc interactions may lead to formation of protein networks at high concentration. The early transients to these network formation may be manifested through peak broadening or peak shift in the 2D NMR spectrum of mAb/mAb fragments. Such insights go beyond rank ordering mAbs based on viscosity behavior, which can be obtained by other methods as well..

Ribosomes are present inside bacterial cells at micromolar concentrations and occupy up to 20% of the cell volume. Under these conditions, even weak quinary interactions between ribosomes and cytosolic proteins can affect protein activity. By using in-cell and in vitro NMR spectroscopy, and biophysical techniques, we show that the enzymes, adenylate kinase and dihydrofolate reductase, and the respective coenzymes, ATP and NADPH, bind to ribosomes with micromolar affinity, and that this interaction suppresses the enzymatic activities of both enzymes. Conversely, thymidylate synthase, which works together with dihydrofolate reductase in the thymidylate synthetic pathway, is activated by ribosomes. We also show that ribosomes impede diffusion of green fluorescent protein in vitro and contribute to the decrease in diffusion in vivo. These results strongly suggest that ribosome-mediated quinary interactions contribute to the differences between in vitro and in vivo protein activities and that ribosomes play a previously under-appreciated nontranslational role in regulating cellular biochemistry.

An understanding of higher order structure (HOS) of monoclonal antibodies (mAbs) could be critical to predicting its function. Amongst the various factors that can potentially affect HOS of mAbs, chemical modifications that are routinely encountered during production and long-term storage are of significant interest. To this end, two Pfizer mAbs were subjected to forced deamidation stress for a period of eight weeks. Samples were aliquoted at various time points and high resolution accurate mass liquid chromatography-mass spectrometry (LC-MS/MS) was performed using low-artifact trypsin digestion (LATD) peptide mapping to identify and quantify chemical modifications. 2D backbone amide and sidechain methyl NMR spectra were acquired to gauge the effect of HOS changes upon chemical modification. Differential scanning calorimetry was also performed to assess the effect of thermal stability of mAbs upon modification. Finally, functional studies via target-binding based ELISA were performed to connect HOS changes to any loss of potency. The extent of deamidation in the mAb domains were quantified by LC-MS/MS. The HOS changes as obtained from 2D NMR were mostly localized around the affected sites leaving the overall structure relatively unchanged. The antigen-antibody binding of the mAbs, in spite of deamidation in the Fab region, remains unchanged. This case study provides an integrated approach of relating chemical modifications in mAb domains with possible changes in HOS. This can be potentially used to assess a possible loss of potency within the structure-function paradigm of proteins in an orthogonal manner.

RNA constitutes up to 20% of a cell’s dry weight, corresponding to ~20 mg/mL. This high concentration of RNA facilitates low-affinity protein–RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein–RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states.

Fc domains are an integral component of monoclonal antibodies (mAbs) and Fc-based fusion proteins. Engineering mutations in the Fc domain is a common approach to achieve desired effector function and clinical efficacy of therapeutic mAbs. It remains debatable, however, whether molecular engineering either by changing glycosylation patterns or by amino acid mutation in Fc domain could impact the higher order structure of Fc domain potentially leading to increased aggregation propensities in mAbs. Here, we use NMR fingerprinting analysis of Fc domains, generated from selected Pfizer mAbs with similar glycosylation patterns, to address this question. Specifically, we use high resolution 2D [13C-1H] NMR spectra of Fc fragments, which fingerprints methyl sidechain bearing residues, to probe the correlation of higher order structure with the storage stability of mAbs. Thermal calorimetric studies were also performed to assess the stability of mAb fragments. Unlike NMR fingerprinting, thermal melting temperature as obtained from calorimetric studies for the intact mAbs and fragments (Fc and Fab), did not reveal any correlation with the aggregation propensities of mAbs. Despite >97% sequence homology, NMR data suggests that higher order structure of Fc domains could be dynamic and may result in unique conformation(s) in solution. The overall glycosylation pattern of these mAbs being similar, these conformation(s) could be linked to the inherent plasticity of the Fc domain, and may act as early transients to the overall aggregation of mAbs.

