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. Compound 21, a derivative with the tetrahydroindazolone cap group, is a potent HDAC6 inhibitor with an IC50 of 18 nM and 217-fold selectivity over HDAC1 and showed favorable oral bioavailability in animals. Compound 21 increases the acetylation level of tubulin without affecting histone acetylation in cutaneous T-cell lymphoma cells and inhibits TNF-α secretion in LPS-stimulated macrophage cells. The anti-arthritic effects of compound 21 were evaluated using a rat adjuvant-induced arthritis (AIA) model. Treatment with compound 21 significantly reduced the arthritis score, and combination treatment with methotrexate showed a synergistic effect in AIA models. We identified a novel HDAC6 inhibitor, compound 21, with excellent in vivo anti-arthritic efficacy, which can lead to the development of oral anti-arthritic drugs.

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. Bromodomain and extra terminal (BET) and PI3K/mTOR inhibitors potentiated ribociclib inhibition of proliferation in an established cell line and freshly dissociated tumor cells from intracranial xenografts of G3 and Sonic hedgehog (SHH) medulloblastomas in vitro. A reverse combination screen using the BET inhibitor JQ1 as anchor, revealed CDK4/6i as the most potentiating drugs. In vivo, ribociclib showed single-agent activity in medulloblastoma models whereas JQ1 failed to show efficacy due to high clearance and insufficient free brain concentration. Despite in vitro synergy, combination of ribociclib with the PI3K/mTOR inhibitor paxalisib did not significantly improve the survival of G3 and SHH medulloblastoma-bearing mice compared with ribociclib alone. Molecular analysis of ribociclib and paxalisib-treated tumors revealed that E2F targets and PI3K/AKT/MTORC1 signaling genes were depleted, as expected. Importantly, in one untreated G3MB model HD-MB03, the PI3K/AKT/MTORC1 gene set was enriched in vitro compared with in vivo suggesting that the pathway displayed increased activity in vitro. Our data illustrate the difficulty in translating in vitro findings in vivo. See related article in Mol Cancer Ther (2022) 21(8):1306-1317.

Preclinical evaluation of proteolytic targeting of LCK as a therapeutic approach in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and there is an unmet need for targeted therapies, especially for patients with relapsed disease. We have recently identified pre-T cell receptor and lymphocyte-specific protein tyrosine kinase (LCK) signaling as a common therapeutic vulnerability in T-ALL. LCK inhibitor dasatinib showed efficacy against T-ALL in preclinical studies and in patients with T-ALL; however, this is transient in most cases. Leveraging the proteolysis targeting chimera (PROTAC) approach, we developed a series of LCK degraders using dasatinib as an LCK ligand and phenyl-glutarimide as a cereblon-directing moiety. Our lead compound SJ11646 exhibited marked efficiency in cereblon-mediated LCK degradation in T-ALL cells. Relative to dasatinib, SJ11646 showed up to three orders of magnitude higher cytotoxicity in LCK-activated T-ALL cell lines and primary leukemia samples in vitro, with drastically prolonged suppression of LCK signaling. In vivo pharmacokinetic and pharmacodynamic profiling indicated a 630% increase in the duration of LCK suppression by SJ11646 over dasatinib in patient-derived xenograft models of T-ALL, which translated into its extended leukemia-free survival over dasatinib in vivo. Last, SJ11646 retained a high binding affinity to 51 human kinases, particularly ABL1, KIT, and DDR1, all of which are known drug targets in other cancers. Together, our dasatinib-based phenyl-glutarimide PROTACs are promising therapeutic agents in T-ALL and valuable tools for developing degradation-based therapeutics for other cancers.

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. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

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. Herein we report the design, synthesis, and characterization of PROTACs of AT7519-7 and AZD5438 and the identification of PROTAC-8, an AZD5438-PROTAC, that exhibits selective, partial CDK2 degradation. Furthermore, PROTAC-8 protects against cisplatin ototoxicity and kainic acid excitotoxicity in zebrafish. Molecular dynamics simulations reveal the structural requirements for CDK2 degradation. Together, PROTAC-8 is among the first-in-class PROTACs with in vivo therapeutic activities and represents a new lead compound that can be further developed for better efficacy and selectivity for CDK2 degradation against hearing loss and cancer.

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. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT-driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context.

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. Taken together, this study offers compound 6 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 in vitro and in vivo, and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.

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. In adult mice, oral delivery of dabrafenib repressed ERK phosphorylation in cochlear cells, and protected from cisplatin- and noise-induced hearing loss. Full protection was achieved in mice with co-treatment with oral AZD5438, a CDK2 kinase inhibitor. Our study explores a previously unidentified cellular pathway and molecular target BRAF kinase for otoprotection and may advance dabrafenib into clinics to benefit patients with cisplatin- and noise-induced ototoxicity.

