Specific Structural Modification Research Articles

Structure modification: a successful tool for prodrug design.

Prodrug strategy is critical for innovative drug development. Structural modification is the most straightforward and effective method to develop prodrugs. Improving drug defects and optimizing the physical and chemical properties of a drug, such as lipophilicity and water solubility, changing the way of administration can be achieved through specific structural modification. Designing prodrugs by linking microenvironment-responsive groups to the prototype drugs is of great help in enhancing drug targeting. In the meantime, making connections between prodrugs and suitable drug delivery systems could realize drug loading increases, greater stability, bioavailability and drug release control. In this paper, lipidic, water-soluble, pH-responsive, redox-sensitive and enzyme-activatable prodrugs are reviewed on the basis of structural modification.

Abstract P218: Annamycin, a novel non-cardiotoxic anthracycline with high activity against sarcomas metastatic to lungs

Abstract Introduction. Annamycin (ANN) is a non-cardiotoxic potent topoisomerase II poison structurally resembling doxorubicin (Dox). Specific structural modification altered ANN properties dramatically when compared with Dox. This novel drug also includes high activity against multidrug-resistant (MDR) tumors, unique subcellular distribution, and different tissue-organ distribution. Especially high differences have been noted in the lung uptake when compared with DOX. ANN is formulated in multilamellar liposomes (L-Annamycin, L-ANN), and such formulation additionally contributes to the high ANN lung uptake. Objective. The objective of this study was to reconfirm the unique tissue-organ distribution pattern in rats and explore ANN high lung uptake to target pulmonary sarcoma metastases by assessing the efficacy of L-ANN in the experimental models of lung metastases. Methods. Pharmacokinetics and biodistribution studies of ANN were performed in rats after bolus injection of L-ANN, free ANN, and Dox. Detection of Dox and ANN in plasma and different organs was accomplished using LC/MS/MS. In vivo efficacy was tested in two experimental lung metastatic models. The tumors were established by intravenous injection of MCA205 or K7M3 cells. The treatment consisted of weekly intravenous injections of 4 mg/kg of L-ANN or Dox. Metastatic nodules were visualized by computer tomography or bioluminescent imaging (BLI). Results. Pharmacokinetic and tissue-organ distribution of L-ANN in rats confirmed unusually high uptake of ANN in lungs: the Cmax of ANN was 30-fold higher than Dox after bolus administration of L-ANN or Dox. A remarkable extension of the survival was observed in two syngeneic sarcoma lung metastasis models. In the MCA205 fibrosarcoma model, the median survival of L-ANN treated mice was 87.5 days vs. 21 days for the vehicle-treated mice (p<0.0001), and 40% of animals were still alive when the experiment has been terminated on day 140. L-ANN also appeared highly efficacious in the osteosarcoma K7M3 model. Median survival has not been reached due to the high efficacy of L-ANN treatment. The experiment has been terminated on day 220 (the median survival for the vehicle-treated was 58 days, p=0.0002). Additionally, L-ANN and Dox were compared in the K7M3-Luc model. The BLI revealed a potent inhibition of the tumor growth by L-ANN-, but not in Dox-treated animals. Consequently, there was no extension of the survival in DOX-treated animals, while no deaths were recorded in L-ANN treated group as of day 90 (MS for Dox-treated mice was 59 days, ongoing study). Conclusion. Annamycin (ANN) is a novel non-cardiotoxic anthracycline that exhibits very high uptake in the lungs compared to Dox. Furthermore, ANN’s rapid cellular uptake and preferential nanomolar cytotoxicity against cancer led to major efficacy in sarcoma lung metastasis models. These promising preclinical results led to the initiation of a multicenter sarcoma-specific clinical study (NCT04887298). Citation Format: Rafal Zielinski, Krzysztof Grela, Roberto Cardenas-Zuniga, Stanislaw Skora, Izabela Fokt, Maria Poimenidou, Salah-Eddine Lamhamedi-Cherradi, Joseph Ludwig, Waldemar Priebe. Annamycin, a novel non-cardiotoxic anthracycline with high activity against sarcomas metastatic to lungs [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P218.

