BIAcore Technology Research Articles

LINC00982-encoded protein PRDM16-DT regulates CHEK2 splicing to suppress colorectal cancer metastasis and chemoresistance.

Metastasis is one of the key factors of treatment failure in late-stage colorectal cancer (CRC). Metastatic CRC frequently develops resistance to chemotherapeutic agents. This study aimed to identify the novel regulators from "hidden" proteins encoded by long noncoding RNAs (lncRNAs) involved in tumor metastasis and chemoresistance. Methods: CRISPR/Cas9 library functional screening was employed to identify the critical suppressor of cancer metastasis in highly invasive CRC models. Western blotting, immunofluorescence staining, invasion, migration, wound healing, WST-1, colony formation, gain- and loss-of-function experiments, in vivo experimental metastasis models, multiplex immunohistochemical staining, immunohistochemistry, qRT-PCR, and RT-PCR were used to assess the functional and clinical significance of FOXP3, PRDM16-DT, HNRNPA2B1, and L-CHEK2. RNA-sequencing, co-immunoprecipitation, qRT-PCR, RT-PCR, RNA affinity purification, RNA immunoprecipitation, MeRIP-quantitative PCR, fluorescence in situ hybridization, chromatin immunoprecipitation and luciferase reporter assay were performed to gain mechanistic insights into the role of PRDM16-DT in cancer metastasis and chemoresistance. An oxaliplatin-resistant CRC cell line was established by in vivo selection. WST-1, colony formation, invasion, migration, Biacore technology, gain- and loss-of-function experiments and an in vivo experimental metastasis model were used to determine the function and mechanism of cimicifugoside H-1 in CRC. Results: The novel protein PRDM16-DT, encoded by LINC00982, was identified as a cancer metastasis and chemoresistance suppressor. The down-regulated level of PRDM16-DT was positively associated with malignant phenotypes and poor prognosis of CRC patients. Transcriptionally regulated by FOXP3, PRDM16-DT directly interacted with HNRNPA2B1 and competitively decreased HNRNPA2B1 binding to exon 9 of CHEK2, resulting in the formation of long CHEK2 (L-CHEK2), subsequently promoting E-cadherin secretion. PRDM16-DT-induced E-cadherin secretion inhibited fibroblast activation, which in turn suppressed CRC metastasis by decreasing MMP9 secretion. Cimicifugoside H-1, a natural compound, can bind to LEU89, HIS91, and LEU92 of FOXP3 and significantly upregulated PRDM16-DT expression to repress CRC metastasis and reverse oxaliplatin resistance. Conclusions: lncRNA LINC00982 can express a new protein PRDM16-DT to function as a novel regulator in cancer metastasis and drug resistance of CRC. Cimicifugoside H-1 can act on the upstream of the PRDM16-DT signaling pathway to alleviate cancer chemoresistance.

Application of Biacore Technology to Detect the Affinity of Hnt3 Protein to Double-stranded DNA

Application of Biacore Technology to Detect the Affinity of Hnt3 Protein to Double-stranded DNA

Study of PDZ-Peptide and PDZ-Lipid Interactions by Surface Plasmon Resonance/BIAcore.

Surface plasmon resonance (SPR)/BIAcore technology enables the characterization of molecular interactions, including determination of affinities and kinetics. InBIAcore, one of the interaction partners (the ligand) is immobilized on a chip and the other (the analyte) is provided in solution. BIAcore allows to study association and dissociation rates in real time without the use of labeling. BIAcore can be applied to molecular interactions involving small compounds and biological macromolecules such as proteins, lipids, nucleic acids, or carbohydrates. Here we describe protocols for the measurements of PDZ domain-peptide (oriented biotinylated peptides), PDZ domain-liposomes (lipid membranes), and PDZ-lipid-peptide tripartite interactions.

