Biology subjects

Spradlin, J. N.

Publications and source records attributed to Spradlin, J. N..

6 recordsLinked to original sources

bioRxiv2022

Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly "druggable" or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.

biochemistry↗

bioRxiv2021

Deubiquitinase-Targeting Chimeras for Targeted Protein Stabilization

Targeted protein degradation is a powerful therapeutic modality that uses heterobifunctional small-molecules to induce proximity between E3 ubiquitin ligases and target proteins to ubiquitinate and degrade specific proteins of interest. However, many proteins are ubiquitinated and degraded to drive disease pathology; in these cases targeted protein stabilization (TPS), rather than degradation, of the actively degraded target using a small-molecule would be therapeutically beneficial. Here, we present the Deubiquitinase-Targeting Chimera (DUBTAC) platform for TPS of specific proteins. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48 ubiquitin-specific deubiquitinase OTUB1. We then developed a heterobifunctional DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds {Delta}F508-CFTR. We demonstrated proof-of-concept of TPS by showing that this DUBTAC robustly stabilized {Delta}F508-CFTR in human cystic fibrosis bronchial epithelial cells in an OTUB1-dependent manner. Our study underscores the utility of chemoproteomics-enabled covalent ligand discovery approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS. Editorial summaryWe have developed the Deubiquitinase Targeting Chimera (DUBTAC) platform for targeted protein stabilization. We have discovered a covalent recruiter against the deubiquitinase OTUB1 that we have linked to the mutant {Delta}F508-CFTR targeting cystic fibrosis drug Lumacaftor to stabilize mutant CFTR protein in cells.

biochemistry↗

bioRxiv2021

Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications

Proteolysis Targeting Chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number of E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand, EN106, that targets FEM1B, an E3 ligase recently discovered as the critical component of the cellular response to reductive stress. By targeting Cys186 in FEM1B, EN106 disrupts recognition of the key reductive stress substrate of FEM1B, FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications, in which we demonstrate that a PROTAC linking EN106 to the BET Bromodomain inhibitor JQ1 leads to specific FEM1B- and proteasome-dependent degradation of BRD4 in cells. Our study showcases a covalent ligand that targets a natural E3 ligase-substrate binding site and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters that can be deployed for TPD applications.

biochemistry↗

bioRxiv2020

Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 80 million cases and 1.7 million deaths to date while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

microbiology↗

bioRxiv2020

A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific protein targets in a proteasome-dependent manner. However, a major limitation to broader TPD applications is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent ligand for the E3 ligase RNF114. When linked to the BET family inhibitor JQ1, the resulting heterobifunctional PROTAC molecule was capable of selectively degrading BRD4 in cancer cells. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or in some cases inactivity. Further contrasting from cereblon or VHL-recruiting degradation, we show that BT1 treatment not only leads to BCR-ABL degradation, but also stabilizes the endogenous RNF114 substrate and tumor suppressor substrate p21. This leads to additional anti-proliferative effects in leukemia cancer cells beyond those observed with cereblon or VHL-recruiting BCR-ABL PROTACs. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs with unique additional benefits for oncology applications. We also further demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing and unpredictable selectivity for the degradation of neo-substrate proteins.

biochemistry↗

bioRxiv2018

Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity across many types of human cancers; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation by disrupting RNF114 substrate recognition, leading to inhibition of ubiquitination and degradation of the tumor-suppressor p21, resulting in its rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the utility of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and drug discovery applications.

cancer biology↗