Discovery of 4-Arylindolines Containing a Thiazole Moiety as Potential Antitumor Agents Inhibiting the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction

Through specific structural modification of a 4-phenylindoline precursor, new 4-arylindolines containing a thiazole moiety were developed and found to be promising modulators of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) axis. Compound A30 exhibited outstanding biochemical activity, with an IC50 of 11.2 nM in a homogeneous time-resolved fluorescence assay. In the cell-based assay, A30 significantly promoted IFN-γ secretion and rescued T-cell proliferation, which were inhibited by PD-1 activation. Furthermore, A30 showed favorable in vivo antitumor activity in a mouse 4T1 breast carcinoma model. Moreover, in mouse CT26 colon carcinoma models, A30 potently suppressed the growth of CT26/PD-L1 tumor but did not obviously affect the growth of CT26/vector tumor. The results of flow cytometry analysis indicated that A30 inhibited tumor growth by activating the immune microenvironment. We concluded that A30 is a new starting point for further development of PD-1/PD-L1 interaction inhibitors as antitumor agents.

Extracellular Oxygen Sensors Based on PtTFPP and Four-Arm Block Copolymers

Three four-arm amphiphilic block copolymers with different chain lengths, consisting of a hydrophilic chain of polyethylene glycol (PEG) and hydrophobic segment of polycaprolactam (PCL), were synthesized and used to encapsulate the high-efficient and hydrophobic oxygen probe of platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) to form polymer micelles. This approach enabled the use of PtTFPP in aqueous solution for biosensing. Experimental results demonstrated that the particle sizes of these nano-oxygen sensors between 40.0 and 203.8 nm depend on the structures of block copolymers. PtTFPP in these micelles showed an effective quantum yield under nitrogen environment, ranging from 0.06 to 0.159. The new sensors are suitable for analyzing dissolved oxygen concentrations in the range of 0.04–39.3 mg/L by using the linear Stern–Volmer equation at room temperature. In addition, it has been shown that these sensors are capable of in situ monitoring the dissolved oxygens in the culture medium of E. coli and Romas cells during the respiration process, and distinguishing the drug activity of antibiotic ampicillin from that of antimycin A. This study showed that the use of these nanostructured multi-arm block copolymer micelles can achieve efficient biological applications without specific structural modification of the hydrophobic PtTFPP probe, which is expected to have broad prospects.

Polyladderane Constructed from a Gemini Monomer through Photoreaction.

Polyladderane, the first polymer to contain the ladderane functional group, was synthesized from a gemini monomer through photoreaction in the solid state. The modular design of the gemini monomers used to create polyladderane allowed specific structural modification, resulting in the formation of two distinct polymer products. Monomers were synthesized by connecting two photoreactive units, either sorbic acids (monomer I) or 2-furanacrylic acids (monomer II), with a 1,4-butanediol linker. Single-crystal X-ray diffraction analysis of the monomers confirmed that they packed in the desired head-to-tail orientation and within a viable distance for photoreaction by electronically complementary interaction. Pre-organized gemini monomers were irradiated with UV light and monitored by FT-IR. Two polyladderanes with cis,anti,cis-[3]-ladderane as a characteristic functional group were constructed stereospecifically in 24-36 hours.

Polyladderane Constructed from a Gemini Monomer through Photoreaction

AbstractPolyladderane, the first polymer to contain the ladderane functional group, was synthesized from a gemini monomer through photoreaction in the solid state. The modular design of the gemini monomers used to create polyladderane allowed specific structural modification, resulting in the formation of two distinct polymer products. Monomers were synthesized by connecting two photoreactive units, either sorbic acids (monomer I) or 2‐furanacrylic acids (monomer II), with a 1,4‐butanediol linker. Single‐crystal X‐ray diffraction analysis of the monomers confirmed that they packed in the desired head‐to‐tail orientation and within a viable distance for photoreaction by electronically complementary interaction. Pre‐organized gemini monomers were irradiated with UV light and monitored by FT‐IR. Two polyladderanes with cis,anti,cis‐[3]‐ladderane as a characteristic functional group were constructed stereospecifically in 24–36 hours.