Effect of Dilong on expression of fibrogenic factors TGF-β1 and α-SMA in lung tissue of mice with pulmonary fibrosis

The aim of this paper was to investigate the effect of Dilong( geosaurus) on the expressions of fibrotic factors TGF-β1 and α-SMA in bleomycin-induced pulmonary fibrosis mice. The binding ability of Dilong to fibrotic factor TGF-β1 was initially detected by Biacore technology and verified by in vivo pharmacodynamics. A total of 60 SPF C57 mice were randomly divided into 6 groups. Except the blank group( injecting 0. 08 m L·kg-1 sodium chloride in the trachea),the other five groups were given bleomycin( 4 mg·kg-1) to replicate the pulmonary fibrosis model. After 14 days of drug treatment,the expressions of TGF-β1 and α-SMA were detected by Masson staining,immunohistochemistry and RT-PCR. The results of Biacore experiment showed that the extract of Dilong was well bound to TGF-β1 protein in vitro,and the binding value reached 619. 3. Compared with the model group,Masson's results showed that cellulose deposition in high-dose,medium-dose and low-dose Dilong groups decreased to varying degrees. RT-PCR results showed that different doses of Dilong could reduce protein and mRNA expressions of TGF-β1 and α-SMA to a certain extent in a dose-dependent manner. In conclusion,Dilong could delay the process of pulmonary fibrosis by binding to target protein TGF-β1 and inhibiting the expression of α-SMA.

Synthesis of Mannosylated Lipopeptides with Receptor Targeting Properties

Present on the surface of antigen presenting cells (APCs), the mannose receptor (MR) has long been recognized as a front-line receptor in pathogen recognition. During the past decade many attempts have been made to target this receptor for applications including vaccine and drug development. In the present study, a library of vaccine constructs comprising fluorescently labeled mannosylated lipid-dendrimers that contained the ovalbumin CD4(+) epitope, OVA(323-339), as the model peptide antigen were synthesized using fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). The vaccine constructs were designed with an alanine spacer between the O-linked mannose moieties to investigate the impact of distance between the mannose units on receptor-mediated uptake and/or binding in APCs. Uptake studies performed on F4/80(+) and CD11c(+) cells showed significant uptake and/or binding for lipopeptides containing mannose, and also the lipopeptide without mannose when compared to the control peptides (peptide with no lipid and peptide with no mannose and no lipid). Furthermore, mannan inhibition assays demonstrated that uptake of the mannosylated and lipidated peptides was receptor mediated. To address the specificity of receptor uptake, surface plasmon resonance studies were performed using biacore technology and confirmed high affinity of the mannosylated and lipidated vaccine constructs toward the MR. These studies confirm that both mannose and lipid moieties play significant roles in receptor-mediated uptake on APCs, potentially facilitating vaccine development.

Spot peptide arrays and SPR measurements: throughput and quantification in antibody selectivity studies.

Antibody selectivity represents a major issue in the development of efficient immuno-therapeutics and detection assays. Its description requires a comparison of the affinities of the antibody for a significant number of antigen variants. In the case of peptide antigens, this task can now be addressed to a significant level of details owing to improvements in spot peptide array technologies. They allow the high-throughput mutational analysis of peptides with, depending on assay design, an evaluation of binding stabilities. Here, we examine the cross-reactive capacity of an antibody fragment using the PEPperCHIP(®) technology platform (PEPperPRINT GmbH, Heidelberg, Germany; >8800 peptides per microarray) combined with the surface plasmon resonance characterization (Biacore(®) technology; GE-Healthcare Biacore, Uppsala, Sweden) of a subset of interactions. ScFv1F4 recognizes the N-terminal end of oncoprotein E6 of human papilloma virus 16. The spot permutation analysis (i.e. each position substituted by all amino acids except cysteine) of the wild type decapeptide (sequence (6)TAMFQDPQER(15)) and of 15 variants thereof defined the optimal epitope and provided a ranking for variant recognition. The SPR affinity measurements mostly validated the ranking of complex stabilities deduced from array data and defined the sensitivity of spot fluorescence intensities, bringing further insight into the conditions for cross-reactivity. Our data demonstrate the importance of throughput and quantification in the assessment of antibody selectivity.

