Research Plans
My lab is driven by leveraging organic chemistry to solve critical challenges and unmet needs at the intersection of chemistry and biology. Driven by fundamental interests in chemical reactivity and molecular recognition with directed applications to cancer/antiviral therapy, my lab’s well-funded and established research program, distinguished by a robust publication record, continues to pursue transformative advancements in 4 distinct areas:
- Radiotheranostic development fueled by our ground-breaking one-step aqueous radiofluorination method;
- Advancing molecular pharmacology of amatoxins to create innovative antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs), and novel amanitin-based payloads for addressing cancer-cell phenotypes;
- Synthesizing functionalized nucleosides for enhancing the activity of aptamers and DNAzymes for targeting anti-gene therapy, molecular imaging, and targeted therapy;
- Engineering peptide macrocycles using novel stapling techniques for lead peptide therapeutic discovery.
Below are detailed descriptions of these ambitious, impact-driven projects.
- Radiotheranostics based on Breakthrough 18F-Labeling:
Background: Positron emission tomography (PET) imaging now provides a means of real-time, non-invasive in-vivo “staining” to quantify pathognomonic targets to inform patient management at all stages of care. More recently, radiotheranostics enable non-invasive imaging of molecular targets with an eye to precision cancer treatments; indeed, the FDA approval of the diagnostic pair 18F-Pylarify/177Lu-Pluvicto highlights the importance of imaging in order to “treat what we can see”. Key to these applications is the use of similar if not identical molecules for both imaging and therapy.[1] Such molecules, or pairs of molecules, are known as theranostics. The growing field of theranostic development depends on several components: a ligand e.g. peptide, nanobody, aptamer, a chelator for radiotherapeutic metal chelation, and a means of imaging, preferably by positron emission tomography (PET), and arguably with 18F-fluoride, the only scalable PET isotope. Of the various PET-isotopes in use, 18F-fluoride is preferred for its high resolution, low toxicity, record of FDA approval, and on-demand cyclotron production in multi-Curie quantities making it the only scalable isotope for PET.[2] Notwithstanding advances in multi-step radiofluorination, the well-known chemical challenges associated with anionic 18F-fluoride along with its short half-life (109 min) have continued to impede the production of 18F-labeled tracers. And thus, for the past 40 years, the challenge of a late-stage, one-step, aqueous radiofluorination reaction for labeling large molecules remained elusive.
In 2005, my lab changed this by thinking “out of the box”. Radically departing from standard methods, we invented a breakthrough technology based on organoboron that enables single-step, aqueous radio-fluorination via an appended organotrifluoroborate (18F-RBF3) prosthetic group.[3] With peptides being a choice scaffold for developing radiotracers, this scientific breakthrough finally enabled the long-sought goal of a one-step, late-stage aqueous radio-fluorination that enables the conversion of essentially any peptide into a PET imaging agent.[4] We created a series of proprietary borate-based radiosynthons that can be click-conjugated to any peptide.[5] In evidence of the user-friendliness of this approach, my lab, along with collaborators at BC Cancer, created over a dozen radiotracers, including 18F-AMB3-octreotate for imaging neuroendocrine cancers, which showed high-contrast images of liver metastases suitable for human translation (Figure 1).[6] This molecule, having undergone first-in-human studies in collaboration at BC Cancer, is now licensed to Alpha9 Oncology, Inc., a spinout that I cofounded in 2019.
- Amatoxin Payload Development and Pharmacology
Background: The potent toxin, α-amanitin enjoys a rich history scientific history.[8] As the principle toxin in the mushroom A. phalloides, it is one of Nature’s most potent toxins. As an allosteric inhibitor of RNA Pol II, it was used as one of the first ligands for affinity chromatography to purify RNA Pol II,[9] and was featured in Kornberg’s crystal structure of RNA Pol II for which the 2006 Nobel prize was awarded.[10] It has now emerged as a promising payload for antibody drug conjugates.[11] Yet, for over 70 years there had been no total synthesis of this toxin owing to inherent synthetic challenges in: i) selective oxidation of a key 6-hydroxytryptathionine sulfoxide crosslink and ii) efficient access of dihydroxyisoleucine.