Discovery of Rapid and Reversible Water Insertion in Rare Earth Sulfates: A New Process for Thermochemical Heat Storage.

Thermal energy storage based on chemical reactions is a prospective technology for the reduction of fossil-fuel consumption by storing and using waste heat. For widespread application, a critical challenge is to identify appropriate reversible reactions that occur below 250 °C, where abundant low-grade waste heat and solar energy might be available. Here, it is shown that lanthanum sulfate monohydrate La2 (SO4 )3 ⋅H2 O undergoes rapid and reversible dehydration/hydration reactions in the temperature range from 50 to 250 °C upon heating/cooling with remarkably small thermal hysteresis (<50 °C), and thus it emerges as a new candidate system for thermal energy storage. Thermogravimetry and X-ray diffraction analyses reveal that the reactions proceed through an unusual mechanism for sulfates: water is removed from, or inserted in La2 (SO4 )3 ⋅H2 O with progressive change in hydration number x without phase change. It is also revealed that only a specific structural modification of La2 (SO4 )3 exhibits this reversible dehydration/hydration behavior.

Spectral characteristics of different structural modifications of Lu1 − x Eu x BO3

The spectral and structural characteristics of polycrystals of Eu3+-doped lutetium borates Lu1 − x Eu x BO3) annealed at different temperatures have been investigated over a wide range of europium concentrations. The conditions for the preparation of Lu1 − x Eu x BO3 in the calcite and vaterite phases have been determined. It has been found that there is a radical difference between the excitation spectra of the main emission bands of the calcite and vaterite phases of the Lu1 − x Eu x BO3 borates. The influence of the europium concentration on the structure of Lu1 − x Eu x BO3 has been analyzed. It has been established that, at europium concentrations of higher than 15 at %, only the vaterite structure is formed independently of the annealing temperature. Thus, by varying the Eu3+ concentration and the annealing temperature of Lu1 − x Eu x BO3, it is possible to directionally synthesize a specific structural modification and, consequently, to control the spectral characteristics of this compound.

Atheroprotective remodelling of vascular dermatan sulphate proteoglycans in response to hypercholesterolaemia in a rat model

Proteoglycan accumulation within the arterial intima has been implicated in atherosclerosis progression in humans. Nevertheless, hypercholesterolaemia is unable to induce intimal thickening and atheroma plaque development in rats. The study was performed to analyse proteoglycans modifications in rats fed with a high-cholesterol diet to understand whether vascular wall remodelling protects against lesions. Sections obtained from rat aortas showed normal features, in intimal-to-media ratio and lipid accumulation. However, focal endothelial hyperplasia and neo-intima rearrangement were observed in high-cholesterol animals. Besides, hypercholesterolaemia induced an inflammatory microenviroment. We determined the expression of different proteoglycans from aortic cells by Western blot and observed a diminished production of decorin and biglycan in high-cholesterol animals compared with control (P<0.01 and P<0.05, respectively). Versican was increased in high-cholesterol animals (P<0.05), whereas perlecan production showed no differences. No modification of the total content of glycosaminoglycans (GAGs) was found between the two experimental groups. In contrast, the chondroitin sulphate/dermatan sulphate ratio was increased in the high-cholesterol group as compared to the control (0.56 and 0.34, respectively). Structural alterations in the disaccharide composition of galactosaminoglycans were also detected by HPLC, as the ratio of 6-sulphate to 4-sulphate disaccharides was increased in high-cholesterol animals (P<0.05). Our results suggest that attenuation of decorin and biglycan expression might be an effective strategy to inhibit the first step in atherogenesis, although specific GAG structural modification associated with the development of vascular disease took place. Results emphasize the potential application of therapies based on vascular matrix remodelling to treat atherosclerosis.