Effect of cleaning agents and additives on Protein A ligand degradation and chromatography performance

Protein A chromatography, employing the recombinant Protein A ligand, is widely used as a capture step for antibody and Fc-fusion proteins manufacture. Protein A ligands in these matrices are susceptible to degradation/loss when exposed to cleaning agents such as sodium hydroxide, resulting in loss of capacity on reuse. In this study, MabSelect Protein A ligand and MabSelect SuRe Protein A ligand were chosen to evaluate the impact of alkaline cleaning solutions on the ligands and the packed columns. The Protein A ligands alone and the Protein A columns were incubated or cycled in different concentrations of sodium hydroxide solutions with and without additives, respectively. Ligand integrity (degradation) and ligand function (binding affinity) were studied using SDS–PAGE and customized Biacore technology, surface plasma resonance (SPR) and were successfully correlated with column performance measurement in terms of static binding capacity (SBC), dynamic binding capacity (DBC) and recovery as a function of exposure to cleaning agents with and without additives. The findings and the methodology presented in this study are not only able to determine appropriate cleaning conditions for Protein A chromatography, but also provided tools to enable systematic and rapid study of the cleaning solutions and conditions.

Abstract 2666: Monoclonal antibodies specific for the oncofetal antigen - immature laminin receptor: Tumor selectivity, rapid internalization and in vivo efficacy

Abstract A series of murine monoclonal antibodies were generated using recombinant oncofetal antigen - immature laminin receptor protein (OFA/iLRP) as the immunogen. Several of the antibodies detected the 37kD OFA/iLRP but did not recognize the structurally related 67kD mature laminin receptor protein (LRP), as determined by western blot analysis. Additional antibodies demonstrated reactivity with both proteins. Those antibodies specific for the 37kD moiety bound to recombinant OFA/iLRP with high affinity (Kaff = 1-2 x 10-10M; Biacore technology). These antibodies also reacted with native OFA/iLRP on the surface of human tumor cells with high affinity (Kaff = 1-2 x 10-9M) but did not show significant reactivity with normal human epithelial cells or fibroblasts. In contrast, antibodies that interacted with both 37kD OFA/iLRP and 67kD mature LRP were reactive with both tumor cells and normal cells. Antibodies specific to OFA/iLRP were internalized upon binding to the target protein on human tumor cells. Two monoclonal antibodies from this group were tested for anti-tumor activity in a murine model of B-cell leukemia. BALB/c mice were injected with A20 leukemia cells either intramuscularly or intravenously. Mice were given a total of six intraperitoneal 100µg antibody injections over a two-week period following tumor initiation. Both antibodies slowed growth of the intramuscular primary tumor by approximately 45%. More importantly, in the intravenous tumor model, the treatments reduced systemic blood tumor levels by greater than 70% and suppressed liver tumor formation by up to 46%. These high affinity tumor-selective antibodies potentially provide a front-line or adjuvant therapy for hematological malignancies. Citation Format: Shannon D. McClintock, Michael K. Dame, Muhammad N. Aslam, Saqib Ali, Randall N. Knibbs, Roscoe L. Warner, James Varani. Monoclonal antibodies specific for the oncofetal antigen - immature laminin receptor: Tumor selectivity, rapid internalization and in vivo efficacy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2666. doi:10.1158/1538-7445.AM2014-2666

From a Laboratory Exercise for Students to a Pioneering Biosensing Technology.

Surface plasmon resonance (SPR) for biosensing was demonstrated 30 years ago. In the present contribution, its general background is described together with the necessary developments both in instrumentation and surface chemistry, leading to the final so-called BIAcore technology. The description is naturally colored by my personal opinion of the developments. SPR for the elucidation of organic mono- and multilayers introduced at the end of the 1970s formed the basis for the first biosensing demonstration of SPR in the beginning of the 1980s. It is pointed out how the need of an up-to-date laboratory exercise for the undergraduate students and the multidisciplinary environment at the Laboratory of Applied Physics at Linköping University led to this demonstration. The initial experiments are touched upon and the further developments at Pharmacia, which led to the BIAcore technology, are described in some details. Some of the present activities in Linköping related to optical biosensing with ubiquitous instrumentation are also described, including SPR detection with a computer screen and a web camera and most recently with a cellular phone.

