Stability-Indicating Method Development and Validation for Busulfan Drug Substance by Gas Chromatography-Flame Ionization Detector.

A new, simple and stability-indicating gas chromatography-flame ionization detection (GC-FID) method was developed and validated for the quantitative determination of busulfan and its organic impurities (OI) in drug substance without derivatization. The chromatographic attributes were achieved on a fused silica capillary column (0.53 mm × 30 m, 1.0 µm, USP Phase G42), using hydrogen as a carrier gas with a split ratio of 1:1. Forced degradation studies were conducted to establish the stability-indicating capability and method specificity that showed the stressed busulfan peak was free from any co-elution. Robustness study demonstrated the chromatograms remained mostly unaffected under deliberate, but small variations of chromatographic parameters, establishing the reliability of the method during routine usage. The method was shown to be reliable, sensitive, specific, linear, accurate, precise and rugged in the 1,4-butanediol concentration range of 1-20 µg/mL. The method, intended for compendial uses, is suitable for quantitative analysis of busulfan and its organic impurities in drug substances.

Encoded libraries of chemically modified peptides.

The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries.

Subunit disassembly and inhibition of TNFα by a semi-synthetic bicyclic peptide.

Macrocyclic peptides are potentially a source of powerful drugs, but their de novo discovery remains challenging. Here we describe the discovery of a high-affinity (Kd = 10 nM) peptide macrocycle (M21) against human tumor necrosis factor-alpha (hTNFα), a key drug target in the treatment of inflammatory disorders, directly from diverse semi-synthetic phage peptide repertoires. The bicyclic peptide M21 (ACPPCLWQVLC) comprises two loops covalently anchored to a 2,4,6-trimethyl-mesitylene core and upon binding induces disassembly of the trimeric TNFα cytokine into dimers and monomers. A 2.9 Å crystal structure of the M21/hTNFα complex reveals the peptide bound to a hTNFα dimer at a normally buried epitope in the trimer interface overlapping the binding site of a previously discovered small molecule ligand (SPD304), which also induces TNF trimer dissociation and synergizes with M21 in the inhibition of TNFα cytotoxicity. The discovery of M21 underlines the potential of semi-synthetic bicyclic peptides as ligands for the discovery of cryptic epitopes, some of which are poorly accessible to antibodies.

Bicyclic peptide inhibitor reveals large contact interface with a protease target.

From a large combinatorial library of chemically constrained bicyclic peptides we isolated a selective and potent (K(i) = 53 nM) inhibitor of human urokinase-type plasminogen activator (uPA) and crystallized the complex. This revealed an extended structure of the peptide with both peptide loops engaging the target to form a large interaction surface of 701 Å(2) with multiple hydrogen bonds and complementary charge interactions, explaining the high affinity and specificity of the inhibitor. The interface resembles that between two proteins and suggests that these constrained peptides have the potential to act as small protein mimics.

Phage-encoded combinatorial chemical libraries based on bicyclic peptides.

Here we describe a phage strategy for the selection of ligands based on bicyclic or linear peptides attached covalently to an organic core. We designed peptide repertoires with three reactive cysteine residues, each spaced apart by several random amino acid residues, and we fused the repertoires to the phage gene-3-protein. Conjugation with tris-(bromomethyl)benzene via the reactive cysteines generated repertoires of peptide conjugates with two peptide loops anchored to a mesitylene core. Iterative affinity selections yielded several enzyme inhibitors; after further mutagenesis and selection, we were able to chemically synthesize a lead inhibitor (PK15; Ki =1.5 nM) specific to human plasma kallikrein that efficiently interrupted the intrinsic coagulation pathway in human plasma tested ex vivo. This approach offers a powerful means of generating and selecting bicyclic macrocycles (or if cleaved, linear derivatives thereof) as ligands poised at the interface of small-molecule drugs and biologics.

Thermodynamically stable aggregation-resistant antibody domains through directed evolution.

