Novel method to specifically determine the structures of non-N297 glycans in IgGs.

Monoclonal antibodies comprise a major class of biologic therapeutics and are also extensively studied in immunology. Given the importance of glycans on antibodies, fluorescent labeling of enzymatically released glycans and their LC/MS analysis is routinely used for in-depth characterization of antibody glycosylation. In this technical note, we propose a method for facile characterization of glycans in the variable region of antibodies using sequential enzymatic digests with Endoglycosidase-S2 and RapidTM Peptide-N-Glycosidase-F followed by labeling with a fluorescent dye carrying an NHS-carbamate moiety. The results and proposed mechanism also suggest that the choice of glycosidases along with the labeling chemistry is critical for accurate glycan analysis for a desired application.

Azaindole-Based Inhibitors of Cdc7 Kinase: Impact of the Pre-DFG Residue, Val 195.

To investigate the role played by the unique pre-DFG residue Val 195 of Cdc7 kinase on the potency of azaindole-chloropyridines (1), a series of novel analogues with various chloro replacements were synthesized and evaluated for their inhibitory activity against Cdc7. X-ray cocrystallization using a surrogate protein, GSK3ß, and modeling studies confirmed the azaindole motif as the hinge binder. Weaker hydrophobic interactions with Met 134 and Val 195 by certain chloro replacements (e.g., H, methyl) led to reduced Cdc7 inhibition. Meanwhile, data from other replacements (e.g., F, O) indicated that loss of such hydrophobic interaction could be compensated by enhanced hydrogen bonding to Lys 90. Our findings not only provide an in-depth understanding of the pre-DFG residue as another viable position impacting kinase inhibition, they also expand the existing knowledge of ligand-Cdc7 binding.

On-column ligand exchange for structure-based drug design: a case study with human 11ß-hydroxysteroid dehydrogenase type 1.

Successfully forming ligand-protein complexes with specific compounds can be a significant challenge in supporting structure-based drug design for a given protein target. In this respect, an on-column ligand- and detergent-exchange method was developed to obtain ligand-protein complexes of an adamantane series of compounds with 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) after a variety of other complexation methods had failed. This report describes the on-column exchange method and an unexpected byproduct of the method in which artificial trimers were observed in the structures.

Iniparib nonselectively modifies cysteine-containing proteins in tumor cells and is not a bona fide PARP inhibitor.

PURPOSE: PARP inhibitors are being developed as therapeutic agents for cancer. More than six compounds have entered clinical trials. The majority of these compounds are ß-nicotinamide adenine dinucleotide (NAD(+))-competitive inhibitors. One exception is iniparib, which has been proposed to be a noncompetitive PARP inhibitor. In this study, we compare the biologic activities of two different structural classes of NAD(+)-competitive compounds with iniparib and its C-nitroso metabolite. EXPERIMENTAL DESIGN: Two chemical series of NAD(+)-competitive PARP inhibitors, iniparib and its C-nitroso metabolite, were analyzed in enzymatic and cellular assays. Viability assays were carried out in MDA-MB-436 (BRCA1-deficient) and DLD1(-/-) (BRCA2-deficient) cells together with BRCA-proficient MDA-MB-231 and DLD1(+/+) cells. Capan-1 and B16F10 xenograft models were used to compare iniparib and veliparib in vivo. Mass spectrometry and the (3)H-labeling method were used to monitor the covalent modification of proteins. RESULTS: All NAD(+)-competitive inhibitors show robust activity in a PARP cellular assay, strongly potentiate the activity of temozolomide, and elicit robust cell killing in BRCA-deficient tumor cells in vitro and in vivo. Cell killing was associated with an induction of DNA damage. In contrast, neither iniparib nor its C-nitroso metabolite inhibited PARP enzymatic or cellular activity, potentiated temozolomide, or showed activity in a BRCA-deficient setting. We find that the nitroso metabolite of iniparib forms adducts with many cysteine-containing proteins. Furthermore, both iniparib and its nitroso metabolite form protein adducts nonspecifically in tumor cells. CONCLUSIONS: Iniparib nonselectively modifies cysteine-containing proteins in tumor cells, and the primary mechanism of action for iniparib is likely not via inhibition of PARP activity.

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design.

The DVD-Ig (TM) protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.

Recombinant kringle 5 from plasminogen antagonises hepatocyte growth factor-mediated signalling.

The blood protein plasminogen is proteolytically cleaved to produce angiostatin and kringle 5 (K5), both of which are known angiogenesis inhibitors. A common structural element between K5, angiostatin and other endogenous angiogenesis inhibitors is the presence of the kringle protein-interacting domain. Another kringle domain-containing protein, hepatocyte growth factor (HGF), promotes angiogenesis by binding to and stimulating the tyrosine kinase receptor Met. HGF binding to Met is dependent on the kringle domains of HGF. Because both K5 and HGF contain kringle motifs and because these proteins have opposite effects on angiogenesis, we hypothesised that K5 can antagonise HGF-mediated signalling in a Met-dependent manner. We determined that K5 binding to H1299 cells is competed by HGF suggesting that these two proteins bind to the same protein. Purified K5 immunoprecipitates with Met and this interaction is abolished by increasing doses of HGF. Using proliferation, phosphorylation of Met and Akt as markers of HGF activity, we determined that K5 inhibits HGF-mediated signalling. Taken together, these data support a model by which K5 binds to Met and functions as a competitive antagonist of HGF signalling and presents a novel mechanism of action of K5.

Synthesis and evaluation of inhibitors of cytochrome P450 3A (CYP3A) for pharmacokinetic enhancement of drugs.

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2-P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.

Structural characterization of a soluble amyloid beta-peptide oligomer.

Alzheimer's disease (AD) is a neurodegenerative disorder that is linked to the presence of amyloid beta-peptides that can form insoluble fibrils or soluble oligomeric assemblies. Soluble forms are present in the brains and tissues of Alzheimer's patients, and their presence correlates with disease progression. Long-lived soluble forms can be generated in vitro by using small amounts of aliphatic hydrocarbon chains of detergents or fatty acids in preparations of amyloid beta-peptides. Using NMR, we have characterized soluble oligomers of Abeta preglobulomer and globulomer that are stable and alter synaptic activity. The NMR data indicate that these soluble forms have a mixed parallel and antiparallel beta-sheet structure that is different from fibrils which contain only parallel beta-sheets. Using the structural data, we engineered a disulfide bond into the soluble Abeta globulomer to give a "new" soluble antigen that is stable, homogeneous, and binds with the same affinity to selective antibodies as the parent wt globulomer.

A potent erythropoietin-mimicking human antibody interacts through a novel binding site.

Recombinant human erythropoietin (rHu-EPO) is used to treat anemia by activating the erythropoietin receptor (EPOR) in erythroid progenitor cells, leading to proliferation and differentiation into mature red blood cells. To allow less frequent dosing, a hyperglycosylated version of EPO has been developed with a longer half-life. In principle, an agonistic antibody targeting EPOR would offer an even longer half-life, support robust monthly dosing, and, unlike EPO products, reduce the risk of pure red cell aplasia. The efficiency of signaling and corresponding potency of previously reported antibody mimics are generally suboptimal compared with EPO and not suitable for clinical use. Here we describe a potent, fully human, agonistic antibody (ABT007) targeting EPOR that supports potent, more sustained, and less pulsatile elevation of hematocrit in a human EPOR-expressing transgenic mouse model compared with standard doses of rHu-EPO while requiring less frequent dosing. Resolution of the crystal structure of the EPOR extracellular domain (ECD) complexed to the ABT007 Fab fragment, determined at 0.32 nm, identifies a binding site that is consistent with a novel mechanism of receptor activation based on a unique antibody-imposed conformational change. These results demonstrate that a symmetric molecule can serve as a potent activator of the EPOR.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.

Design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery.

Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.

Structure of MurF from Streptococcus pneumoniae co-crystallized with a small molecule inhibitor exhibits interdomain closure.

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.

A highly potent and selective farnesyltransferase inhibitor ABT-100 in preclinical studies.

Ras mutation has been detected in approximately 20-30% of all human carcinomas, primarily in pancreatic, colorectal, lung and bladder carcinomas. The indirect inhibition of Ras activity by inhibiting farnesyltransferase (FTase) function is one therapeutic intervention to control tumor growth. Here we report the preclinical anti-tumor activity of our most advanced FTase inhibitor (FTI), ABT-100, and a direct comparison with the current clinical candidates. ABT-100 is a highly selective, potent and orally bioavailable FTI. It broadly inhibits the growth of solid tumors in preclinical animal models. Thus, ABT-100 is an attractive candidate for further clinical evaluation. In addition, our results provide plausible insights to explain the impressive potency and selectivity of ABT-100. Finally, we have demonstrated that ABT-100 significantly suppresses the expression of vascular endothelial growth factor (VEGF) mRNA and secretion of VEGF protein, as well as inhibiting angiogenesis in the animal model.

Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization.

Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.25) have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization. Screening of a 10 000 compound random library provided several low affinity leads and their corresponding X-ray crystal structures bound to the enzyme. The presence of a common structural feature in each of the leads suggested a strategy for the construction of a directed library of approximately 1000 compounds that were screened for inhibitory activity in a traditional enzyme assay. Several lead compounds with IC(50) values of about 1 microM against DHNA were identified, and crystal structures of their enzyme-bound complexes were obtained by cocrystallization. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar IC(50) values.