Generation and Evaluation of Bispecific Affibody Molecules for Simultaneous Targeting of EGFR and HER2

Coexpression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here, we have generated six such bispecific targeting proteins, each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly(4)Ser)(3) or (Ser(4)Gly)(3), and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays, it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the inherent monomeric affibody molecules was favorable to promote the binding to the respective target. Interestingly, bispecific constructs containing the novel (Ser(4)Gly)(3) linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly(4)Ser)(3). It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimer formation and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed.

Abstract 4294: GE152: In vivo detection of tumor apoptosis as a tool for assessment of therapeutic efficacy

Abstract A major challenge in personalized healthcare is predicting how effective a drug treatment is. This is particularly the case in oncology, where there is a call for better therapy monitoring to maximize drug treatment effectiveness leading to improved patient survival and help reduce healthcare costs. GE152, a nuclear imaging agent under development at GE Healthcare, is being evaluated preclinically as a tool to assess therapeutic efficacy by detecting tumor apoptosis. GE152 is based on a 99mTechnetium radiolabelled peptide that shows nanomolar affinity for a specific cell death target, as demonstrated by studies using Biacore technology. Biodistribution using the murine lymphoma (EL4) tumour therapy model has shown increased tumor uptake and retention of GE152 following chemotherapy, with positive tumor:muscle and tumor:blood ratios. Correlation of tumor apoptosis levels (determined by caspase activity) with GE152 tumor retention suggest a trend of increasing agent retention with rising levels of apoptosis (GE152 retention in low apoptotic tumors is 4.9%ID/g; GE152 retention in high apoptotic tumors is 8.2%ID/g).Further validation of GE152 was carried out using an apoptosis-specific inducible cell death model whereby HT29 colorectal cancer cell xenografts engineered to inducibly express either a constitutively active form of caspase-3 (which causes synchronous cell death in vivo when exposed to doxycycline) or an inactive point mutant. GE152 demonstrated greater uptake in tumors undergoing apoptosis (3.6% ID/g) than in controls (1.2% ID/g), correlating with caspase activity levels (and subsequent apoptosis) as determined both enzymatically and by IHC and blood-borne biomarkers of cell death. These results are in agreement with preliminary preclinical imaging studies using SPECT/CT, where region of interest analysis has demonstrated increased post-therapy tumor retention of GE152. We are currently optimizing the performance of GE152 by assessing different radiolabelling precursors to improve imaging agent pharmacokinetics. In addition to the current 99mTechnetium-based approach, we are exploring 18F radiolabelling options that would allow expansion of the agent's utility to PET imaging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4294. doi:1538-7445.AM2012-4294