Bispecific antibodies represent a promising avenue whereby 2 different binding specificities of a single-chain antibody can be grafted into a common Fc fragment to generate one antibody-like molecule. Despite the promising efficacy of such modalities, they may lack manufacturability because of stability and aggregation issues. Herein, we performed a systematic buffer screening for an aggregation-prone therapeutic bispecific antibody (BsAb) during early stage development. To this end, various buffers (histidine, glutamate, acetate, and arginine) and buffer combinations, including arginine and glutamate (Arg + Glu), were evaluated for their stabilizing effects on BsAb. Specifically, we identified an equimolar combination of histidine and glutamate (His + Glu at pH 5.0) buffer that showed enhanced colloidal stability as measured by dynamic light scattering interaction parameter (kD). This implies a role of net protein-protein interaction in mediating aggregation propensity of the protein. Two-dimensional nuclear magnetic resonance and multiangle light scattering experiments suggest the formation of a reversible dimer as a potential precursor to overall aggregation process. Furthermore, 1D nuclear magnetic resonance studies suggest a unique mode of interaction of histidine with BsAb that can be modulated by other buffer components or ionic strength.

Distinct differences between how model proteins interact in-cell and in vitro suggest that the cytosol might have a profound effect in modulating protein-protein and/or protein-ligand interactions that are not observed in vitro. Analyses of in-cell NMR spectra of target proteins interacting with physiological partners are further complicated by low signal-to-noise ratios, and the long overexpression times used in protein-protein interaction studies may lead to changes in the in-cell spectra over the course of the experiment. To unambiguously resolve the principal binding mode between two interacting species against the dynamic cellular background, we analyzed in-cell spectral data of a target protein over the time course of overexpression of its interacting partner by using single-value decomposition (SVD). SVD differentiates between concentration-dependent and concentration-independent events and identifies the principal binding mode between the two species. The analysis implicates a set of amino acids involved in the specific interaction that differs from previous NMR analyses but is in good agreement with crystallographic data.

Here, we report the karyotypes and mean haploid idiograms of the ten Asian Corchorus species (2n = 2x = 14). Chromosomes were small, with a mean chromosome length of 2.30 μm. The largest chromosome was recorded in C. pseudo-olitorius (3.50 μm) and the shortest in C. pseudocapsularis (1.60 μm). The karyotypes of the two cultivated species (C. capsularis and C. olitorius) and C. pseudo-olitorius were the most diverse and specialized, whereas those of C. depressus and C. trilocularis were the least diverse. C. fascicularis had the most asymmetrical and C. urticifolius the most symmetrical karyotypes. An increase in genome size was accompanied by increasing karyotype diversity in terms of morphologically distinct chromosome types and interchromosomal asymmetry, with uneven distribution of additional DNA throughout the karyotype. A positive correlation between interchromosomal asymmetry and dispersion index suggested that size differences between chromosomes were mainly associated with karyotype asymmetry. Karyotypes of the Corchorus species became progressively asymmetrical in the course of evolution. Relationships among the ten Corchorus species were defined by using a neighbor-joining tree inferred from inter-simple sequence repeat data. C. fascicularis and C. pseudocapsularis, with shorter karyotypes and smaller genomes, were closely related to C. pseudo-olitorius and C. capsularis, respectively, which were characterized by relatively longer karyotypes and larger genomes. However, the two cultivated species with different levels of interchromosomal asymmetries, dispersion indices and genome sizes were distantly related. Taking this molecular evidence into consideration, we have discussed chromosomal evolution in relation to karyological data including genome size.

We report for the first time the design and synthesis of a novel cyclotide able to activate the unique receptor of angiotensin-(1–7) (AT1–7), the MAS1 receptor. This was accomplished by grafting an AT 1–7 peptide analog onto loop 6 of cyclotide MCoTI-I using isopeptide bonds to preserve the α-amino and C-terminal carboxylate groups of AT1–7, which are required for activity. The resulting cyclotide construct was able to adopt a cyclotide-like conformation and showed similar activity to that of AT1–7. This cyclotide also showed high stability in human serum thereby providing a promising lead compound for the design of a novel type of peptide-based in the treatment of cancer and myocardial infarction.

Historically introduced by McConkey to explain the slow mutation rate of highly abundant proteins, weak protein (quinary) interactions are an emergent property of living cells. The protein complexes that result from quinary interactions are transient and thus difficult to study biochemically in vitro. Cross-correlated relaxation-induced polarization transfer-based in-cell nuclear magnetic resonance allows the characterization of protein quinary interactions with atomic resolution inside live prokaryotic and eukaryotic cells. We show that RNAs are an important component of protein quinary interactions. Protein quinary interactions are unique to the target protein and may affect physicochemical properties, protein activity, and interactions with drugs.

