Protease-Cleavable Linkers Modulate the Anticancer Activity of Noninternalizing Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) represent an attractive class of biopharmaceutical agents, with the potential to selectively deliver potent cytotoxic agents to tumors. It is generally assumed that ADC products should preferably bind and internalize into cancer cells in order to liberate their toxic payload, but a growing body of evidence indicates that also ADCs based on noninternalizing antibodies may be potently active. In this Communication, we investigated dipeptide-based linkers (frequently used for internalizing ADC products) in the context of the noninternalizing F16 antibody, specific to a splice isoform of tenascin-C. Using monomethyl auristatin E (MMAE) as potent cytotoxic drug, we observed that a single amino acid substitution of the Val-Cit dipeptide linker can substantially modulate the in vivo stability of the corresponding ADC products, as well as the anticancer activity in mice bearing the human epidermoid A431 carcinoma. In these settings, the linker based on the Val-Ala dipeptide exhibited better performances, compared to Val-Cit, Val-Lys, and Val-Arg analogues. Mass spectrometric analysis revealed that the four linkers displayed not only different stability in vivo but also differences in cleavage sites. Moreover, the absence of anticancer activity for a F16-MMAE conjugate featuring a noncleavable linker indicated that drug release modalities, based on proteolytic degradation of the immunoglobulin moiety, cannot be exploited with noninternalizing antibodies. ADC products based on the noninternalizing F16 antibody may be useful for the treatment of several human malignancies, as the cognate antigen is abundantly expressed in the extracellular matrix of several tumors, while being virtually undetectable in most normal adult tissues.

A Specific and Covalent JNK-1 Ligand Selected from an Encoded Self-Assembling Chemical Library.

We describe the construction of a DNA-encoded chemical library comprising 148 135 members, generated through the self-assembly of two sub-libraries, containing 265 and 559 members, respectively. The library was designed to contain building blocks potentially capable of forming covalent interactions with target proteins. Selections performed with JNK1, a kinase containing a conserved cysteine residue close to the ATP binding site, revealed the preferential enrichment of a 2-phenoxynicotinic acid moiety (building block A82) and a 4-(3,4-difluorophenyl)-4-oxobut-2-enoic acid moiety (building block B272). When the two compounds were joined by a short PEG linker, the resulting bidentate binder (A82-L-B272) was able to covalently modify JNK1 in the presence of a large molar excess of glutathione (0.5 mm), used to simulate intracellular reducing conditions. By contrast, derivatives of the individual building blocks were not able to covalently modify JNK1 in the same experimental conditions. The A82-L-B272 ligand was selective over related kinases (BTK and GAK), which also contain targetable cysteine residues in the vicinity of the active site.

Hit-Validation Methodologies for Ligands Isolated from DNA-Encoded Chemical Libraries.

DNA-encoded chemical libraries (DECLs) are large collections of compounds linked to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of interest. In typical DECL selections, preferential binders are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified in this procedure need to be confirmed, by resynthesis and by performing affinity measurements. In this article we present new methods based on hybridization of oligonucleotide conjugates with fluorescently labeled complementary oligonucleotides; these facilitate the determination of affinity constants and kinetic dissociation constants. The experimental procedures were demonstrated with acetazolamide, a binder to carbonic anhydrase IX with a dissociation constant in the nanomolar range. The detection of binding events was compatible not only with fluorescence polarization methodologies, but also with Alphascreen technology and with microscale thermophoresis.

A 99mTc-Labeled Ligand of Carbonic Anhydrase IX Selectively Targets Renal Cell Carcinoma In Vivo.