Binding of FVIII to Recombinant VWF

AbstractAbstract 1408Von Willebrand factor (VWF) forms a non-covalent complex with FVIII in the circulation, which not only stabilizes the heterodimeric structure of FVIII but also protects this coagulation protein from early proteolytic degradation and cellular uptake. By convention, the physiological ratio in plasma between VWF and FVIII is 1 IU to 1 IU, corresponding to a stoichiometry of approximately 1 FVIII molecule bound per 50 VWF monomers. However, since each VWF monomer contains one FVIII binding site, this ratio does not seem to reflect the genuine binding capacity of VWF for FVIII.We therefore investigated the interaction between FVIII and a recombinant VWF (rVWF) drug candidate under different conditions. The FVIII binding capacity of VWF (VWF:FVIIIB) was measured by an ELISA system, where the VWF-bound FVIII was quantified by a FVIII chromogenic assay. Under this condition, VWF:FVIIIB of rVWF was similar to that of VWF derived from human normal plasma. The binding affinity of VWF for FVIII was determined using a Biacore system in which VWF is immobilized onto the sensor chip surface. The FVIII binding affinities were similar for rVWF KD=0.50) and pdVWF (KD=0.49 nM) and agreed with data from the literature.Plasma-derived VWF was reported to show increased FVIII binding when in solution (instead of being immobilized to a surface). To find out whether this also holds true for rVWF, we developed a new assay system based on Biacore technology which measures the binding of FVIII to VWF in solution without any surface binding step of the VWF-FVIII complex. A mixture of rVWF and rFVIII is thereby injected into the flow cell of a Biacore sensor chip with immobilized rVWF. In this indirect assay system, the rFVIII that forms a complex with rVWF in solution is not available for binding to the immobilized rVWF. Under these conditions, rVWF was endowed with a 30-times higher FVIII binding capacity than could be anticipated from the physiological ratio and similar results were obtained for pdVWF.The combined results showed that the ability to serve as the physiological chaperone protein for FVIII is fully preserved in rVWF, which can therefore be expected to stabilize FVIII in the circulation in a similar manner to VWF in plasma. Results from an ongoing phase 1 study show the good FVIII stabilizing properties of rVWF also when administered in humans with type 3 VWD. Disclosures:Schrenk:Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter BioScience: Employment.

Biosensing applications of surface plasmon resonance-based Biacore technology

In the past, surface plasmons have been used to improve the surface sensitivity of several spectroscopic measurements. Surface plasmon resonance reflectivity measurements can be used to detect biomolecules and their interactions by changes in the local index of refraction upon adsorption of the target molecule to the metal surface. Using this principle several SPR-based biosensors have been generated in the past with wide range of applications in the field of biology, biomedicine and biochemistry. Among several techniques available for biomolecular interactions, SPR-based Biacore is one of the widely used real-time monitoring systems and proved to be a convenient system for wide range of molecular sizes from small ligands to whole cells. Biacore system can be employed for biomolecular interactions, selection procedures, epitope mapping, molecular discriminations, kinetic analyses, screening processes and other applications. There were several successes reported using the versatility of Biacore system. In this report, several biosensing applications of this system were overviewed.

In-depth analysis by Biacore technology to evaluate the affinity versus avidity of the binding of the oligomeric pattern recognition molecule mannan-binding lectin

In-depth analysis by Biacore technology to evaluate the affinity versus avidity of the binding of the oligomeric pattern recognition molecule mannan-binding lectin

Identification and characterization of a novel heparin‐binding peptide for promoting osteoblast adhesion and proliferation by screening an Escherichia coli cell surface display peptide library

Heparin/heparan sulfate (HS) plays a key role in cellular adhesion. In this study, we utilized a 12-mer random Escherichia coli cell surface display library to identify the sequence, which binds to heparin. Isolated insert analysis revealed a novel heparin-binding peptide sequence, VRRSKHGARKDR, designated as HBP12. Our analysis of the sequence alignment of heparin-binding motifs known as the Cardin-Weintraub consensus (BBXB, where B is a basic residue) indicates that the HBP12 peptide sequence contains two consecutive heparin-binding motifs (i.e. RRSK and RKDR). SPR-based BIAcore technology demonstrated that the HBP12 peptide binds to heparin with high affinity (KD = 191 nM). The HBP12 peptide is found to bind the cell surface HS expressed by osteoblastic MC3T3 cells and promote HS-dependent cell adhesion. Moreover, the surface-immobilized HBP12 peptide on titanium substrates shows significant increases in the osteoblastic MC3T3-E1 cell adhesion and proliferation.

Structural and Functional Investigations of Active Site Binding Regions in Factor VIIa.

