In vivo bioluminescence imaging of labile iron in xenograft models and liver using FeAL-1, an iron-activatable form of D-luciferin.

Dysregulated iron homeostasis underlies diverse pathologies, from ischemia-reperfusion injury to epithelial-mesenchymal transition and drug-tolerant "persister" cancer cell states. Here, we introduce ferrous iron-activatable luciferin-1 (FeAL-1), a small-molecule probe for bioluminescent imaging of the labile iron pool (LIP) in luciferase-expressing cells and animals. We find that FeAL-1 detects LIP fluctuations in cells after iron supplementation, depletion, or treatment with hepcidin, the master regulator of systemic iron in mammalian physiology. Utilizing FeAL-1 and a dual-luciferase reporter system, we quantify LIP in mouse liver and three different orthotopic pancreatic ductal adenocarcinoma tumors. We observed up to a 10-fold increase in FeAL-1 bioluminescent signal in xenograft tumors as compared to healthy liver, the major organ of iron storage in mammals. Treating mice with hepcidin further elevated hepatic LIP, as predicted. These studies reveal a therapeutic index between tumoral and hepatic LIP and suggest an approach to sensitize tumors toward LIP-activated therapeutics.

Modification of Cysteine-Substituted Antibodies Using Enzymatic Oxidative Coupling Reactions.

Cysteines are routinely used as site-specific handles to synthesize antibody-drug conjugates for targeted immunotherapy applications. Michael additions between thiols and maleimides are some of the most common methods for modifying cysteines, but these functional groups can be difficult to prepare on scale, and the resulting linkages have been shown to be reversible under some physiological conditions. Here, we show that the enzyme tyrosinase, which oxidizes conveniently accessed phenols to afford reactive ortho-quinone intermediates, can be used to attach phenolic cargo to cysteines engineered on antibody surfaces. The resulting linkages between the thiols and ortho-quinones are shown to be more resistant than maleimides to reversion under physiological conditions. Using this approach, we construct antibody conjugates bearing cytotoxic payloads, which exhibit targeted cell killing, and further demonstrate this method for the attachment of a variety of cargo to antibodies, including fluorophores and oligonucleotides.

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-XL Inhibitor.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.

Cyclodextrin Reduces Intravenous Toxicity of a Model Compound.

Solubilization of new chemical entities for toxicity assessment must use excipients that do not negatively impact drug pharmacokinetics and toxicology. In this study, we investigated the tolerability of a model freebase compound, GDC-0152, solubilized by pH adjustment with succinic acid and complexation with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) to enable intravenous use. Solubility, critical micelle concentration, and association constant with HP-ß-CD were determined. Blood compatibility and potential for hemolysis were assessed in vitro. Local tolerability was assessed after intravenous and subcutaneous injections in rats. A pharmacokinetic study was conducted in rats after intravenous bolus administration. GDC-0152 exhibited pH-dependent solubility that was influenced by self-association. The presence of succinic acid increased solubility in a concentration-dependent manner. HP-ß-CD alone also increased solubility, but the extent of solubility enhancement was significantly lower than succinic acid alone. Inclusion of HP-ß-CD in the solution of GDC-0152 improved blood compatibility, reduced hemolytic potential by ∼20-fold in vitro, and increased the maximum tolerated dose to 80 mg/kg.

An Anti-CLL-1 Antibody-Drug Conjugate for the Treatment of Acute Myeloid Leukemia.

PURPOSE: The treatment of acute myeloid leukemia (AML) has not significantly changed in 40 years. Cytarabine- and anthracycline-based chemotherapy induction regimens (7 + 3) remain the standard of care, and most patients have poor long-term survival. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has demonstrated ADCs as a clinically validated option to enhance the effectiveness of induction therapy. We are interested in developing a next-generation ADC for AML to improve upon the initial success of Mylotarg. EXPERIMENTAL DESIGN: The expression pattern of CLL-1 and its hematopoietic potential were investigated. A novel anti-CLL-1-ADC, with a highly potent pyrrolobenzodiazepine (PBD) dimer conjugated through a self-immolative disulfide linker, was developed. The efficacy and safety profiles of this ADC were evaluated in mouse xenograft models and in cynomolgus monkeys. RESULTS: We demonstrate that CLL-1 shares similar prevalence and trafficking properties that make CD33 an excellent ADC target for AML, but lacks expression on hematopoietic stem cells that hampers current CD33-targeted ADCs. Our anti-CLL-1-ADC is highly effective at depleting tumor cells in AML xenograft models and lacks target independent toxicities at doses that depleted target monocytes and neutrophils in cynomolgus monkeys. CONCLUSIONS: Collectively, our data suggest that an anti-CLL-1-ADC has the potential to become an effective and safer treatment for AML in humans, by reducing and allowing for faster recovery from initial cytopenias than the current generation of ADCs for AML.

Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes.

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

Discovery of Peptidomimetic Antibody-Drug Conjugate Linkers with Enhanced Protease Specificity.

Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.

Pyrrolobenzodiazepine Dimer Antibody-Drug Conjugates: Synthesis and Evaluation of Noncleavable Drug-Linkers.

Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 µg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.

Decoupling stability and release in disulfide bonds with antibody-small molecule conjugates.

Disulfide 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. We examined this by attaching a small molecule directly to engineered cysteines in an antibody. At certain sites this unhindered disulfide was stable in circulation yet cellular internalization and antibody catabolism generated a disulfide catabolite that was rapidly reduced. We demonstrated that this stable connection and facile release is applicable to a variety of payloads. The ability to reversibly stabilize a labile functional group with an antibody may offer a way to improve targeted probes and therapeutics.

Modulating Therapeutic Activity and Toxicity of Pyrrolobenzodiazepine Antibody-Drug Conjugates with Self-Immolative Disulfide Linkers.

A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody-drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871-8. ©2017 AACR.

Activity-based probes for the ubiquitin conjugation-deconjugation machinery: new chemistries, new tools, and new insights.

The reversible post-translational modification of proteins by ubiquitin and ubiquitin-like proteins regulates almost all cellular processes, by affecting protein degradation, localization, and complex formation. Deubiquitinases (DUBs) are proteases that remove ubiquitin modifications or cleave ubiquitin chains. Most DUBs are cysteine proteases, which makes them well suited for study by activity-based probes. These DUB probes report on deubiquitinase activity by reacting covalently with the active site in an enzyme-catalyzed manner. They have proven to be important tools to study DUB selectivity and proteolytic activity in different settings, to identify novel DUBs, and to characterize deubiquitinase inhibitors. Inspired by the efficacy of activity-based probes for DUBs, several groups have recently reported probes for the ubiquitin conjugation machinery (E1, E2, and E3 enzymes). Many of these enzymes, while not proteases, also posses active site cysteine residues and can be targeted by covalent probes. In this review, we will discuss how features of the probe (cysteine-reactive group, recognition element, and reporter tag) affect reactivity and suitability for certain experimental applications. We will also review the diverse applications of the current probes, and discuss the need for new probe types to study emerging aspects of ubiquitin biology.

Activity-based probes for the multicatalytic proteasome.

Proteasomes are multisubunit protease complexes responsible for degrading most intracellular proteins. In addition to removing damaged proteins, they regulate many important cellular processes through the controlled degradation of transcription factors, cell cycle regulators, and enzymes. Eukaryotic proteasomes have three catalytic subunits, ß1, ß2, and ß5, that each has different substrate specificities. Additionally, although we know that diverse cell types express proteasome variants with distinct activity and specificity profiles, the functions of these different pools of proteasomes are not fully understood. Covalent inhibitors of the protease activity of the proteasome have been developed as drugs for hematological malignancies and are currently under investigation for other diseases. Therefore, there is a need for tools that allow direct monitoring of proteasome activity in live cells and tissues. Activity-based probes have proven valuable for biochemical and cell biological studies of the role of individual proteasome subunits, and for evaluating the efficacy and selectivity of proteasome inhibitors. These probes react covalently with the protease active sites, and contain a reporter tag to identify the probe-labeled proteasome subunits. This review will describe the development of broad-spectrum and subunit-specific proteasome activity-based probes, and discuss how these probes have contributed to our understanding of proteasome biology, and to the development of proteasome inhibitors.

Targeted drug delivery through the traceless release of tertiary and heteroaryl amines from antibody-drug conjugates.