Protein aggregates are usually formed by interactions between unfolded or partially unfolded species, and often occur when a protein is denatured by, for example, heat or low pH. In earlier work, we used a Darwinian selection strategy to create human antibody variable domains that resisted heat aggregation. The repertoires of domains were displayed on filamentous phage and denatured (at 80 degrees C in pH 7.4), and folded domains were selected by binding to a generic ligand after cooling. This process appeared to select for domains with denatured states that resisted aggregation, but the domains only had low free energies of folding (Delta G(N-D)(o)=15-20 kJ/mol at 25 degrees C in pH 7.4). Here, using the same phage repertoire, we have extended the method to the selection of domains resistant to acid aggregation. In this case, however, the thermodynamic stabilities of selected domains were higher than those selected by thermal denaturation (under both neutral and acidic conditions; Delta G(N-D)(o)=26-47 kJ/mol at 25 degrees C in pH 7.4, or Delta G(N-D)(o)=27-34 kJ/mol in pH 3.2). Furthermore, we identified a key determinant (Arg28) that increased the aggregation resistance of the denatured states of the domains at low pH without compromising their thermodynamic stabilities. Thus, the selection process yielded domains that combined thermodynamic stability and aggregation-resistant unfolded states. We suggest that changes to these properties are controlled by the extent to which the folding equilibrium is displaced during the process of selection.

Repertoires of aggregation-resistant human antibody domains.

We recently described a method for the generation of a large human domain antibody repertoire involving combinatorial assembly of CDR building blocks from a smaller repertoire comprising a high frequency of aggregation-resistant antibody domains. Here we show that the frequency of aggregation-resistant domains in the combinatorial repertoire remained high. Furthermore, one of the antigen-binding domains selected from the combinatorial repertoire retained its binding properties through 25 cycles of thermal denaturation, suggesting that antibody domains can be created that rival the heat-resistance of thermophilic proteins such as Taq polymerase.

Beta-edge interactions in a pentadecameric human antibody V kappa domain.

Antibodies are the archetypal molecules of the Ig-fold superfamily. Their highly conserved beta-sheet architecture has evolved to avoid aggregation by protecting edge strands. However, the crystal structure of a human V kappa domain described here, reveals an exposed beta-edge strand which mediates assembly of a helical pentadecameric oligomer. This edge strand is highly conserved in V kappa domains but is both shortened and capped by the use of two sequential trans-proline residues in V lambda domains. We suggest that the exposure of this beta-edge in V kappa domains may explain why light-chain deposition disease is mediated predominantly by kappa antibodies.

Early protein evolution: building domains from ligand-binding polypeptide segments.

It has been suggested that in the early evolution of proteins, segments of polypeptide, unable to fold in isolation, may have collapsed together to form folded proto-domains. We wondered whether the incorporation of segments with a pre-existing binding activity into a folded domain could, by fixing the ligand binding conformation and/or providing additional contacts, lead to large affinity improvements and provide an evolutionary advantage. As a model, we took a segment of polypeptide from hen egg lysozyme that in the native protein forms the binding interface with the monoclonal antibodies HyHEL5 and F10 (KD=60 pM). When expressed in bacteria the isolated segment was unfolded, readily proteolysed and only bound weakly to the antibodies (KD>1 microM). We then combined the segment with random genomic segments to create a repertoire of chimaeric polypeptides displayed on filamentous bacteriophage. By use of proteolysis (to select folded polypeptide) and anti-lysozyme antibodies (to select an active conformation) we isolated a folded dimeric protein with an enhanced antibody affinity (KD=400 pM). Unexpectedly the dimer also incorporated a single heme molecule (KD=33 nM) that stabilised the dimer (Tm=59 degrees C with heme, 35 degrees C without heme). These results show that the binding affinities of flexible polypeptide segments can be greatly enhanced on protein folding, and that the folding can be stabilised by prosthetic groups. This supports the hypothesis that sub-domain polypeptide segments with functional activities may have contributed to domain creation in early evolution.

Tapping diversity lost in transformations--in vitro amplification of ligation reactions.