UNLABELLED: Small organic ligands, selective for tumor-associated antigens, are increasingly being considered as alternatives to monoclonal antibodies for the targeted delivery of diagnostic and therapeutic payloads such as radionuclides and drugs into neoplastic masses. We have previously described a novel acetazolamide derivative, a carbonic anhydrase ligand with high affinity for the tumor-associated isoform IX (CAIX), which can transport highly potent cytotoxic drugs into CAIX-expressing solid tumors. The aim of the present study was to quantitatively investigate the biodistribution properties of said ligand and understand whether acetazolamide conjugates merit further development as drug carriers and radioimaging agents. METHODS: The conjugate described in this study, consisting of a derivative of acetazolamide, a spacer, and a peptidic (99m)Tc chelator, was labeled using sodium pertechnetate under reducing conditions and injected intravenously into CAIX-expressing SKRC-52 xenograft-bearing mice. Animals were sacrificed, and organ uptake as percentage injected activity of radiolabeled ligand per gram of tissues (%IA/g) was evaluated between 10 min and 24 h. Additionally, postmortem imaging by SPECT was performed. RESULTS: The acetazolamide conjugate described in this study could be labeled to high radiochemical purity (>95%, 2.2-4.5 MBq/nmol). Analysis of organ uptake at various time points revealed that the ligand displayed a maximal tumor accumulation 3 h after intravenous injection (22 %IA/g), with an excellent tumor-to-blood ratio of 70:1 at the same time point. The ligand accumulation in the tumor was more efficient than in any other organ, but a residual uptake in the kidney, lung, and stomach (9, 16, and 10 %IA/g, respectively) was also observed, in line with patterns of carbonic anhydrase isoform expression in those tissues. Interestingly, tumor-to-organ ratios improved on administration of higher doses of radiolabeled ligand, suggesting that certain binding sites in normal organs can be saturated in vivo. CONCLUSION: The (99m)Tc-labeled acetazolamide conjugate exhibits high tumor uptake and favorable tumor-to-kidney ratios of up to 3 that may allow imaging of tumors in the kidney and distant sites at earlier time points than commonly possible with antibody-based products. These data suggest that the described molecule merit further development as a radioimaging agent for CAIX-expressing renal cell carcinoma.

Chimeric RNA Oligonucleotides with Triazole and Phosphate Linkages: Synthesis and RNA Interference.

Chimeric RNA oligonucleotides with an artificial triazole linker were synthesized using solution-phase click chemistry and solid-phase automated synthesis. Scalable synthesis methods for jointing units for the chimeric structure have been developed, and after click-coupling of the jointing units with triazole linkers, a series of chimeric oligonucleotides was prepared by utilizing the well-established phosphoramidite method for the elongation. The series of chimeric 21-mer oligonucleotides that possessed the triazole linker at different strands and positions allowed for a screening study of the RNA interference to clarify the preference of the triazole modifications in small-interfering RNA molecules.

Synthesis of Bioconjugated sym-pentasubstituted corannulenes: experimental and theoretical investigations of supramolecular architectures.

Applications of supramolecular architectures span a broad range of fields from medicinal chemistry to materials science and gas storage, making the design and synthesis of such structures a goal of high interest. The unique structural and symmetric properties of corannulene and the recent synthetic developments of C5-symmetric pentafunctionalized derivatives motivate efforts to synthesize bioconjugated-corannulene systems and investigate their supramolecular assembly properties. Herein is presented the synthesis of sym-pentasubstituted corannulenes functionalized with sugar (galactose and ribose), oligopeptide, nucleosides (thymidine and deoxyadenosine), and palindromic oligonucleotide strands. Experimental and theoretical results demonstrate capability of supramolecular assembly formation in these constructs. Ab initio theoretical modeling enables further evaluation of structure and energetics of oligonucleotide-functionalized corannulene formation. Results indicate formation of aggregates, although icosahedral supramolecular formation is not observed. Analyses suggest future improvements to synthetic routes to achieve icosahedral architectures.

Five-fold symmetric penta-substituted corannulene with gelation properties and a liquid-crystalline phase.

Five-fold symmetric substituted corannulene derivatives that display liquid-crystalline behavior and organogelation properties were prepared by coupling of N-azidoethyl long-chain fatty acid amides to sym-pentabutynyl corannulenes via dipolar cycloaddition chemistry.

Nanomolar cholera toxin inhibitors based on symmetrical pentavalent ganglioside GM1os-sym-corannulenes.

Eight symmetric and pentavalent corannulene derivatives were functionalized with galactose and the ganglioside GM1-oligosaccharide (GM1os) via copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions. The compounds were evaluated for their ability to inhibit the binding of the pentavalent cholera toxin to its natural ligand, ganglioside GM1. In this assay, all ganglioside GM1os-sym-corannulenes proved to be highly potent nanomolar inhibitors of cholera toxin.

Sym-(CH2X)5-corannulenes: molecular pentapods displaying functional group and bioconjugate appendages.

Pentapodal ω-functional derivatives of corannulene have been synthesized from sym-pentachlorocorannulene by iron-catalyzed aryl-alkyl cross coupling reactions. Click chemistry gives access to pentapods with bioconjugate appendages.