Factor VII (FVII) consists of an N-terminal γ-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor-like (EGF1 and EGF2) domains and the C-terminal protease domain. Activation of FVII results in a two-chain FVIIa molecule consisting of a light chain (Gla-EGF1-EGF2 domains) and a heavy chain (protease domain) held together by a single disulfide bond. The complex of tissue factor (TF) and FVIIa activates FIX and FX during coagulation. FVIIa on its own is structurally more “zymogen-like” and when bound to TF it is more “active enzyme-like.” We obtained crystal structures of EGR-VIIa/soluble (s) TF (2.9 Å resolution), dansyl-EGR-VIIa/sTF (1.9 Å resolution) and benzamidine-VIIa/sTF (1.6 Å resolution). We also investigated the effect of TF binding on the S1, S2, and S3/S4 subsites (Schechter and Berger, BBRC , 27:157-162, 1967) in FVIIa. The affinity of variously inhibited FVIIa to sTF was also measured using Biacore technology. For obtaining second order inhibition rate constants, FVIIa ± soluble (s) TF was incubated with each inhibitor for various times, diluted several fold and assayed for the residual FVIIa activity. The second order rate constants were obtained by plotting the first order rate constants versus the inhibitor concentrations. These data are summarized in the table below. From these data it appears that all subsites are affected upon FVIIa binding to sTF. Since in the crystal structure of EGR-VIIa/sTF the P1 Arg residue is the only residue that makes contact with FVIIa, it follows that the S1 site is affected ~10-fold upon binding to sTF. Adding a dansyl group that partially occupies the S3/S4 position (1.9 Å structure) increases the second order rate constant 7-fold (2.41 versus 0.35) over that of EGR-ck. Moreover the addition of Pro (DFPR-ck) or Phe (DFFR-ck) residue occupying the S2 position increases the second order rate constant 357-fold and 1500- fold, respectively (125 and 525 versus 0.35). Thus, comparison of dEGR, DFPR, DFFR inhibition suggests that FVIIa prefers Phe at S2 and at S3/S4 positions, and that TF opens up the S1/S2/S3/S4 sites for substrate or inhibitor occupancy. These data are consistent with LTR (P3/P2/P1) residues in FX at its activation cleavage site as well as with LTR (P3/P2/P1) residues and FTR (P3/P2/P1) residues at the 145-146 and 180-181 FIX activation cleavage sites, respectively. Thus, these studies with chloromethylketone inhibitors have biologic relevance. For Biacore studies, sTF was amine coupled to a CM5 chip. The binding of unoccupied active site FVIIa in 5 mM calcium to sTF was characterized by a KD of 7 nM. Benzamidine (10 mM)-VIIa, p-aminobenzamidine (pAB, 1 mM)-VIIa, EGR-VIIa, dEGR-VIIa, DFPR-VIIa and DFFR-VIIa each bound to sTF with KD values ranging from 1- 2 nM. These affinity measurements indicate that the S1 site occupied FVIIa molecule (benzamidine-FVIIa, pAB-VIIa) has essentially the same conformation as the S1/S2/S3/S4 occupied FVIIa. This conclusion is consistent with similar crystal structures of variously inhibited FVIIa molecules complexed with sTF. The differential rates of incorporation of various chloromethylketone inhibitors could be due to the interaction of various residues (P1, P2, P3, P4) with the corresponding active subsites (S1/S2/S3/S4) of FVIIa. Additionally, the rate of incorporation of chloromethylketone inhibitors into FVIIa also involves the irreversible alkylation step, which could be faster for DFFR-ck and DFPR-ck. Once these inhibitors are incorporated, it appears that they induce the same conformation in FVIIa as achieved by S1 site occupancy alone. Thus S1 site occupancy in FVIIa induces the required conformation to modestly increase the affinity for TF.Second Order Rate Constants for Inhibition of FVIIa ± sTF with Various Chloromethylketone (ck) InhibitorsInhibitorMinus sTF k (min−1 mM−1)Plus sTF k (min−1 mM−1)Fold DifferenceEGR-ck0.040.358.8dansyl EGR-ck0.072.4134.4(D)FPR-ck2.312554.3D)FFR-ck5.652593.8