Molecular evolution is a powerful means of engineering proteins. It usually requires the generation of a large recombinant DNA library of variants for cloning into a phage or plasmid vector, and the transformation of a host organism for expression and screening of the variant proteins. However, library size is often limited by the low yields of circular DNA and the poor transformation efficiencies of linear DNA. Here we have overcome this limitation by amplification of recombinant circular DNA molecules directly from ligation reactions. The amplification by bacteriophage Phi29 polymerase increased the number of transformants; thus from a nanogram-scale ligation of DNA fragments comprising two sub-libraries of variant antibody domains, we succeeded in amplifying a highly diverse and large combinatorial phage antibody library (>10(9) transformants in Escherichia coli and 10(5)-fold more transformants than without amplification). From the amplified library, but not from the smaller un-amplified library, we could isolate several antibody fragments against a target antigen. It appears that amplification of ligations with Phi29 polymerase can help recover clones and molecular diversity otherwise lost in the transformation step. A further feature of the method is the option of using PCR-amplified vectors for ligations.

Mapping epitopes and antigenicity by site-directed masking.

Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (beta-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to beta-lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with beta-lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops.

Identification of protein domains by shotgun proteolysis.

The identification of protein domains within multi-domain proteins is a persistent problem. Here, we describe an experimental method (shotgun proteolysis) based on random DNA fragmentation and protease selection of the encoded polypeptides on phage for this purpose. We applied the method to the Escherichia coli genome and identified 124 protease-resistant fragments; several were re-cloned for expression as soluble fragments in bacteria, and corresponded to autonomously folding units with folding energies similar to natural protein domains (DeltaG(u)=3.8-6.6 kcal/mol). Structural information was available for approximately half of the selected proteins, which corresponded to compact, globular and domain-sized units that had been derived from a wide range of protein superfamilies. Furthermore, boundaries of the selected fragments correlated with domain boundaries as defined by bioinformatics predictions (R2=0.82; p=0.016). However, predictions were incomplete or entirely lacking for the remaining fragments, reflecting the limited proteome coverage of current bioinformatics methods. Shotgun proteolysis therefore provides a means to identify domains and other autonomously folding units on a genome-wide scale, without any prior knowledge of sequence or structure. Shotgun proteolysis should be particularly valuable for structural studies of proteins and represents a high-throughput alternative to the classical limited proteolysis method for the isolation of stable components of multi-domain proteins.

Inhibition of papillomavirus protein function in cervical cancer cells by intrabody targeting.

Papillomaviruses (HPVs) are a major cause of human disease, and are responsible for approximately half a million cases of cervical cancer each year. HPVs also cause genital warts, and are the most common sexually transmitted disease in many countries. Despite their importance, there are currently no specific antivirals that are active against HPVs. Papillomavirus protein function is mediated largely by protein-protein interactions, which are difficult to inhibit using conventional approaches. To circumvent these problems, we have prepared an scFv library, and have used this to isolate high-affinity binding molecules that may stearically hinder the association of E6 with p53 and prevent E6-mediated p53 degradation in cervical cancer cells. One of the molecules isolated from the library (GTE6-1), had an affinity for 16E6 of 60nM, and bound within the first zinc finger of the protein. GTE6-1 was able to associate with non-denatured E6 following expression in mammalian cells and could inhibit E6-mediated p53 degradation in in vitro assays. E6-mediated p53 degradation is essential for the continuous growth of cervical cancer cells caused by HPV16. To examine the potential of GTE6-1 as an inhibitor of E6 function in such cells, the molecule was expressed in scFv, diabody and triabody formats in a number of cell lines that are driven to proliferate by the HPV16 oncogenes E6 and E7, including the cervical cancer cell line SiHa. In contrast to small E6-binding peptides containing the ELLG E6-binding motif, GTE6-1 expression lead to changes in nuclear structure, the appearance of apoptosis markers, and an elevation in the levels of p53. No effects were seen with a control scFv molecule, or when GTE6-1 was expressed in cells that are driven to proliferate by simian virus 40 (SV40) T-antigen. Given the accessibility of HPV-associated lesions to topical therapy, our results suggest that large interfering molecules such as intrabodies may be useful inhibitors of viral protein-protein interactions and be particularly appropriate for the treatment of HPV-associated disease.

Generating molecular diversity by homologous recombination in Escherichia coli.