A study on AB2O6 compounds, Part II: the branches of the hcp family

Abstract The crystal chemistry of many AB2O6 compounds is summarized in family trees comparing the crystal symmetry of several structures in group-subgroup relations. In this second part of a series the largest family derived from a hexagonally close packed arrangement of anions is described. On reducing the symmetry, different choices of octahedral sites for A and B cations and voids lead to four branches – the Rosiaite-, the ZnTa2O6-, the columbite- and the rutile-branch. We trace the reasons for the specific distribution patterns of the cations in the individual compounds by comparing long range contributions, i.e. the Madelung Part of Lattice Energy (MAPLE), and local effects represented by bond valence calculations according to I. D. Brown. Long range effects largely determine the choice of sites in the respective space groups, however, small local adjustments play a decisive role for the stability of a specific structural modification. Such mechanisms ultimately impose how the structure of an individual compound deviates from an ideally packed arrangement. We discuss the difference between the ideal structures and the real examples given in the family tree by defining a sort of deformation tensor and a measure of similarity, and we describe and depict the anisotropic deformation by a strain tensor.

Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

The 5′untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5′UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5′UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5′UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5′UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5′UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5′UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.

Advanced biorefinery in lower termite-effect of combined pretreatment during the chewing process

BackgroundCurrently the major barrier in biomass utilization is the lack of an effective pretreatment of plant cell wall so that the carbohydrates can subsequently be hydrolyzed into sugars for fermentation into fuel or chemical molecules. Termites are highly effective in degrading lignocellulosics and thus can be used as model biological systems for studying plant cell wall degradation.ResultsWe discovered a combination of specific structural and compositional modification of the lignin framework and partial degradation of carbohydrates that occurs in softwood with physical chewing by the termite, Coptotermes formosanus, which are critical for efficient cell wall digestion. Comparative studies on the termite-chewed and native (control) softwood tissues at the same size were conducted with the aid of advanced analytical techniques such as pyrolysis gas chromatography mass spectrometry, attenuated total reflectance Fourier transform infrared spectroscopy and thermogravimetry. The results strongly suggest a significant increase in the softwood cellulose enzymatic digestibility after termite chewing, accompanied with utilization of holocellulosic counterparts and an increase in the hydrolysable capacity of lignin collectively. In other words, the termite mechanical chewing process combines with specific biological pretreatment on the lignin counterpart in the plant cell wall, resulting in increased enzymatic cellulose digestibility in vitro. The specific lignin unlocking mechanism at this chewing stage comprises mainly of the cleavage of specific bonds from the lignin network and the modification and redistribution of functional groups in the resulting chewed plant tissue, which better expose the carbohydrate within the plant cell wall. Moreover, cleavage of the bond between the holocellulosic network and lignin molecule during the chewing process results in much better exposure of the biomass carbohydrate.ConclusionCollectively, these data indicate the participation of lignin-related enzyme(s) or polypeptide(s) and/or esterase(s), along with involvement of cellulases and hemicellulases in the chewing process of C. formosanus, resulting in an efficient pretreatment of biomass through a combination of mechanical and enzymatic processes. This pretreatment could be mimicked for industrial biomass conversion.

Electrodeposition and Corrosion Properties of Zn-V2O5 Composite Coatings

Composite coatings were obtained by electrochemical codeposition of V2O5 nanoparticles with zinc, from an additive free acid sulfate bath. The corrosion behavior of electrodeposited Zn and Zn-V2O5 composite coatings was investigated in 3.5% NaCl solution using polarization and electrochemical impedance spectroscopy (EIS). The decrease in I corr values and an increase in R ct values show the higher corrosion resistant nature of Zn-V2O5 coatings. The observed textural modifications of composite coatings are associated with the specific structural modification of Zn crystallites provoked by the adsorption-desorption phenomena occurring on the metal surface, induced by the presence of V2O5 nanoparticles. It has been observed that the presence of V2O5 nanoparticles favors the [1 0 2] and [1 1 2] texture of zinc matrix. Moreover, the codeposition of V2O5 nanoparticles with zinc was found to be favored at pH 3.5 and applied current density 4 A dm−2. A considerable grain refinement of the deposit occurred due to incorporation of V2O5 nanoparticles and hence improved the preferred orientation of the zinc crystallites.

Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure.