In 2018, my lab reported the first total synthesis of this notorious toxin;[12] to achieve this we tackled the challenge of the first enantioselective synthesis of the deceptively simple dihydroxyisoleucine as well as elaboration of the 6-hydroxy-tryptathionine sulfoxide, which required a sequential three-fold oxidation of tryptophan (Figure 2). With access to this toxin, we have generated a number of “clickable” toxins for bioconjugation to generate a platform payload for generating targeted chemotherapeutics.[13] In validation of this enabling methodology, we have validated clickable toxins in the context of a peptide drug conjugate. Excitingly, we exploited our synthetic know-how to prepare proprietary super-toxins that show enhanced cytotoxicity against certain cell lines.[14] We are now pursuing these toxins as new payloads in the design of ADCs.
- Functionalized Nucleic Acids for Catalysis and Protein Recognition
Background: Nucleic acids offer significant opportunities in biotechnology, most notably as nucleic acid catalysts and as aptamers that are discovered by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), a combinatorial technique used to identify active sequences that specifically bind target proteins, or catalyze mRNA cleavage. SELEX begins with a large, random pool of ~1015th sequences of DNA or RNA; over an iterative process, sequences that show exalted affinity and specificity are amplified, sequenced, and engineered for diagnostic, therapeutic, and biosensing applications. Aptamers provide notable advantages over antibodies: high thermal stability, ready bioconjugation to toxins and radioisotopes, non-immunogenicity, biocompatibility, and synthetic scalability.[16] In the past 3 decades, hundreds of reports have featured aptamers as in-vitro reagents for multiplexed detection of cell-surface proteins[17] and as preclinical in-vivo imaging agents with high potential in oncology for imaging and therapy, as reviewed.[18]
However, the limited chemical diversity of aptamers diminishes their activity compared to antibodies; to wit, nucleic acids critically lack cationic amines (e.g. Lys, Arg) for full charge complementarity, a hydrophobic group (Trp, Phe), a multivalent group (e.g. Tyr) and a group for acid-base catalysis (e.g. His) for accessing the full panoply of non-covalent interactions. These added functionalities can be introduced using chemically modified bases for use in SELEX to enhance binding affinity, stability, or catalytic activity of selected nucleic acids. Contemporaneously with the Joyce, Benner, and Gold labs,[19] I and my lab pioneered the expansion of chemical functionality for selected DNAzymes and aptamers (Figure 3, next page).[20] Noting the unrealized potential of ribozymes (and DNAzymes) to catalytically destroy pathogenic mRNAs, their clinical advancement has been impeded by their reliance on Mg2+, which is limited in cells. To address this, we pioneered the selection of M2+-independent DNAzymes by incorporating three different nucleosides e.g. dA, dC, dT, each modified with imidazoles, amines and guanidinium cations. Next, we appreciated that tyrosines are overrepresented in the antigen-combining domains of antibodies. Hence, we introduced a phenol-modified dUTP to select modified aptamers against gram negative bacteria.[21] Excitingly, we have developed a “baited-SELEX” approach to select modified aptamers appended with a small-molecule ligand directed against the prostate specific membrane antigen (PSMA) and have pursued both whole-cell and whole-animal selections to discover high-affinity aptamers that selectively bind PSMA in-vivo.
- Peptide Stapling for New Macrocycle Discovery
Background: Peptides are valuable drug leads for recognizing receptors implicated in cancer, inflammation, and immune checkpoint therapy, and for disrupting protein-protein interactions (PPIs). While linear peptides recognize undruggable targets, their lack of defined structure, particularly in short peptides, accounts for low affinity and poor humoral stability. One common chemical approach to address these deficiencies is peptide stapling, which pre-organizes peptide secondary structure, thereby enhancing affinity, rigidity, stability, and even membrane permeability. With over 150 peptides in clinical trials as of 2023, new stapling methods represent platform technologies that can improve peptide drugability and pharmacological performance. Numerous reports have disclosed new stapling methods, as reviewed.[23] As stapling typically occurs late stage, exceptional chemoselectivity is required and may be achieved with non-canonical amino acids. Of further interest are fluorogenic stapling reactions, where emergent fluorescence signals reaction progress and generates a fluorescent peptide that allows for direct visualization of its association with biological targets thereby obviating the need to append a fluorophore that may alter affinity.[24] Appreciating the novel bicyclic stapled architecture of amanitin, we expanded our interest in seeking new, high-affinity peptides with tryptathionine staples compatible with a prototypical OBOC library format for on- and off-bead screening,[25] discovering and controlling the synthesis of 2,2’-bis-indole staples,[26] and exploiting the synthesis of highly fluorescent isoindole staples [24b] in the context of a number of high-affinity octreotate and melanocortins with Kd’s in the nanomolar range or lower (Figure 4).
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