Toxin-Targeted Design for Anticancer Therapy. I: Synthesis and Biological Evaluation of New Thioimidate Heterobifunctional Reagents

Delprino, Laura; Giacomotti, M.; Dosio, Franco; Brusa, Paola; Ceruti, Maurizio; Grosa, Giorgio; Cattel, Luigi

doi:10.1002/jps.2600820515

Cited by 14 publications

(11 citation statements)

References 39 publications

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“…Both linkers can be used for controlled reactions because of their protected thiol moiety. [119,120] Disulfide bridges with sulfosuccinimidyl N-[3-(acetylthio)-3-methylbytyryl-b-alaninate (sNHS-ATMBA) are very stable due to a sterically hindered a-carbon atom. [121] Thiol-and Carbonyl-Reactive Linkers: Due to its pyridyl disulfide end, 3-(2-pyridyldithio)propionyl hydrazide (PDPH) undergoes disulfide exchanges (Table 3).…”

Section: Heterobifunctional Linkersmentioning

confidence: 99%

Delivery of Nucleic Acids via Disulfide‐Based Carrier Systems

et al. 2009

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Nucleic acids are not only expected to assume a pivotal position as "drugs" in the treatment of genetic and acquired diseases, but could also act as molecular cues to control the microenvironment during tissue regeneration. Despite this promise, the efficient delivery of nucleic acids to their side of action is still the major hurdle. One among many prerequisites for a successful carrier system for nucleic acids is high stability in the extracellular environment, accompanied by an efficient release of the cargo in the intracellular compartment. A promising strategy to create such an interactive delivery system is to exploit the redox gradient between the extra- and intracellular compartments. In this review, emphasis is placed on the biological rationale for the synthesis of redox sensitive, disulfide-based carrier systems, as well as the extra- and intracellular processing of macromolecules containing disulfide bonds. Moreover, the basic synthetic approaches for introducing disulfide bonds into carrier molecules, together with examples that demonstrate the benefit of disulfides at the individual stages of nucleic acid delivery, will be presented.

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Section: Heterobifunctional Linkersmentioning

confidence: 99%

Delivery of Nucleic Acids via Disulfide‐Based Carrier Systems

et al. 2009

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“…Disulfide Bond Stability in Modified AR-3. The protein (33.3 µM, 1 mL) was reacted with the ligands M-AMPT, Ph-AMPT, M-CDPT, and Ph-CDCT and with the analogous unhindered thioimidates AMPT and CDPT, previously prepared in our laboratory (19), so as to incorporate an average of 1-1.2 mol of linkers/mol of protein.…”

Section: Determination Of Aryldithio Group Reactivitymentioning

confidence: 99%

“…Kinetic Studies. To verify the relationship between steric hindrance and stability of the disulfide bond in the (aryldithio)thioimidate linker series, we studied the reactivity toward thiol-disulfide exchange in the precursor nitrile derivatives 4, 6, 7, and 8 using cysteine as thiol reagent (19).…”

Section: Chemistrymentioning

confidence: 99%

“…Stability of Model Disulfides in Derivatized AR-3. The relative stability of the disulfide bond introduced into AR-3 by derivatization with ligands M-AMPT 9, Ph-AMPT 10, M-CDPT 13, and Ph-CDCT 14 was analyzed by comparison with the parent unhindered linkers AMPT and CDPT (19) using GSH as reducing agent.…”

Section: Chemistrymentioning

confidence: 99%

“…In preceding papers, we have reported the preparation of two new acyclic thioimidate cross-linking reagents, 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT) and ethyl S-acetylpropionthioimidate (AMPT) (19,20). We used these reagents to prepare immunotoxins made from the RIP-1 gelonin (21) and the AR-3 mAb that recognizes the CAR-3 antigen widely expressed among * Author to whom correspondence should be addressed (telephone ++39.11.6707697; fax ++39.11.6707695; e-mail cattel@ ch.unito.it).…”

Section: Introductionmentioning

confidence: 99%

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New Coupling Reagents for the Preparation of Disulfide Cross-Linked Conjugates with Increased Stability

Berlier¹,

Dosio²,

Brusa³

et al. 1997

Bioconjugate Chem.

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To improve the in vivo stability of disulfide-linked immunotoxins (ITs), a series of sterically hindered cross-linking reagents were designed and synthesized. These ligands are characterized by a thioimidate group linked to an S-acetyl thiol or a substituted aryldithio group. To select the reagent of choice, several aryldithio thioimidates, substituted with a methyl or a phenyl group adjacent to the disulfide, were analyzed in thiol-disulfide exchange reactions. Also analyzed were the following: (i) the stability and solubility of the linkers in aqueous solution, (ii) the rate of protein derivatization, and (iii) the steric hindrance due to methyl or phenyl group substituents toward cleavage of the disulfide bond by glutathione. Ethyl S-acetyl 3-mercaptobutyrothioimidate (M-AMPT) was chosen as reagent to prepare two types of stable disulfide-containing AR-3-gelonin conjugates (IT2 and IT3). IT2 was prepared by a 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT)-derivatized antibody coupled to the M-AMPT-derivatized gelonin to afford a conjugate characterized by the presence of a methyl group adjacent to the sulfide bond. In the IT3 conjugate, an M-AMPT-derivatized toxin was coupled to the antibody thiolated with M-AMPT and then activated with Ellman's reagent (DNTB). The in vitro and in vivo stabilities of the three immunoconjugates were assayed, respectively, (i) by adding an excess of glutathione and monitoring protein release and (ii) by studying their pharmacokinetic behaviors. The specificity and cytotoxicity of all ITs were analyzed on target and unrelated cell lines, and no significant differences in activity were observed. IT3, consisting of a symmetrical dimethyl-substituted disulfide bond, was substantially more stable in vivo (t1/2 beta = 88.3 h) than the corresponding IT2, characterized by a disulfide-protected monomethyl substituent bond (t1/2 beta = 60.2 h) compared to the unhindered conjugate IT1 (t1/2 beta = 27.9 h). This family of cross-linking reagents therefore offers advantages, such as minimal perturbation of the protein structure and controlled reactivity due to the thioimidate moiety, as well as the capacity to yield immunotoxins possessing substantial stability in vivo.

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