Discovery of quinazoline HPK1 inhibitors with high cellular potency.

Hematopoietic progenitor kinase 1 (HPK1) is regarded as a highly validated target in pre-clinical immune oncology. HPK1 has been described as regulating multiple critical signaling pathway in both adaptive and innate cells. In support of this role, HPK1 KO T cells show enhanced sensitivity to TCR activation and HPK1 KO mice display enhanced anti-tumor activity. Taken together, inhibition of HPK1 has the potential to induce enhanced anti-tumor immune response. Herein, we described the discovery of highly potent HPK1 inhibitors starting form a weak HTS hit. Using a structure-based drug design, HPK1 inhibitors exhibiting excellent cellular single-digit nanomolar potency in both proximal (pSLP76) and distal (IL-2) biomarkers along with sustained elevation of IL-2 cytokine secretion were discovered.

Design, synthesis and biological evaluation of Proteolysis Targeting Chimeras (PROTACs) as a BTK degraders with improved pharmacokinetic properties.

A new series of Proteolysis Targeting Chimeras (PROTACs) targeting Bruton's Tyrosine Kinase (BTK) was synthesized, with the goal of improving the pharmacokinetic properties of our previously reported PROTAC, MT802. We recently described the ability of MT802 to induce degradation of both wild-type and C481S mutant BTK in immortalized cells and patient-derived B-lymphocytes. However, the pharmacokinetic properties of MT802 were not suitable for further in vivo development. Therefore, we undertook a systematic medicinal chemistry campaign to overcome this issue and made a series of PROTACs with structural modifications to the linker and E3-recruiting ligand; more specifically, the new PROTACs were synthesized with different von Hippel-Lindau (VHL) and cereblon (CRBN) ligands while keeping the BTK ligand and linker length constant. This approach resulted in an equally potent PROTAC, SJF620, with a significantly better pharmacokinetic profile than MT802. This compound may hold promise for further in vivo exploration of BTK degradation.

Investigation of the rhodium-catalyzed hydroboration of NHC-boranes: the role of alkene coordination and the origin of enantioselectivity.

The mechanism of the intramolecular enantioselective rhodium(i)-catalyzed hydroboration of NHC-boranes is investigated by experimental reactivity measurements and molecular electronic structure calculations, within the framework of the Density Functional Theory and the Random Phase Approximation methods. The crucial role of alkene coordination and the origin of enantioselectivity are discussed. Two possible mechanisms are considered, via either prior hydride migratory insertion or boron migratory insertion. The minimum energy computed pathway leads to the enantiomer experimentally observed, therefore supporting the hydride migratory insertion mechanism. Calculations of the final steps of the catalytic cycle, i.e. regeneration of the catalyst and release of the product, give us further insights into the mechanism and rationalize the experimental results.

Targeting the C481S Ibrutinib-Resistance Mutation in Bruton's Tyrosine Kinase Using PROTAC-Mediated Degradation.

Inhibition of Bruton's tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has emerged as a transformative treatment option for patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies, yet >80% of CLL patients develop resistance due to a cysteine to serine mutation at the site covalently bound by ibrutinib (C481S). Currently, an effective treatment option for C481S patients exhibiting relapse to ibrutinib does not exist, and these patients have poor outcomes. To address this, we have developed a PROteolysis TArgeting Chimera (PROTAC) that induces degradation of both wild-type and C481S mutant BTK. We selected a lead PROTAC, MT-802, from several candidates on the basis of its potency to induce BTK knockdown. MT-802 recruits BTK to the cereblon E3 ubiquitin ligase complex to trigger BTK ubiquitination and degradation via the proteasome. MT-802 binds fewer off-target kinases than ibrutinib does and retains an equivalent potency (>99% degradation at nanomolar concentrations) against wild-type and C481S BTK. In cells isolated from CLL patients with the C481S mutation, MT-802 is able to reduce the pool of active, phosphorylated BTK whereas ibrutinib cannot. Collectively, these data provide a basis for further preclinical study of BTK PROTACs as a novel strategy for treatment of C481S mutant CLL.

Targeted protein unfolding uncovers a Golgi-specific transcriptional stress response.

In eukaryotic cells, organelle-specific stress-response mechanisms are vital for maintaining cellular homeostasis. The Golgi apparatus, an essential organelle of the secretory system, is the major site of protein modification and sorting within a cell and functions as a platform for spatially regulated signaling. Golgi homeostasis mechanisms that regulate organelle structure and ensure precise processing and localization of protein substrates remain poorly understood. Using a chemical biology strategy to induce protein unfolding, we uncover a Golgi-specific transcriptional response. An RNA-sequencing profile of this stress response compared with the current state-of-the-art Golgi stressors, nigericin and xyloside, demonstrates the enhanced precision of Golgi targeting achieved with our system. The data set further reveals previously uncharacterized genes that we find to be essential for Golgi structural integrity. These findings highlight the Golgi's ability to sense misfolded proteins and establish new aspects of Golgi autoregulation.

Efficient Synthesis of Immunomodulatory Drug Analogues Enables Exploration of Structure-Degradation Relationships.

The immunomodulatory drugs (IMiDs) thalidomide, pomalidomide, and lenalidomide have been approved for the treatment of multiple myeloma for many years. Recently, their use as E3 ligase recruiting elements for small-molecule-induced protein degradation has led to a resurgence in interest in IMiD synthesis and functionalization. Traditional IMiD synthesis follows a stepwise route with multiple purification steps. Herein we describe a novel one-pot synthesis without purification that provides rapid access to a multitude of IMiD analogues. Binding studies with the IMiD target protein cereblon (CRBN) reveals a narrow structure-activity relationship with only a few compounds showing sub-micromolar binding affinity in the range of pomalidomide and lenalidomide. However, anti-proliferative activity as well as Aiolos degradation could be identified for two IMiD analogues. This study provides useful insight into the structure-degradation relationships for molecules of this type as well as a rapid and robust method for IMiD synthesis.

