Antibody-drug conjugates (ADCs) for the treatment of cancer aim to achieve selective delivery of a cytotoxic payload to tumor cells while sparing normal tissue. In vivo, multiple tumor-dependent and -independent processes act on ADCs and their released payloads to impact tumor-versus-normal delivery, often resulting in a poor therapeutic window. An ADC with a labeled payload would make synchronous correlations between distribution and tissue-specific pharmacological effects possible, empowering preclinical and clinical efforts to improve tumor-selective delivery; however, few methods to label small molecules without destroying their pharmacological activity exist.
View Article and Find Full Text PDFHeterobifunctional compounds that direct the ubiquitination of intracellular proteins in a targeted manner via co-opted ubiquitin ligases have enormous potential to transform the field of medicinal chemistry. These chimeric molecules, often termed proteolysis-targeting chimeras (PROTACs) in the chemical literature, enable the controlled degradation of specific proteins via their direction to the cellular proteasome. In this report, we describe the second phase of our research focused on exploring antibody-drug conjugates (ADCs), which incorporate BRD4-targeting chimeric degrader entities.
View Article and Find Full Text PDFThe biological and medicinal impacts of proteolysis-targeting chimeras (PROTACs) and related chimeric molecules that effect intracellular degradation of target proteins via ubiquitin ligase-mediated ubiquitination continue to grow. However, these chimeric entities are relatively large compounds that often possess molecular characteristics, which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. We therefore explored the conjugation of such molecules to monoclonal antibodies using technologies originally developed for cytotoxic payloads so as to provide alternate delivery options for these novel agents.
View Article and Find Full Text PDFWe are interested in developing a second generation of antibody-drug conjugates (ADCs) for the treatment of non-Hodgkin lymphoma (NHL) that could provide a longer duration of response and be more effective in indolent NHL than the microtubule-inhibiting ADCs pinatuzumab vedotin [anti-CD22-vc-monomethyl auristatin E (MMAE)] and polatuzumab vedotin (anti-CD79b-vc-MMAE). Pinatuzumab vedotin (anti-CD22-vc-MMAE) and polatuzumab vedotin (anti-CD79b-vc-MMAE) are ADCs that contain the microtubule inhibitor MMAE. Clinical trial data suggest that these ADCs have promising efficacy for the treatment of NHL; however, some patients do not respond or become resistant to the ADCs.
View Article and Find Full Text PDFFull-length antibodies lack ideal pharmacokinetic properties for rapid targeted imaging, prompting the pursuit of smaller peptides and fragments. Nevertheless, studying the disposition properties of antibody-based imaging agents can provide critical insight into the pharmacology of their therapeutic counterparts, particularly for those coupled with potent payloads. Here, we evaluate modulation of binding to the neonatal Fc receptor (FcRn) as a protein engineering-based pharmacologic strategy to minimize the overall blood pool background with directly labeled antibodies and undesirable systemic click reaction of radiolabeled tetrazine with circulating pretargeted -cyclooctene (TCO)-modified antibodies.
View Article and Find Full Text PDFChimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry.
View Article and Find Full Text PDFThe ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g.
View Article and Find Full Text PDFAntibody-drug conjugates (ADCs) contain a disease-receptor antibody and a payload drug connected via a linker. The payload delivery depends on both tumor properties and ADC characteristics. In this study, we used different linkers, attachment sites, and doses to modulate payload delivery of several ADCs bearing maytansinoids (e.
View Article and Find Full Text PDFIn cells, catalytic disulfide cleavage is an essential mechanism in protein folding and synthesis. However, detailed enzymatic catalytic mechanism relating cleavage of disulfide bonds in xenobiotics is not well understood. This study reports an enzymatic mechanism of cleavage of disulfide bonds in xenobiotic small molecules and antibody conjugate (ADC) linkers.
View Article and Find Full Text PDFThe valine-citrulline (Val-Cit) dipeptide and p-aminobenzyl (PAB) spacer have been commonly used as a cleavable self-immolating linker in ADC design including in the clinically approved ADC, brentuximab vedotin (Adcetris). When the same linker was used to connect to the phenol of the cyclopropabenzindolone (CBI) (P1), the resulting ADC1 showed loss of potency in CD22 target-expressing cancer cell lines (e.g.
View Article and Find Full Text PDFAntibody-drug conjugates (ADC) have become important scaffolds for targeted cancer therapies. However, ADC exposure-response correlation is not well characterized. We demonstrated that intratumor payload exposures correlated well with the corresponding efficacies of several disulfide-linked ADCs, bearing an DNA alkylating agent, pyrrolo[2,1-c][1,4]benzodiazepine-dimer (PBD), in HER2-expressing xenograft models.
View Article and Find Full Text PDFConjugation of small molecule payloads to cysteine residues on proteins via a disulfide bond represents an attractive strategy to generate redox-sensitive bioconjugates, which have value as potential diagnostic reagents or therapeutics. Advancement of such "direct-disulfide" bioconjugates to the clinic necessitates chemical methods to form disulfide connections efficiently, without byproducts. The disulfide connection must also be resistant to premature cleavage by thiols prior to arrival at the targeted tissue.
View Article and Find Full Text PDFDisulfide bonds provide a bioactivatable connection with applications in imaging and therapy. The circulation stability and intracellular release of disulfides are problematically coupled in that increasing stability causes a corresponding decrease in cleavage and payload release. However, an antibody offers the potential for a reversible stabilization.
View Article and Find Full Text PDFDisulfide bonds could be valuable linkers for a variety of therapeutic applications requiring tunable cleavage between two parts of a molecule (e.g., antibody-drug conjugates).