We explored the use of recE-mediated homologous recombination to generate molecular diversity in Escherichia coli. Two homologous genes were placed on different phagemid vectors each comprising multiple EcoRI restriction sites and overlapping N- and C-terminal portions of beta-lactamase. By co-infection of these phage into RecE+ EcoRI+ E.coli, we were able to introduce double-strand breaks into these vectors, allowing efficient homologous recombination (in up to 10% of bacteria) by the recE pathway and selection of the recombinants by resistance to ampicillin. Recombination gave single crossovers; these were more frequent near the EcoRI sites and the recombination frequency increased with the target length and degree of homology. The system was used to create a large combinatorial chicken antibody library (10(10)) for display on filamentous phage and to isolate several antibody fragments with binding affinities in the 10-100 nM range.

Folding and stability of a primitive protein.

We have previously attempted to simulate domain creation in early protein evolution by recombining polypeptide segments from non-homologous proteins, and we have described the structure of one such de novo protein, 1b11, a segment-swapped tetramer with novel architecture. Here, we have analyzed the thermodynamic stability and folding kinetics of the 1b11 tetramer and its monomeric and dimeric intermediates, and of 1b11 mutants with changes at the domain interface. Denatured 1b11 polypeptides fold into transient, folded monomers with marginal stability (DeltaG<1kcalmol(-1)) which convert rapidly ( approximately 6x10(4)M(-1)s(-1)) into dimers (DeltaG=9.8kcal/mol) and then more slowly ( approximately 3M(-1)s(-1)) into tetramers (DeltaG=28kcalmol(-1)). Segment swapping takes place during dimerization, as suggested by mass spectroscopic analysis of covalently linked peptides derived from proteolysis of a disulfide-linked dimer. Our results confirm that segment swapping and associated oligomerization are both powerful ways of stabilizing proteins, and we suggest that this may have been a feature of early protein evolution.

A segment of cold shock protein directs the folding of a combinatorial protein.

It has been suggested that protein domains evolved by the non-homologous recombination of building blocks of subdomain size. In earlier work we attempted to recapitulate domain evolution in vitro. We took a polypeptide segment comprising three beta-strands in the monomeric, five-stranded beta-barrel cold shock protein (CspA) of Escherichia coli as a building block. This segment corresponds to a complete exon in homologous eukaryotic proteins and includes residues that nucleate folding in CspA. We recombined this segment at random with fragments of natural proteins and succeeded in generating a range of folded chimaeric proteins. We now present the crystal structure of one such combinatorial protein, 1b11, a 103-residue polypeptide that includes segments from CspA and the S1 domain of the 30S ribosomal subunit of E. coli. The structure reveals a segment-swapped, six-stranded beta-barrel of unique architecture that assembles to a tetramer. Surprisingly, the CspA segment retains its structural identity in 1b11, recapitulating its original fold and deforming the structure of the S1 segment as necessary to complete a barrel. Our work provides structural evidence that (i) random shuffling of nonhomologous polypeptide segments can lead to folded proteins and unique architectures, (ii) many structural features of the segments are retained, and (iii) some segments can act as templates around which the rest of the protein folds.

Selection of optical biosensors from chemisynthetic antibody libraries.

We describe a method for creating antibodies with a fluorescent reporter integrated into the antigen-binding site. A reporter molecule was chemically linked to a hypervariable loop of an antibody repertoire displayed on phage, and this repertoire was selected for antigen binding. In one selected antibody, the fluorescence of the probe responded quantitatively to antigen binding. The method may have application for the engineering of homogeneous immunoassays.

Aggregation-resistant domain antibodies selected on phage by heat denaturation.

We describe a method for selecting aggregation-resistant proteins by heat denaturation. This is illustrated with antibody heavy chain variable domains (dAbs), which are prone to aggregate. The dAbs were displayed multivalently at the infective tip of filamentous bacteriophage, and heated transiently to induce unfolding and to promote aggregation of the dAbs. After cooling, the dAbs were selected for binding to protein A (a ligand common to these folded dAbs). Phage displaying dAbs that unfold reversibly were thereby enriched with respect to those that do not. From a repertoire of phage dAbs, six dAbs were characterized after selection; they all resisted aggregation, and were soluble, well expressed in bacteria and could be purified in good yields. The method should be useful for making aggregation-resistant proteins and for helping to identify features that promote or prevent protein aggregation, including those responsible for misfolding diseases.