The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study.

Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach.

Assessing Different E3 Ligases for Small Molecule Induced Protein Ubiquitination and Degradation.

Proteolysis targeting chimera (PROTAC) technology, the recruitment of E3 ubiquitin ligases to induce the degradation of a protein target, is rapidly impacting chemical biology, as well as modern drug development. Here, we explore the universality of this approach by evaluating different E3 ubiquitin ligases, engineered in their substrate binding domains to accept a recruiting ligand. Five out of six E3 ligases were found to be amenable to recruitment for target degradation. Taking advantage of the tight spatiotemporal control of inducing ubiquitination on a preselected target in living cells, we focused on two of the engineered E3 ligases, ßTRCP and parkin, to unravel their ubiquitination characteristics in comparison with the PROTAC-recruited endogenous E3 ligases VHL and cereblon.

Straightforward and Controlled Shape Access to Efficient Macrocyclic Imidazolylboronium Anion Receptors.

A straightforward synthesis of air- and water-stable bis-cationic macrocyclic imidazolylboronium anion receptors is described herein. By taking advantage of the bulky and rigid 9-borabicyclo[3.3.1]-nonane (9-BBN) attaching point and a well-designed bis-imidazolylaryl, highly stable dimeric imidazolylboronium macrocycles were synthesized. Additionally, NMR spectroscopy ((1) H, DOSY, and HOESY), mass spectrometry (MS), and X-ray diffraction studies revealed that these macrocyclic scaffolds can bind several monoanions with high association constants in DMSO, and are particularly sensitive for the MS detection of anions (with concentrations in the nm range). This anion/receptor interaction involves eight C-H binding sites, which include Csp2 -H and unusual Csp3 -H hydrogen-bonding donors.

Small-Molecule PROTACS: New Approaches to Protein Degradation.

The current inhibitor-based approach to therapeutics has inherent limitations owing to its occupancy-based model: 1) there is a need to maintain high systemic exposure to ensure sufficient in vivo inhibition, 2) high in vivo concentrations bring potential for off-target side effects, and 3) there is a need to bind to an active site, thus limiting the drug target space. As an alternative, induced protein degradation lacks these limitations. Based on an event-driven model, this approach offers a novel catalytic mechanism to irreversibly inhibit protein function by targeting protein destruction through recruitment to the cellular quality control machinery. Prior protein degrading strategies have lacked therapeutic potential. However, recent reports of small-molecule-based proteolysis-targeting chimeras (PROTACs) have demonstrated that this technology can effectively decrease the cellular levels of several protein classes.

Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL.

Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome. The synthesis of PROTAC compounds that mediate the degradation of c-ABL and BCR-ABL by recruiting either Cereblon or Von Hippel Lindau E3 ligases is reported. During the course of their development, we discovered that the capacity of a PROTAC to induce degradation involves more than just target binding: the identity of the inhibitor warhead and the recruited E3 ligase largely determine the degradation profiles of the compounds; thus, as a starting point for PROTAC development, both the target ligand and the recruited E3 ligase should be varied to rapidly generate a PROTAC with the desired degradation profile.

Expeditious Synthesis of Isoquinolones and Isocoumarins with a Vinyl Borane as an Acetylene Equivalent.

An innovative and simple expeditious synthesis of 3,4-unsubstituted isoquinolones and isocoumarins starting from safe and easy to handle two-carbon acetylene equivalent was developed. The synthetic potential of this new method was further demonstrated in the facile total synthesis of two naturally occurring alkaloids: corydaldine and doryanine.

Synthesis and structure of Ag(I), Pd(II), Rh(I), Ru(II) and Au(I) NHC-complexes with a pendant Lewis acidic boronic ester moiety.

Bifunctional Ag(I), Pd(II), Rh(I), Ru(II) and Au(I) complexes containing a NHC ligand and a pendant trivalent boron moiety have been synthesized in high yields. Fine-tuned reaction conditions were used to prevent potential ligand self-quenching or polymerization due to the eventual co-existence in situ of free NHC (Lewis base) and boronic ester (Lewis acid) in the same molecule.

Asymmetric rhodium-directed anti-Markovnikov regioselective boracyclopentannulation.

A Shimoi-type activation of B-H bond of NHC-boranes by a diphosphane-ligated cationic Rh complex was applied in an unprecedented intramolecular hydroboration reaction of simple olefins. The use of NHC-boranes as hydroborating reagents is still undisclosed due to their nonreactivity toward alkenes which could be explained by the high stability of this complex rendering it unable to provide a "free" borane hydroborating reagent. B-H bond Rh activation of NHC-borane circumvents this limitation, and asymmetric Rh-directed anti-Markovnikov boracyclopentannulation reaction led to a library of enantioenriched cyclic boranes in high yield (up to 94%) with high regio- (up to 100%) and enantioselectivity (er up to 99.2:0.8). This new activation mode of NHC-boranes highlights their use in organometallic chemistry and offers a very good approach to access chiral cyclic NHC-boranes.

Chemical functionalization of electrodes for detection of gaseous nerve agents with carbon nanotube field-effect transistors.

An innovative sensor for the detection of nerve agents in the gas phase based on a carbon nanotube field-effect transistor was developed. A high sensitivity to organophosphorus gases was obtained by modifying gold electrodes with specific tailor-made self-assembled monolayers.