Enzymatic Modification of Heparan Sulfate on a Biochip Promotes Its Interaction with Antithrombin III

A heparan sulfate glycosaminoglycan chain, biotinylated at its reducing-end, was bound to a streptavidin-coated biochip. Surface plasmon resonance spectroscopy showed a low affinity interaction with antithrombin III (ATIII) when it was flowed over a surface containing heparan sulfate. ATIII bound tightly with high affinity when the same surface was enzymatically modified to using 3-O-sulfotransferase isoform 1 (3-OST-1) in the presence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). The 3-OST-1 enzyme is involved in heparan sulfate biosynthesis and introduces a critical 3-O-sulfo group into this glycosaminoglycan affording the appropriate pentasaccharide sequence capable of high affinity binding to ATIII. This experiment demonstrates the specific structural modification of a glycosaminoglycan bound to a biochip using a biosynthetic enzyme, suggesting a new approach to rapid screening glycosaminoglycan-protein interactions.

Inhibition of proprotein convertases-1, -7 and furin by diterpines of Andrographis paniculata and their succinoyl esters

Studies were performed to investigate the prohormone/proprotein convertase (PC)-inhibitory properties of chemical constituents of the medicinally active plant Andrographis paniculata (AP; from the family Acanthaceae), also known as ‘King of Bitters ’. Among the individual components tested against the clinically important convertases, furin and PC1, neoandrographolide (a C3 O-glucoside derivative of the major constituent andrographolide) exhibited the highest inhibitory action with an IC50 of 53.5 µM against furin. The data further revealed that although andrographolide, the major bitter principle of AP, exhibited a relatively small enzyme inhibition (IC50 = 1.0 mM and Ki = 200 µM against furin), upon succinoylation, its inhibitory action against the above convertases was enhanced significantly with a Ki in the low micromolar range (< 30 µM), suggesting that a specific structural modification of the andrographolide skeleton may be exploited to develop a new class of non-peptide inhibitors of PCs. When tested against PC7, these succinoylated derivatives of andrographolide also displayed strong inhibitory action, with Ki values again in the low micromolar range. This potentially interesting observation may be attributed to the reported anti-HIV property of 14-dehydroandrographolide succinic acid monoester (DASM). It is suggested here that DASM, by virtue of this protease inhibitory property, possibly acts by suppressing the proteolytic cleavage of envelope glycoprotein gp160 of HIV, which is known to be PC-mediated, particularly by furin and PC7.

Inhibition of proprotein convertases-1, -7 and furin by diterpines of Andrographis paniculata and their succinoyl esters

Studies were performed to investigate the prohormone/proprotein convertase (PC)-inhibitory properties of chemical constituents of the medicinally active plant Andrographis paniculata (AP; from the family Acanthaceae), also known as 'King of Bitters'. Among the individual components tested against the clinically important convertases, furin and PC1, neoandrographolide (a C3 O-glucoside derivative of the major constituent andrographolide) exhibited the highest inhibitory action with an IC50 of 53.5 microM against furin. The data further revealed that although andrographolide, the major bitter principle of AP, exhibited a relatively small enzyme inhibition (IC50=1.0 mM and Ki=200 microM against furin), upon succinoylation, its inhibitory action against the above convertases was enhanced significantly with a Ki in the low micromolar range (<30 microM), suggesting that a specific structural modification of the andrographolide skeleton may be exploited to develop a new class of non-peptide inhibitors of PCs. When tested against PC7, these succinoylated derivatives of andrographolide also displayed strong inhibitory action, with Ki values again in the low micromolar range. This potentially interesting observation may be attributed to the reported anti-HIV property of 14-dehydroandrographolide succinic acid monoester (DASM). It is suggested here that DASM, by virtue of this protease inhibitory property, possibly acts by suppressing the proteolytic cleavage of envelope glycoprotein gp160 of HIV, which is known to be PC-mediated, particularly by furin and PC7.