View Article and Find Full Text PDFDespite recent technological advances in quantifying antibody drug conjugate (ADC) species, such as total antibody, conjugated antibody, conjugated drug, and payload drug in circulation, the correlation of their exposures with the efficacy of ADC outcomes in vivo remains challenging. Here, the chemical structures and concentrations of intratumor catabolites were investigated to better understand the drivers of ADC in vivo efficacy. Anti-CD22 disulfide-linked pyrrolobenzodiazepine (PBD-dimer) conjugates containing methyl- and cyclobutyl-substituted disulfide linkers exhibited strong efficacy in a WSU-DLCL2 xenograft mouse model, whereas an ADC derived from a cyclopropyl linker was inactive.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
December 2014
There is great interest in developing selective protein kinase inhibitors by targeting allosteric sites, but these sites often involve protein-protein or protein-peptide interfaces that are very challenging to target with small molecules. Here we present a systematic approach to targeting a functionally conserved allosteric site on the protein kinase PDK1 called the PDK1-interacting fragment (PIF)tide-binding site, or PIF pocket. More than two dozen prosurvival and progrowth kinases dock a conserved peptide tail into this binding site, which recruits them to PDK1 to become activated.
View Article and Find Full Text PDFLike many coactivators, the GACKIX domain of the master coactivator CBP/p300 recognizes transcriptional activators of diverse sequence composition via dynamic binding surfaces. The conformational dynamics of GACKIX that underlie its function also render it especially challenging for structural characterization. We have found that the ligand discovery strategy of Tethering is an effective method for identifying small-molecule fragments that stabilize the GACKIX domain, enabling for the first time the crystallographic characterization of this important motif.
View Article and Find Full Text PDFThere is significant interest in identifying and characterizing allosteric sites in enzymes such as protein kinases both for understanding allosteric mechanisms as well as for drug discovery. Here, we apply a site-directed technology, disulfide trapping, to interrogate structurally and functionally how an allosteric site on the Ser/Thr kinase, 3-phosphoinositide-dependent kinase 1 (PDK1)--the PDK1-interacting-fragment (PIF) pocket--is engaged by an activating peptide motif on downstream substrate kinases (PIFtides) and by small molecule fragments. By monitoring pairwise disulfide conjugation between PIFtide and PDK1 cysteine mutants, we defined the PIFtide binding orientation in the PIF pocket of PDK1 and assessed subtle relationships between PIFtide positioning and kinase activation.
View Article and Find Full Text PDFGet into the groove: The first high-resolution structure of a foldamer bound to a protein target is described (see picture; foldamer in sticks). The foldamer consists of alpha- and beta-amino acid residues and is bound to the anti-apoptotic protein Bcl-x(L). The overall binding mode and key interactions observed in the foldamer/Bcl-x(L) complex mimic those seen in complexes of Bcl-x(L) with natural alpha-peptide ligands.
View Article and Find Full Text PDFAlanine scanning has been widely employed as a method of identifying side chains that play important roles in protein-protein and protein-peptide interactions. Here we show how an analogous and complementary technique, hydrophile scanning, can provide additional insight on such interactions. Mutation of a wild-type residue to alanine removes most of the side-chain atoms, and the effect of this removal is typically interpreted to indicate contribution of the deleted side chain to the stability of the complex.
View Article and Find Full Text PDFVascular endothelial growth factor (VEGF) is a homodimeric proangiogenic protein that induces endothelial cell migration and proliferation primarily through interactions with its major receptors, VEGFR-1 and VEGFR-2. Inhibitors of one or both of these VEGF-receptor interactions could be beneficial as therapeutics for diseases caused by dysfunctional angiogenesis (e.g.
View Article and Find Full Text PDFWe describe the use of parallel and split-and-mix library synthesis strategies for exploration of structure-activity relationships among peptidic foldamer ligands for the BH3-recognition cleft of the anti-apoptotic protein Bcl-xL. This effort began with a chimeric (alpha/beta+alpha)-peptide oligomer (composed of an alpha/beta-peptide segment and an alpha-peptide segment) that we previously identified to bind tightly to the target cleft on Bcl-xL. The side chains that interact with Bcl-xL were varied in a 1000-member one-bead-one-compound library.
View Article and Find Full Text PDFThe equilibrium unfolding properties of four model protein systems were characterized using SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is an H/D exchange- and mass spectrometry-based technique for measuring the free energy (DeltaGf) and m-value (deltaDeltaGf/delta[denaturant]) associated with the folding/unfolding reaction of a protein. The model proteins in this study (calmodulin, carbonic anhydrase II, RmlB, Bcl-xL) were chosen to test the applicability of SUPREX to the thermodynamic analysis of larger (> approximately 15 kDa) or multidomain proteins.
View Article and Find Full Text PDFProtein-protein interactions play crucial roles in cell-signaling events and are often implicated in human disease. Molecules that bind tightly to functional protein-surface sites and show high stability to degradative enzymes could be valuable pharmacological tools for dissection of cell-signaling networks and might ultimately lead to therapeutic agents. We recently described oligomers containing both alpha- and beta-amino acid residues that bind tightly to the BH3 recognition site of the anti-apoptotic protein Bcl-x(L).
View Article and Find Full Text PDFThe development of molecules that bind to specific protein surface sites and inhibit protein-protein interactions is a fundamental challenge in molecular recognition. New strategies for approaching this challenge could have important long-term ramifications in biology and medicine. We are exploring the concept that unnatural oligomers with well-defined conformations ("foldamers") can mimic protein secondary structural elements and thereby block specific protein-protein interactions.
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