Small‐Molecule Negative Modulators of Adrenomedullin: Design, Synthesis, and 3D‐QSAR Study

Adrenomedullin (AM) is a peptidic hormone that was isolated in 1993, the function of which is related to several diseases such as diabetes, hypertension, and cancer. Compound 1 is one of the first nonpeptidic small-molecule negative modulators of AM, identified in a high-throughput screen carried out at the National Cancer Institute. Herein we report the synthesis of a series of analogues of 1. The ability of the synthesized compounds to disrupt the binding between AM and its monoclonal antibody has been measured, together with surface plasmon resonance (SPR)-based binding assays as implemented with Biacore technology. These data were used to derive a three-dimensional quantitative structure-activity relationship (3D-QSAR) model, with a q(2) (LOO) value of 0.8240. This study has allowed us to identify relevant features for effective binding to AM: the presence of a hydrogen-bond donor group and an aromatic ring. Evaluation of the ability of selected compounds to modify cAMP production in Rat2 cells showed that the presence of a free carboxylic acid is essential for negative AM modulation.

Thermodynamic characterization of pyrazole and azaindole derivatives binding to p38 mitogen-activated protein kinase using Biacore T100 technology and van’t Hoff analysis

Biacore T100 technology was used in conjunction with a van’t Hoff analysis to characterize the thermodynamic binding parameters of 85 small-molecule inhibitors of adenosine triphosphate (ATP) binding to p38 mitogen-activated protein (MAP) kinase. The compounds were selected from a large panel of azaindole and pyrazole derivatives for which IC 50 data exist. We showed a strong relationship between the K D and IC 50 of a compound, but only a modest relationship between k off and IC 50 was detected and an apparent relationship between a compound’s k on and its IC 50 could not be discerned. Similarly, a correlation between a compound’s IC 50 and its thermodynamic parameters ΔH° and ΔS° could not be established. The lack of a predominant kinetic or thermodynamic signature associated with the inhibitory potential of these compounds demonstrates that there exists, even within a single well-defined system, a library of kinetic routes or, alternatively, a library of initial and final enthalpic and entropic states from which to effect inhibition. As a complement to these studies, selected double mutant thermodynamic cycles were performed to probe the energetic coupling, if any, between common sites of fluorination in both the azaindole and pyrazole classes and two different substituents. Although both cycles indicated negligible coupling free energies, both revealed significant coupling enthalpies, an observation made in other similarly dissected systems. The possible significance and caveats associated with these findings along with the advantages of using Biacore technology to derive thermodynamic parameters in drug discovery efforts are discussed.

Detection and quantification of affinity ligand leaching and specific antibody fragment concentration within chromatographic fractions using surface plasmon resonance

Rapid analyses of chromatographic steps within a biopharmaceutical manufacturing process are often desirable to evaluate column performance, provide mass balance data and to permit accurate calculations of yields and recoveries. Using SPR (surface plasmon resonance) biosensor (Biacore) technology, we have developed a sandwich immunoassay to quantify polyclonal anti-digoxin Fab fragments used for the production of the FDA (Food and Drug Administration)-approved biotherapeutic DigiFab. The results show that specific Fab may be quantified in all affinity process streams and accurate yield and mass balance data calculated. Control experiments using sheep Fab and Fc indicate that the assay is specific to DigiFab. The quantification of potential leached ligand within chromatographic fractions may also be technically challenging, particularly when low-molecular-mass ligands are covalently coupled with an affinity absorbent. Typical methods to assess ligand leakage such as DDMA (digoxin-dicarboxymethoxylamine; digoxin analogue) often involve the use of labelled ligands and relatively complex and labour-intensive analytical techniques. Using the same analytical methodologies, an assay to detect leached or eluted ligand off the column was developed. The results indicate minimal levels of leached ligand in all chromatographic fractions, with total levels of leached DDMA calculated to be 1.52 microg. This is less than 0.01% of the total amount of DDMA coupled with the laboratory-scale affinity column. The SPR methods described in the present study may be applicable for the rapid in-process analysis of specific polyclonal Fab fragments (within a polyclonal mixture) and to rapidly assess leakage of small molecule ligands covalently attached to chromatographic supports.

The Middle and Distal Carboxyl Terminal Domains of ADAMTS13 Are Critical for Docking ADAMTS13 to von Willebrand Factor.

ADAMTS13 cleaves von Willebrand factor (VWF) at the Tyr1605-Met1606 bond of the central A2 domain. However, the domains of ADAMTS13 required for recognition and cleavage of VWF remain controversial. On the one hand, we and others have shown that the proximal carboxyl terminal domains (i.e. Cys-rich and spacer domains) of ADAMTS13 appeared to be sufficient for recognition of VWF under the static and denatured conditions (Zheng et al, JBC, 2003; Soejima et al, Blood, 2003, Ai et al, JBC, 2005). On the other hand, the distal carboxyl terminus (i.e. the first CUB domain) of ADAMTS13 has been proposed to dock ADAMTS13 to VWF under flow (Tao et al, Blood, 2005). To determine the interaction between ADAMTS13 and native VWF in real time, we employed Biacore technology based on surface plasmon resonance. To avoid conformational change or activation of VWF during the ligand immobilization, we chose to flow purified VWF or A2 fragment (VWF73) over the carboxymethylated dextran (CM5) surface to which was covalently attached via amine coupling either purified full-length recombinant ADAMTS13 (FL-A13) (~8,200 RU), the variant truncated after the 8th thrombospondin type 1 repeat (delCUB) (~7,200 RU), or that truncated after the spacer domain (MDTCS) (~4,200 RU). The control surface was prepared similarly by activation and deactivation processes without protein being coupled. The VWF at various concentrations in 20 mM HEPES buffer, pH 7.5, 150 mM NaCl, 0.02% Tween 20 and 0.05% BSA was flown at 20 μl/min over the surfaces as prepared above. The sensorgrams were recorded simutaneouly in all four microfluid cells and the kinetic parameters were determined. We showed that VWF bound immobilized FL-A13 in a dose and time-dependent manner with an equilibrium dissociation constant (Kd) of 50.0 ± 21.8 nM (n=6). Removal of the CUB domains (construct delCUB) reduced its affinity for VWF by ~5.5 fold (Kd=274 ± 158 nM (n=6). Further removal of the TSP1 2~8 repeats (construct MDTCS) almost completely abolished its binding to VWF (n=4) (Kd not determined). The affinity between ADAMTS13 (or variants) and VWF was not significantly altered by addition of 10 mM EDTA. Pre-denaturization of VWF with 1.5 M guanidine-HCl at 37 °C for 1 h, however, modestly enhanced VWF binding to immobilized delCUB (Kd=185 ± 96 nM, n=4) and MDTCS (Kd not determined), but reduced VWF binding to FL-A13 by ~2 fold, perhaps due to the mild denaturization of FL-A13 by 0.15 M guanidine-HCl in the flowing buffer. Interestingly, VWF73 bound FL-A13, delCUB and MDTCS with the Kd (s) of 2.6 ± 0.5 nM, 4.9 ± 1.0 nM and 3.0 ± 0.5 nM, respectively, which was at least 20~55 fold higher than native VWF did, suggesting that the protein structure surrounding the cleavage site at the central A2 domain of VWF negatively regulates ADAMTS13-VWF interaction. Moreover, the Michael-Menton constant Km (s) for cleavage of the denatured VWF (or VWF73) by FL-A13, delCUB and MDTCS were 260 ± 117 nM, 276 ± 118 nM and 534 ± 657 nM (or 1.8 ± 1.5 μM, 0.3 ± 0.1 μM and 0.6 ± 0.2 μM), respectively. These data demonstrate that the interaction between ADAMTS13 and native VWF is a high affinity one and that the middle and distal carboxyl terminal domains of ADAMTS13 may be critical for initial docking of ADAMTS13 to the exosites of native VWF in solution.