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. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific 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. Rational X-ray co-structure enabled optimization afforded a 25-fold improvement in potency relative to the initial screening hit. The potency gains are largely attributed to additional hydrophobic interactions mimicking the N-terminal acetyl group that drives binding of UBE2M to DCN1. The compounds inhibit the protein-protein interaction, block NEDD8 transfer in biochemical assays, engage DCN1 in cells, and selectively reduce the steady-state neddylation of Cul1 and Cul3 in two squamous carcinoma cell lines harboring DCN1 amplification.

Mechanistic Insight on the Mode of Action of Colletoic Acid.

The natural product colletoic acid (CA) is a selective inhibitor of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-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. 11ß-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 11ß-HSD1 bound to CA at 2.6 Å resolution revealed the nature of those interactions, namely, a close-fitting and favorable interactions between the constrained CA spirocycle and the catalytic triad of 11ß-HSD1. Structure-activity relationship studies culminated in the development of a superior CA analogue with improved target engagement. Furthermore, we demonstrate that CA selectively inhibits preadipocyte differentiation through 11ß-HSD1 inhibition, suppressing other relevant key drivers of adipogenesis (i.e., PPARγ, PGC-1α), presumably by negatively modulating the glucocorticoid signaling pathway. The combined findings provide an in-depth evaluation of the mode of action of CA and its potential as a tool compound to study adipose tissue and its implications in metabolic syndrome.

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. Moreover, we screened 36 analogues of AT7519 and identified analogue 7, which exhibited an improved therapeutic index. When delivered locally, analogue 7 and AZD5438 both provided significant protection against cisplatin-induced ototoxicity in mice. Thus, we have identified two additional compounds that prevent cisplatin-induced ototoxicity in vivo and provided further evidence that CDK2 is a druggable target for treating cisplatin-induced ototoxicity.

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 (CLint = 170 mL/min/kg). This work lays out the discovery of an orally bioavailable analogue, NAcM-OPT (67). Compound 67 retains the favorable biochemical and cellular activity of compound 7 but is significantly more stable both in vitro and in vivo. Compound 67 is orally bioavailable, well tolerated in mice, and currently used to study the effects of acute pharmacologic inhibition of the DCN1-UBE2M interaction on the NEDD8/CUL pathway.

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. The optimized compounds inhibit the DCN1-UBE2M protein-protein interaction in our TR-FRET binding assay and inhibit cullin neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous cell carcinomas and other cancers.

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. CDK2-deficient mice displayed enhanced resistance to cisplatin toxicity in cochlear explants and to cisplatin- and noise-induced hearing loss in vivo. Mechanistically, we showed that kenpaullone directly inhibits CDK2 kinase activity and reduces cisplatin-induced mitochondrial production of reactive oxygen species, thereby enhancing cell survival. Our experiments have revealed the proapoptotic function of CDK2 in postmitotic cochlear cells and have identified promising therapeutics for preventing 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. Many therapies have been successful in preclinical work, but only a few have shown any protection in the clinic, and none have been approved by the FDA. This review summarizes the clinical and preclinical studies of the most effective small-molecule candidates currently in clinical trials, while also detailing their molecular mechanisms of action, to gain insight for future drug development in the field.

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. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 µM. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for in vitro absorption, distribution, metabolism, excretion, and in vivo pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC50) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to Leishmania species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant in vivo efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug.

Ventromorphins: A New Class of Small Molecule Activators of the Canonical BMP Signaling Pathway.

Here, we describe three new small-molecule activators of BMP signaling found by high throughput screening of a library of ∼600 000 small molecules. Using a cell-based luciferase assay in the BMP4-responsive human cervical carcinoma clonal cell line, C33A-2D2, we identified three compounds with similar chemotypes that each ventralize zebrafish embryos and stimulate increased expression of the BMP target genes, bmp2b and szl. Because these compounds ventralize zebrafish embryos, we have termed them "ventromorphins." As expected for a BMP pathway activator, they induce the differentiation of C2C12 myoblasts to osteoblasts. Affymetrix RNA analysis confirmed the differentiation results and showed that ventromorphins treatment elicits a genetic response similar to BMP4 treatment. Unlike isoliquiritigenin (SJ000286237), a flavone that maximally activates the pathway after 24 h of treatment, all three ventromorphins induced SMAD1/5/8 phosphorylation within 30 min of treatment and achieved peak activity within 1 h, indicating that their responses are consistent with directly activating BMP signaling.

Performance of a docking/molecular dynamics protocol for virtual screening of nutlin-class inhibitors of Mdmx.

A virtual screening protocol involving docking and molecular dynamics has been tested against the results of fluorescence polarization assays testing the potency of a series of compounds of the nutlin class for inhibition of the interaction between p53 and Mdmx, an interaction identified as a driver of certain cancers. The protocol uses a standard docking method (AutoDock) with a cutoff based on the AutoDock score (ADscore), followed by molecular dynamics simulation with a cutoff based on root-mean-square-deviation (RMSD) from the docked pose. An analysis of the experimental and computational results shows modest performance of ADscore alone, but dramatically improved performance when RMSD is also used.