The Interaction of Candida boidinii Formate Dehydrogenase with a New Family of Chimeric Biomimetic Dye-Ligands

Seven chimeric biomimetic dye-ligands (BM) are purpose-designed and synthesized by specific structural modification of the parent anthraquinone dichlorotriazine dye Vilmafix blue A-R (VBAR). Each BM dye is composed of two enzyme-recognition moieties. The terminal biomimetic moiety bears a variable carboxylated structure linked to the triazine ring, thus mimicking the substrate of formate dehydrogenase (FDH). The anthraquinone moiety remains the same as that of the parent dye and recognizes the nucleotide-binding area of the target enzyme. Dyes are purified by liquid column chromatography (typically 99%), analyzed by liquid-paper chromatography, thin-layer chromatography, and high-performance liquid chromatography, and their λmax and ϵ values are determined. The ability of dyes to act as affinity ligands versus Candida boidinii FDH is evaluated by kinetic studies and determining KD values from both difference spectra and enzyme inactivation studies. The parent dichlorotriazine dye VBAR binds specifically and irreversibly to FDH (k3 0.19 min−1; KD 19.3 μM). The inactivation of the NAD+-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH, and ADP. Quantitatively inhibited FDH contained approx 1 mol of dye per mole of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM each of NAD+, NADH, and ADP or by extensive dialysis or gel filtration chromatography. The monochlorotriazine BM dyes do not inactivate FDH but inhibit competitively the inactivation by VBAR. When compared to VBAR and Cibacron blue 3GA (CB3GA), all BM dye-ligands exhibited lower KD values. FDH generally preferred binding to BM ligands which bore an aromatic terminal biomimetic moiety substituted with a monocarboxyl group rather than an α-ketoacid. Dye binding to FDH is accompanied by a characteristic spectral change in the range 550-800 nm. This phenomenon is perturbed after titration by increasing amounts of NAD+. Electrostatic interactions appeared to play a dominant role in the dye · FDH complex. The BM dye-ligand bearing a m-aminobenzoate at its terminal biomimetic moiety (BM1) exhibited the highest affinity (KD 1.6 μM, 8.0-fold decrease over CB3GA). BM1 differentiated between the binding sites of FDH, displaying uncompetitive inhibition with respect to NAD+ (Ki 15.6 μM) and competitive with respect to formate (Ki 18.1 μM).

Enhancement of tyrosine kinase activity of the Drosophila epidermal growth factor receptor homolog by alterations of the transmembrane domain

The Drosophila epidermal growth factor receptor homolog (DER) displays sequence similarity to both the epidermal growth factor (EGF) receptor and the neu vertebrate proteins. We have examined the possibility of deregulating the tyrosine kinase activity of DER by introducing structural changes which mimic the oncogenic alterations in the vertebrate counterparts. Substitution of valine by glutamic acid in the transmembrane domain, in a position analogous to the oncogenic mutation in the rat neu gene, elevated the in vivo kinase activity of DER in Drosophila Schneider cells sevenfold. A chimera containing the oncogenic neu extracellular and transmembrane domains and the DER kinase region, also showed a threefold elevated activity relative to a similar chimera with normal neu sequences. Double truncation of DER in the extracellular and cytoplasmic domains, mimicking the deletions in the v-erbB oncogene, did not however result in stimulation of in vivo kinase activity. The chimeric constructs were also expressed in monkey COS cells, and similar results were obtained. The ability to enhance the DER kinase activity by a specific structural modification of the transmembrane domain demonstrates the universality of this activation mechanism and strengthens the notion that this domain is intimately involved in signal transduction. These results also support the inclusion of DER within the tyrosine-kinase receptor family.

A simple method for increasing hapten immunogenicity by a specific structural modification of the carrier

A simple procedure to bind haptens, drugs or peptides selectively through their amino or carboxylic group to a spacer arm modified non-immunogenic polypeptide is described. Gelatin, a well known non-immunogenic carrier, was modified by blocking its amino groups. Spacer arms with primary amino groups such as β-alanine or ethylenediamine were conjugated to this protein by carbodiimide resulting in spacer modified gelatin. These modified polypeptides were tested for their ability to selectively bind haptens through their amino or carboxylic groups. Three probes were used and the results obtained confirm the hypothesis. Three conjugates obtained were further used to induce an immune response in mice. Enhancement of the immunogenicity of these spacer-arm supported haptens was observed. Overall, this study provides a rational approach to the production of well defined antigens using a simple conjugation technique.