Intrinsically disordered proteins (IDPs) or unstructured segments within proteins play an important role in cellular physiology and pathology. Low cellular concentration, multiple binding partners, frequent post-translational modifications and the presence of multiple conformations make it difficult to characterize IDP interactions in intact cells. We used peptide aptamers selected by using the yeast-two-hybrid scheme and in-cell NMR to identify high affinity binders to transiently structured IDP and unstructured segments at atomic resolution. Since both the selection and characterization of peptide aptamers take place inside the cell, only physiologically relevant conformations of IDPs are targeted. The method is validated by using peptide aptamers selected against the prokaryotic ubiquitin-like protein, Pup, of the mycobacterium proteasome. The selected aptamers bind to distinct sites on Pup and have vastly different effects on rescuing mycobacterial proteasome substrate and on the survival of the Bacille-Calmette-Guèrin, BCG, strain of M. bovis. This technology can be applied to study the elusive action of IDPs under near physiological conditions.

We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with a highly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human α-synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in a ratio of 1:5×10(4). This demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells.

The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum, and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein–protein interactions.

Herein, we report for the first time the design and synthesis of a novel cyclotide able to efficiently inhibit HIV-1 viral replication by selectively targeting cytokine receptor CXCR4. This was accomplished by grafting a series of topologically modified CVX15 based peptides onto the loop 6 of cyclotide MCoTI-I. The most active compound produced in this study was a potent CXCR4 antagonist (EC50 ≈ 20 nM) and an efficient HIV-1 cell-entry blocker (EC50 ≈ 2 nM). This cyclotide also showed high stability in human serum thereby providing a promising lead compound for the design of a novel type of peptide-based anti-cancer and anti-HIV-1 therapeutics.

Cyclotides are fascinating natural plant micro-proteins ranging from 28 to 37 amino acid residues long and exhibit various biological actions including anti-microbial, insecticidal, cytotoxic, antiviral (against HIV), protease inhibitory, and hormone-like activities.[1–4] They share a unique head-to-tail circular knotted topology of three disulfide bridges; one disulfide penetrates through a macrocycle formed by the other two disulfides, thereby inter-connecting the peptide backbone to form what is called a cystine knot topology (Fig. 1). This cyclic cystine knot (CCK) framework gives the cyclotides exceptional rigidity[5], resistance to thermal and chemical denaturation, and enzymatic stability against degradation.[4, 6] In fact, some cyclotides have been shown to be orally bioavailable. For example, the first cyclotide to be discovered, kalata B1, was found to be an orally effective uterotonic,[7] and other cyclotides have been shown to cross the cell membrane through macropinocytosis.[8–10] All of these features make cyclotides ideal tools for drug development.[11–14]

We report an efficient approach for the chemical synthesis of Rhesus θ-defensin-1 (RTD-1) using Fmoc-based solid-phase peptide synthesis in combination with an intramolecular version of native chemical ligation. The corresponding linear thioester precursor was cyclized and folded in a one-pot reaction using reduced glutathione. The reaction was extremely efficiently yielding natively folded RTD-1 with minimal or no purification at all. This approach is fully compatible with the high throughput production of chemical libraries using this peptide scaffold.

Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. In contrast to mammalian α- and β-defensins, rhesus theta defensin-1 (RTD-1) comprises only 18 amino acids stabilized by three disulfide bonds and an unusual backbone cyclic topology. In this work we report for the first time the recombinant expression of the fully folded θ-defensin RTD-1 using a bacterial expression system. This was accomplished using an intramolecular native chemical ligation in combination with a modified protein-splicing unit. RTD-1 was produced either in vitro or in vivo. In-cell production of RTD-1 was estimated to reach an intracellular concentration of ≈ 4 μM. Recombinant RTD-1 was shown to be correctly folded as characterized by heteronucelar-NMR and by its ability to specifically inhibit Lethal Factor protease. The recombinant production of folded θ-defensins opens the possibility to produce peptide libraries based on this peptide scaffold that could be used to develop in-cell screening and directed evolution technologies.