The reversible attachment of a small-molecule drug to a carrier for targeted delivery can improve pharmacokinetics and the therapeutic index. Previous studies have reported the delivery of molecules that contain primary and secondary amines via an amide or carbamate bond; however, the ability to employ tertiary-amine-containing bioactive molecules has been elusive. Here we describe a bioreversible linkage based on a quaternary ammonium that can be used to connect a broad array of tertiary and heteroaryl amines to a carrier protein. Using a concise, protecting-group-free synthesis we demonstrate the chemoselective modification of 12 complex molecules that contain a range of reactive functional groups. We also show the utility of this connection with both protease-cleavable and reductively cleavable antibody-drug conjugates that were effective and stable in vitro and in vivo. Studies with a tertiary-amine-containing antibiotic show that the resulting antibody-antibiotic conjugate provided appropriate stability and release characteristics and led to an unexpected improvement in activity over the conjugates previously connected via a carbamate.

Targeting LGR5+ cells with an antibody-drug conjugate for the treatment of colon cancer.

Cancer stem cells (CSCs) are hypothesized to actively maintain tumors similarly to how their normal counterparts replenish differentiated cell types within tissues, making them an attractive therapeutic target for the treatment of cancer. Because most CSC markers also label normal tissue stem cells, it is unclear how to selectively target them without compromising normal tissue homeostasis. We evaluated a strategy that targets the cell surface leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a well-characterized tissue stem cell and CSC marker, with an antibody conjugated to distinct cytotoxic drugs. One antibody-drug conjugate (ADC) demonstrated potent tumor efficacy and safety in vivo. Furthermore, the ADC decreased tumor size and proliferation, translating to improved survival in a genetically engineered model of intestinal tumorigenesis. These data demonstrate that ADCs can be leveraged to exploit differences between normal and cancer stem cells to successfully target gastrointestinal cancers.

Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

A Novel Anti-CD22 Anthracycline-Based Antibody-Drug Conjugate (ADC) That Overcomes Resistance to Auristatin-Based ADCs.

PURPOSE: We are interested in identifying mechanisms of resistance to the current generation of antibody-drug conjugates (ADC) and developing ADCs that can overcome this resistance. EXPERIMENTAL DESIGN: Pinatuzumab vedotin (anti-CD22-vc-MMAE) and polatuzumab vedotin (anti-CD79b-vc-MMAE) are ADCs that contain the microtubule inhibitor monomethyl auristatin E (MMAE) attached to the antibody by the protease-cleavable linker maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). Early clinical trial data suggest that these ADCs have promising efficacy for the treatment of non-Hodgkin lymphoma (NHL); however, some patients do not respond or become resistant to the ADCs. Anthracyclines are very effective in NHL, but ADCs containing the anthracycline doxorubicin were not clinically efficacious probably due to the low drug potency and inadequate linker technology. The anthracycline analogue PNU-159682 is thousands of times more cytotoxic than doxorubicin, so we used it to develop a new class of ADCs. We used the same MC-vc-PAB linker and antibody in pinatuzumab vedotin but replaced the MMAE with a derivative of PNU-159682 to make anti-CD22-NMS249 and tested it for in vivo efficacy in xenograft tumors resistant to MMAE-based ADCs. RESULTS: We derived cell lines from in vivo xenograft tumors that were made resistant to anti-CD22-vc-MMAE and anti-CD79b-vc-MMAE. We identified P-gp (ABCB1/MDR1) as the major driver of resistance to the vc-MMAE-based conjugates. Anti-CD22-NMS249 was at least as effective as anti-CD22-vc-MMAE in xenograft models of the parental cell lines and maintained its efficacy in the resistant cell lines. CONCLUSIONS: These studies provide proof of concept for an anthracycline-based ADC that could be used to treat B-cell malignancies that are resistant to vc-MMAE conjugates.

Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity.

A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.

Site-specific trastuzumab maytansinoid antibody-drug conjugates with improved therapeutic activity through linker and antibody engineering.

Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.

Structure-Guided Rescaffolding of Selective Antagonists of BCL-XL.

Because of the promise of BCL-2 antagonists in combating chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), interest in additional selective antagonists of antiapoptotic proteins has grown. Beginning with a series of selective, potent BCL-XL antagonists containing an undesirable hydrazone functionality, in silico design and X-ray crystallography were utilized to develop alternative scaffolds that retained the selectivity and potency of the starting compounds.

Tailoring small molecules for an allosteric site on procaspase-6.

Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules.