Development of a tetrahydroindazolone-based HDAC6 inhibitor with in-vivo anti-arthritic activity.

Histone deacetylase 6 (HDAC6) induces the expression of pro-inflammatory cytokines in macrophages; therefore, HDAC inhibitors may be beneficial for the treatment of macrophage-associated immune disorders and chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis. Structure-activity relationship studies were conducted on various phenyl hydroxamate HDAC6 inhibitors with indolone/indazolone-based bi- or tricyclic ring moieties as the cap group aiming to develop novel anti-arthritic drug candidates. Several compounds exhibited nanomolar activity and HDAC6 selectivity greater than 500-fold over HDAC1.

Protein-protein interfaces in molecular glue-induced ternary complexes: classification, characterization, and prediction.

Molecular glues are a class of small molecules that stabilize the interactions between proteins. Naturally occurring molecular glues are present in many areas of biology where they serve as central regulators of signaling pathways. Importantly, several clinical compounds act as molecular glue degraders that stabilize interactions between E3 ubiquitin ligases and target proteins, leading to their degradation.

Combination of Ribociclib with BET-Bromodomain and PI3K/mTOR Inhibitors for Medulloblastoma Treatment In Vitro and In Vivo.

Despite improvement in the treatment of medulloblastoma over the last years, numerous patients with MYC- and MYCN-driven tumors still fail current therapies. Medulloblastomas have an intact retinoblastoma protein RB, suggesting that CDK4/6 inhibition might represent a therapeutic strategy for which drug combination remains understudied. We conducted high-throughput drug combination screens in a Group3 (G3) medulloblastoma line using the CDK4/6 inhibitor (CDK4/6i) ribociclib at IC20, referred to as an anchor, and 87 oncology drugs approved by FDA or in clinical trials.

Ca-mediated mitochondrial inner membrane permeabilization induces cell death independently of Bax and Bak.

The ability of mitochondria to buffer a rapid rise in cytosolic Ca is a hallmark of proper cell homeostasis. Here, we employed m-3M3FBS, a putative phospholipase C (PLC) agonist, to explore the relationships between intracellular Ca imbalance, mitochondrial physiology, and cell death. m-3M3FBS induced a potent dose-dependent Ca release from the endoplasmic reticulum (ER), followed by a rise in intra-mitochondrial Ca.

Target and tissue selectivity of PROTAC degraders.

Targeted protein degradation (TPD) strategies have revolutionized how scientists tackle challenging protein targets deemed undruggable with traditional small molecule inhibitors. Many promising campaigns to inhibit proteins have failed due to factors surrounding inhibition selectivity and targeting of compounds to specific tissues and cell types. One of the major improvements that PROTAC (proteolysis targeting chimera) and molecular glue technology can exert is highly selective control of target inhibition.

Development of Potent and Selective Janus Kinase 2/3 Directing PG-PROTACs.

Aberrant activation of the JAK-STAT signaling pathway has been implicated in the pathogenesis of a range of hematological malignancies and autoimmune disorders. Here we describe the design, synthesis, and characterization of JAK2/3 PROTACs utilizing a phenyl glutarimide (PG) ligand as the cereblon (CRBN) recruiter. SJ10542 displayed high selectivity over GSPT1 and other members of the JAK family and potency in patient-derived ALL cells containing both JAK2 fusions and CRLF2 rearrangements.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy.

AZD5438-PROTAC: A selective CDK2 degrader that protects against cisplatin- and noise-induced hearing loss.

Cyclin-dependent kinase 2 (CDK2) is a potential therapeutic target for the treatment of hearing loss and cancer. Previously, we identified AZD5438 and AT7519-7 as potent inhibitors of CDK2, however, they also targeted additional kinases, leading to unwanted toxicities. Proteolysis Targeting Chimeras (PROTACs) are a new promising class of small molecules that can effectively direct specific proteins to proteasomal degradation.

Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia.

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs.

Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators.

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice.

BRAF inhibition protects against hearing loss in mice.

Hearing loss caused by noise, aging, antibiotics, and chemotherapy affects 10% of the world population, yet there are no Food and Drug Administration (FDA)-approved drugs to prevent it. Here, we screened 162 small-molecule kinase-specific inhibitors for reduction of cisplatin toxicity in an inner ear cell line and identified dabrafenib (TAFINLAR), a BRAF kinase inhibitor FDA-approved for cancer treatment. Dabrafenib and six additional kinase inhibitors in the BRAF/MEK/ERK cellular pathway mitigated cisplatin-induced hair cell death in the cell line and mouse cochlear explants.

Development of BODIPY FL Thalidomide As a High-Affinity Fluorescent Probe for Cereblon in a Time-Resolved Fluorescence Resonance Energy Transfer Assay.

Ligands for cereblon, a component of a functional E3 ligase complex that targets proteins for proteolysis, are critical for developing molecular glues and proteolysis-targeting chimeras (PROTACs), which have therapeutic implications for various diseases. However, the lack of sensitivity of previously reported assays limits characterization of cereblon ligands. To address this shortcoming, we developed BODIPY FL thalidomide () as a high-affinity fluorescent probe for the human cereblon protein, with a value of 3.

Structural basis for substrate recognition and chemical inhibition of oncogenic MAGE ubiquitin ligases.

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics.

Discovery of Novel Pyrazolo-pyridone DCN1 Inhibitors Controlling Cullin Neddylation.

Chemical control of cullin neddylation is attracting increased attention based largely on the successes of the NEDD8-activating enzyme (E1) inhibitor pevonedistat. Recently reported chemical probes enable selective and time-dependent inhibition of downstream members of the neddylation trienzymatic cascade including the co-E3, DCN1. In this work, we report the optimization of a novel class of small molecule inhibitors of the DCN1-UBE2M interaction.

Mechanistic Insight on the Mode of Action of Colletoic Acid.

The natural product colletoic acid (CA) is a selective inhibitor of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which primarily converts cortisone to the active glucocorticoid (GC) cortisol. Here, CA's mode of action and its potential as a chemical tool to study intracellular GC signaling in adipogenesis are disclosed. 11β-HSD1 biochemical studies of CA indicated that its functional groups at C-1, C-4, and C-9 were important for enzymatic activity; an X-ray crystal structure of 11β-HSD1 bound to CA at 2.

Identification of Toll-like receptor signaling inhibitors based on selective activation of hierarchically acting signaling proteins.

Toll-like receptors (TLRs) recognize various pathogen- and host tissue-derived molecules and initiate inflammatory immune responses. Exaggerated or prolonged TLR activation, however, can lead to etiologically diverse diseases, such as bacterial sepsis, metabolic and autoimmune diseases, or stroke. Despite the apparent medical need, no small-molecule drugs against TLR pathways are clinically available.

Development of Second-Generation CDK2 Inhibitors for the Prevention of Cisplatin-Induced Hearing Loss.

There are currently no FDA-approved therapies to prevent the hearing loss associated with the usage of cisplatin in chemotherapeutic regimens. We recently demonstrated that the pharmacologic inhibition with kenpaullone or genetic deletion of CDK2 preserved hearing function in animal models treated with cisplatin, which suggests that CDK2 is a promising therapeutic target to prevent cisplatin-induced ototoxicity. In this study, we identified two lead compounds, AT7519 and AZD5438, from a focused library screen of 187 CDK2 inhibitors, performed in an immortalized cell line derived from neonatal mouse cochleae treated with cisplatin.

Piperidinyl Ureas Chemically Control Defective in Cullin Neddylation 1 (DCN1)-Mediated Cullin Neddylation.

We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure-activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1-UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit.

Discovery of an Orally Bioavailable Inhibitor of Defective in Cullin Neddylation 1 (DCN1)-Mediated Cullin Neddylation.

We previously reported the discovery, validation, and structure-activity relationships of a series of piperidinyl ureas that potently inhibit the DCN1-UBE2M interaction. We demonstrated that compound 7 inhibits both the DCN1-UBE2M protein-protein interaction and DCN1-mediated cullin neddylation in biochemical assays and reduces levels of steady-state cullin neddylation in a squamous carcinoma cell line harboring DCN1 amplification. Although compound 7 exhibits good solubility and permeability, it is rapidly metabolized in microsomal models (CL = 170 mL/min/kg).

CDK2 inhibitors as candidate therapeutics for cisplatin- and noise-induced hearing loss.

Hearing loss caused by aging, noise, cisplatin toxicity, or other insults affects 360 million people worldwide, but there are no Food and Drug Administration-approved drugs to prevent or treat it. We screened 4,385 small molecules in a cochlear cell line and identified 10 compounds that protected against cisplatin toxicity in mouse cochlear explants. Among them, kenpaullone, an inhibitor of multiple kinases, including cyclin-dependent kinase 2 (CDK2), protected zebrafish lateral-line neuromasts from cisplatin toxicity and, when delivered locally, protected adult mice and rats against cisplatin- and noise-induced hearing loss.

Progress in the Development of Preventative Drugs for Cisplatin-Induced Hearing Loss.

Cisplatin is a highly effective treatment for malignant cancers and has become a cornerstone in chemotherapeutic regimens. Unfortunately, its use in the clinic is often coupled with a high incidence of severe hearing loss. Over the past few decades, enormous effort has been put forth to find protective agents that selectively protect against the ototoxic side effects of cisplatin and do not interfere with its antitumoral activity.

Discovery of novel, orally bioavailable, antileishmanial compounds using phenotypic screening.

Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection.