Additional Inhibitory Activity Research Articles

Connecting GSK-3β Inhibitory Activity with IKK-β or ROCK-1 Inhibition to Target Tau Aggregation and Neuroinflammation in Alzheimer's Disease-Discovery, In Vitro and In Cellulo Activity of Thiazole-Based Inhibitors.

GSK-3β, IKK-β, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid β (Aβ) and tau proteins, as well as inflammatory processes. Among these kinases, GSK-3β plays the most crucial role. In this study, we present compound 62, a novel, remarkably potent, competitive GSK-3β inhibitor (IC50 = 8 nM, Ki = 2 nM) that also exhibits additional ROCK-1 inhibitory activity (IC50 = 2.3 µM) and demonstrates anti-inflammatory and neuroprotective properties. Compound 62 effectively suppresses the production of nitric oxide (NO) and pro-inflammatory cytokines in the lipopolysaccharide-induced model of inflammation in the microglial BV-2 cell line. Furthermore, it shows neuroprotective effects in an okadaic-acid-induced tau hyperphosphorylation cell model of neurodegeneration. The compound also demonstrates the potential for further development, characterized by its chemical and metabolic stability in mouse microsomes and fair solubility.

A phase 1 single-arm, open-label study of emavusertib (CA-4948) in combination with azacitidine and venetoclax in patients (pts) with acute myeloid leukemia (AML) in complete response (CR) with measurable residual disease (MRD).

TPS6587 Background: Emavusertib is a novel potent oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) with additional inhibitory activity against FMS-like tyrosine kinase 3 (FLT3) and CDC-like kinases (CLK1/2/4). Inhibition of these 3 onco-proteins by emavusertib may address the unmet need for novel therapies in AML. Clinical studies with emavusertib monotherapy have demonstrated a significant reduction in AML blasts with clinical and molecular responses in pts with relapsed or refractory AML (Metzeler 2022). Azacitidine + venetoclax (aza+ven) is an approved therapy for newly diagnosed pts with AML, unfit for intensive chemotherapy. Pre-clinical studies have demonstrated that emavusertib in combination with aza+ven has synergistic and antileukemic effects. In the VIALE-A study, composite CR (CRc) (CR, CRh, or CRi) in the absence of MRD of <1 residual blast/1000 leukocytes (MRD negative [MRD−]) resulted in longer duration of response (DOR), event-free survival, and overall survival (OS) compared with AML pts who achieved CRc but were MRD+ (Pratz, 2022). Therefore, adding emavusertib to aza+ven in CR or CRh, MRD+ pts may convert MRD status without significant toxicity and become a potential new regimen in front-line therapy. Methods: This is a single-arm, open-label Phase 1 study of emavusertib in combination with first line aza+ven in AML pts ≥60 years of age who achieved CR or CRh with MRD+ status based on local testing. The primary objective is to determine a safe and tolerable dosing schedule for the triple combination. Secondary objectives include determining the MRD conversion rate, pharmacokinetics, DOR, and OS. The study will enroll approximately 24 pts at 5 to 10 sites globally. Pts will have received aza+ven as first line therapy and achieved CR or CRh after 1-6 cycles of aza+ven. If MRD status remains positive, emavusertib will be added to the existing aza+ven regimen. Key exclusion criteria include residual toxicities and significant comorbidities. In the first cohort, the starting emavusertib dose is 200 mg BID for 7 days per cycle of 28 days. The duration of emavusertib treatment will extend to 14 and 21 days, in subsequent cohorts. The pts will continue triple treatment (emavusertib+aza+ven) until consent withdrawal, disease progression, intolerable toxicity, or not achieving MRD- after 6 cycles of treatment. Enrollment to cohort 1 began in December 2023. (EU CT Number 2023-505828-58). Clinical trial information: 2023-505828-58.

In-Cell Testing of Zinc-Dependent Histone Deacetylase Inhibitors in the Presence of Class-Selective Fluorogenic Substrates: Potential and Limitations of the Method.

The development of anticancer drugs based on zinc-dependent histone deacetylase inhibitors (HDACi) has acquired great practical significance over the past decade. The most important HDACi characteristics are selectivity and strength of inhibition since they determine the mechanisms of therapeutic action. For in-cell testing of the selectivity of de novo-synthesized HDACi, Western blot analysis of the level of acetylation of bona fide protein substrates of HDACs of each class is usually used. However, the high labor intensity of this method prevents its widespread use in inhibitor screening. We developed an in-cell high-throughput screening method based on the use of three subtype-selective fluorogenic substrates of the general structure Boc-Lys(Acyl)-AMC, which in many cases makes it possible to determine the selectivity of HDACi at the class level. However, we found that the additional inhibitory activity of HDACi against metallo-β-lactamase domain-containing protein 2 (MBLAC2) leads to testing errors.

N-Benzylated 5-Hydroxybenzothiophene-2-carboxamides as Multi-Targeted Clk/Dyrk Inhibitors and Potential Anticancer Agents.

Numerous studies have reported that Dyrk1A, Dyrk1B, and Clk1 are overexpressed in multiple cancers, suggesting a role in malignant disease. Here, we introduce a novel class of group-selective kinase inhibitors targeting Dyrk1A, Dyrk1B, and Clk1. This was achieved by modifying our earlier selective Clk1 inhibitors, which were based on the 5-methoxybenzothiophene-2-carboxamide scaffold. By incorporating a 5-hydroxy group, we increased the potential for additional hydrogen bond interactions that broadened the inhibitory effect to include Dyrk1A and Dyrk1B kinases. Within this series, compounds 12 and 17 emerged as the most potent multi-kinase inhibitors against Dyrk1A, Dyrk1B, and Clk1. Furthermore, when assessed against the most closely related kinases also implicated in cancer, the frontrunner compounds revealed additional inhibitory activity against Haspin and Clk2. Compounds 12 and 17 displayed high potency across various cancer cell lines with minimal effect on non-tumor cells. By examining the effect of these inhibitors on cell cycle distribution, compound 17 retained cells in the G2/M phase and induced apoptosis. Compounds 12 and 17 could also increase levels of cleaved caspase-3 and Bax, while decreasing the expression of the antiapoptotic Bcl-2 protein. These findings support the further study and development of these compounds as novel anticancer therapeutics.

Abstract CT150: Trial in progress: A phase 1b single-arm, open-label, study of emavusertib (CA-4948) in combination with azacitidine and venetoclax in acute myeloid leukemia patients in complete response with measurable residual disease

Abstract Background: Emavusertib is a novel potent oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) with additional inhibitory activity against FMS-like tyrosine kinase 3 (FLT3) and CDC-like kinases (CLK1/2/4). Inhibition of these onco-proteins may induce remission thereby addressing a critical unmet need for novel therapies in acute myeloid leukemia (AML). Clinical studies with emavusertib monotherapy have demonstrated a significant reduction in AML blasts with clinical and molecular responses, including patients with relapsed or refractory AML, previously treated with an HMA and/or FLT3 inhibitors (Metzeler 2022). Azacitidine + venetoclax (aza+ven) has been approved in newly diagnosed, unfit patients with AML. In the VIALE-A study, composite complete response (CRc) (CR, CRh, or CRi) in the absence of measurable residual disease (MRD) of <1 residual blast/1000 leukocytes (MRD negative [MRD−]) resulted in longer duration of response (DOR), event-free survival, and overall survival (OS), and better HSCT outcome compared with patients who achieved CRc but were MRD+ (Pratz, 2022). In pre-clinical studies, emavusertib in combination with aza+ven demonstrated synergistic antileukemic effects in AML cell lines, including azacitidine- or venetoclax-resistant cell lines. Adding emavusertib to aza+ven in MRD+ patients at the time of CR may convert MRD status without adding significant toxicity and confirm that emavusertib can be safely added to aza+ven as a potential new regimen in front-line therapy. Study Design: This is a single-arm, open-label Phase 1b trial evaluating safety and tolerability, PK, and conversion of MRD status with emavusertib as an add-on agent to aza+ven in AML patients who achieved CR or CRh with MRD+ based on local testing (EU CT Number 2023-505828-58-00). The primary objective of the study is to determine a safe and tolerable dosing schedule for the triple combination. Secondary objectives include MRD conversion rate, DOR, OS, and pharmacokinetics. The study will enroll approximately 24 patients at 5 to 10 sites globally. Patients will have received azacitidine and venetoclax as first line therapy and achieved CR or CRh after 1-6 cycles of aza/ven. If MRD status remains positive, emavusertib will be added to the existing well tolerated aza+ven regimen. The starting emavusertib dose is 200 mg BID for 7 days per cycle of 28 days. If well tolerated, duration of emavusertib treatment will be extended to 14 and 21, respectively; no intra-patient change of emavusertib dosing duration is planned. The patients will continue triple treatment (emavusertib+aza+ven) until consent withdrawal, disease progression, intolerable toxicity, or not achieving MRD- within 6 cycles of triple treatment. In this Phase 1b trial, MRD can be evaluated by local testing of bone marrow. Key exclusion criteria include residual toxicities and significant comorbidities. Citation Format: Reinhard von Roemeling, Adolfo De La Fuente, Claudio Cerchione, Sebastian Scholl, Jan Moritz Middeke, Gaurav S. Choudhary, Maria Lamar, Steven Angelides, Uwe Platzbecker. Trial in progress: A phase 1b single-arm, open-label, study of emavusertib (CA-4948) in combination with azacitidine and venetoclax in acute myeloid leukemia patients in complete response with measurable residual disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT150.

Trial in Progress: A Phase 1b Single-Arm, Open-Label Study of Emavusertib (CA-4948) in Combination with Azacitidine and Venetoclax in Acute Myeloid Leukemia Patients in Complete Response with Measurable Residual Disease

Background Emavusertib is a novel potent oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) with additional inhibitory activity against FMS-like tyrosine kinase 3 (FLT3) and CDC-like kinases (CLK1/2/4). Inhibition of these onco-proteins may induce remission thereby addressing a critical unmet need for novel therapies in acute myeloid leukemia (AML). Clinical studies with emavusertib monotherapy have demonstrated a significant reduction in AML blasts with clinical and molecular responses, including patients with relapsed or refractory AML, previously treated with an HMA and/or FLT3 inhibitors (Metzeler 2022). Azacitidine + venetoclax (aza+ven) has been approved in newly diagnosed, unfit patients with AML. In the VIALE-A study, composite complete response (CRc) (CR, CRh, or CRi) in the absence of measurable residual disease (MRD) of <1 residual blast/1000 leukocytes (MRD negative [MRD−]) resulted in longer duration of response (DOR), event-free survival, and overall survival (OS), and better HSCT outcome compared with patients who achieved CRc but were MRD+ (Pratz, 2022). In pre-clinical studies, emavusertib in combination with aza+ven demonstrated synergistic antileukemic effects in AML cell lines, including azacitidine- or venetoclax-resistant cell lines. Adding emavusertib to aza+ven in MRD+ patients at the time of CR may convert MRD status without adding significant toxicity and confirm that emavusertib can be safely added to aza+ven as a potential new regimen in front-line therapy. Study Design: This is a single-arm, open-label Phase 1b trial evaluating safety and tolerability, PK, and conversion of MRD status with emavusertib as an add-on agent to aza+ven in AML patients who achieved CR or CRh with MRD+ based on local testing (EU CT Number 2023-505828-58-00). The primary objective of the study is to determine a safe and tolerable dosing schedule for the triple combination. Secondary objectives include MRD conversion rate, DOR, OS, and pharmacokinetics. The study will enroll approximately 24 patients at 5 to 10 sites globally. Patients will have received azacitidine and venetoclax as first line therapy and achieved CR or CRh after 1-6 cycles of aza/ven. If MRD status remains positive, emavusertib will be added to the existing well tolerated aza+ven regimen. The starting emavusertib dose is 200 mg BID for 7 days per cycle of 28 days. If well tolerated, duration of emavusertib treatment will be extended to 14 and 21, respectively; no intra-patient change of emavusertib dosing duration is planned. The patients will continue triple treatment (emavusertib+aza+ven) until consent withdrawal, disease progression, intolerable toxicity, or not achieving MRD- within 6 cycles of triple treatment. In this Phase 1b trial, MRD can be evaluated by local testing of bone marrow. Key exclusion criteria include residual toxicities and significant comorbidities.

Takeaim Lymphoma: An Open-Label, Dose Escalation and Expansion Trial of Emavusertib (CA-4948) in Combination with Ibrutinib in Patients with Relapsed or Refractory Hematologic Malignancies

Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4), which is essential for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in B cell proliferation. Additional inhibitory activity against FMS-like tyrosine kinase 3 (FLT3) and CDC-like kinases (CLK1/2/4) is also present in emavusertib. IRAK4 forms a Myddosome complex with MYD88 adaptor protein and drives overactivation ofnuclear factor-kappa B (NF-κB), causing inflammation and tumor growth. Emavusertib has been reported to be well tolerated and active as monotherapy in heavily pretreated patients with relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL) (Nowakowski 2020). Preclinical studies demonstrated that tumor resistance and survival via IRAK4 activation could be delayed or reversed. Emavusertib crossed the blood-brain barrier in a murine PDX model of PCNS lymphoma, blocked the activated IRAK4 pathway, and induced tumor response and prolonged survival (von Roemeling 2022). In combination with Bruton tyrosine kinase (BTK) inhibitors, emavusertib showed in vitro synergy and re-sensitization of resistant cell lines, overcoming BTK resistance (Guidetti 2023a and 2023b). In vivo synergy in B-cell NHL was also demonstrated. Adaptive chemotherapy treatment resistance through IRAK4 upregulation and activation was also described in other disease models (Melgar 2019, Li 2019). This abstract presents preliminary efficacy and safety data of emavusertib + ibrutinib in R/R NHL patients. Aim:Assessment of safety and clinical activity of emavusertib in combination with ibrutinib. Methods: This is an ongoing open-label trial (NCT03328078) of emavusertib as monotherapy and in combination with ibrutinib. Part A1 - completed; dose escalation of emavusertib as monotherapy; the recommended phase 2 dose (RP2D) is 300 mg BID with continuous oral dosing (Nowakowski 2020). Part A2 - completed; dose escalation of emavusertib in combination with ibrutinib at 420 and 560 mg QD (Joffe 2022). Part B - ongoing; an expansion cohort of PCNSL patients with resistance to prior BTK inhibitors for additional safety and efficacy related to exposure: emavusertib at 100 or 200 mg BID + ibrutinib at 560 mg QD in 28-Day cycles. Results:As of 19 July 2023, 18 heavily pretreated NHL (including five PCNSL) patients received emavusertib at 100, 200, or 300 mg BID in combination with full ibrutinib doses per label. Median age was 63.5 years (range 50-92). Median number of prior lines of anti-cancer therapies was 3 (range 1-10). Eleven of 18 patients failed prior BTK inhibitors. Median treatment duration was 95.5 days (21-587 days), suggesting acceptable safety and tolerability. No DLTs were observed at the 200 mg BID dose level, and two reversible DLTs (stomatitis and syncope) were observed at the 300 mg BID dose level. The preliminary efficacy data of 16 evaluable patients in combination with ibrutinib showed 5 CRs (2 MCL, 3 PCNSL) and 1 PR (1 CLL). All 3 PCNSL patients with CR were previously treated with BTK inhibitors. Conclusion: The combination ofemavusertib plus ibrutinib (ema+ibr) is well tolerated with an acceptable long-term safety profile and promising efficacy, showing several objective responses in heavily pretreated and/or BTK inhibitor resistant patients. Emavusertib may have the potential to overcome BTK inhibitor resistance and the combination of ema+ibr has the potential to show increased anti-cancer activity compared to ibrutinib monotherapy.

Trial in Progress: An Open-Label Expansion Trial Evaluating the Safety, PK/PD, and Clinical Activity of Emavusertib (CA-4948) + Ibrutinib in R/R Primary CNS Lymphoma

Background: Primary central nervous system lymphoma (PCNSL) is a rare and aggressive form of NHL isolated to the central nervous system or vitreoretinal space. Despite high initial rates of responses to available therapies, most PCNLS relapse in two years, presenting a very poor prognosis. Combination with novel therapies may overcome resistance (Guidetti 2023a). Upregulation of the B-cell receptor (BCR/NF-κB) and toll-like receptor (TLR)/NF-κB) signaling axis have been identified as key pathways in the pathogenesis of PCNSL. MYD88 L265P, a widely expressed somatic mutation in more than 70% of PCNSL patients, activates NF-κB signaling through stimulation of BTK and IRAK4. Emavusertib (CA-4948) is a novel, potent oral inhibitor of interleukin 1 receptor-associated kinase 4 (IRAK4), with additional inhibitory activity against FMS-like tyrosine kinase 3 (FLT3) and CDC-like kinases (CLK1/2/4). Targeted inhibition of IRAK4 with emavusertib in PCNSL xenografts indicated sufficient blood brain barrier penetration of emavusertib, target inhibition, and antitumor activity (von Roemeling 2022). Further preclinical studies have demonstrated that emavusertib synergizes with ibrutinib and can re-sensitize and overcome resistance to BTK inhibitors (Guidetti 2023a and 2023b). Initial clinical data have reported responses in BTK-resistant PCNSL and good tolerance of the combination (Iqbal 2022; Grommes 2023). Study Design: This is an ongoing multi-center, open-label trial (NCT03328078) in adult patients with hematologic malignancies to evaluate the safety, PK, and anti-cancer activity of oral administration of emavusertib alone or in combination with ibrutinib. Part Bis an open trial expansion cohort with BTKi relapsed or refractory PCNSL. About 20 evaluable PCNSL patients will be enrolled globally. Patients will receive oral CA-4948 at 100 or 200 mg BID in combination with ibrutinib at 560 mg QD in 28-day cycles until disease progression or intolerable toxicity. The primary endpoint is expanded safety/tolerability; secondary endpoints include ORR, DOR, PFS, OS, and pharmacokinetics. Key inclusion criteria for this expansion cohort: pathologically confirmed diagnosis of PCNSL with evidence of disease progression, measurable disease, relapsed or refractory with no more than 3 lines of prior anti-PCNSL therapy (including BTKi), and absence of residual Grade ≥2 toxicity from prior therapy. Key exclusion criteria: intraocular PCNSL without brain lesion or CSF involvement or significant co-morbidity. Key clinical assessments include brain imaging, CSF- and ocular examinations.

TAS0612, a Novel RSK, AKT, and S6K Inhibitor, Exhibits Antitumor Effects in Preclinical Tumor Models.

The MAPK and PI3K pathways are involved in cancer growth and survival; however, the clinical efficacy of single inhibitors of each pathway is limited or transient owing to resistance mechanisms, such as feedback signaling and/or reexpression of receptor-type tyrosine kinases (RTK). This study identified a potent and novel kinase inhibitor, TAS0612, and characterized its properties. We found that TAS0612 is a potent, orally available compound that can inhibit p90RSK (RSK), AKT, and p70S6K (S6K) as a single agent and showed a strong correlation with the growth inhibition of cancer cells with PTEN loss or mutations, regardless of the presence of KRAS and BRAF mutations. Additional RSK inhibitory activity may differentiate the sensitivity profile of TAS0612 from that of signaling inhibitors that target only the PI3K pathway. Moreover, TAS0612 demonstrated broad-spectrum activity against tumor models wherein inhibition of MAPK or PI3K pathways was insufficient to exert antitumor effects. TAS0612 exhibited a stronger growth-inhibitory activity against the cancer cell lines and tumor models with dysregulated signaling with the genetic abnormalities described above than treatment with inhibitors against AKT, PI3K, MEK, BRAF, and EGFR/HER2. In addition, TAS0612 demonstrated the persistence of blockade of downstream growth and antiapoptotic signals, despite activation of upstream effectors in the signaling pathway and FoxO-dependent reexpression of HER3. In conclusion, TAS0612 with RSK/AKT/S6K inhibitory activity may provide a novel therapeutic strategy for patients with cancer to improve clinical responses and overcome resistance mechanisms.

Inhibition of Both IRAK1 and IRAK4 Is Required for Complete Suppression of NF-Kb Signaling across Multiple Receptor-Mediated Pathways in MDS and AML

There is increasing interest in the role of the IRAK kinases as targets in the treatment of MDS and AML (reviewed in J Bennett and DT Starczynowski, Curr Opin Hematol 2022). IRAK1 and IRAK4 lie downstream of multiple receptors that stimulate the canonical NF-κB signaling pathway upstream of TRAF6 via the myddosome complex. While TRAF6 is necessary to preserve the tonic NF-κB signaling that is required for hematopoietic stem cell (HSC) homeostasis (J Fang et. al. Cell Reports 2018), overactivity of the NF-κB signaling pathway has been implicated in both MDS and AML (reviewed in MCJ Bosman et. al., Crit Rev Oncol/Hematol 2016; JJ Trowbridge and DT Starczynowski, J Exp Med 2021). The requirement for both IRAK1 and IRAK4 in this pathway has been explored using both genetic and pharmacologic technologies. The use of genetic loss-of-function approaches revealed that deletion and/or inhibition of IRAK4 results in a compensatory increase in IRAK1 protein and activation (data unpublished), suggesting that high potency antagonism of both kinases will be required for optimal inhibition of NF-κB-mediated transcriptional responses in disease-propagating MDS and AML cells. In this report, we use a pharmacological approach in which a series of IRAK inhibitors of varying relative potency at IRAK1:IRAK4 were examined for both inhibition of NF-κB activation in AML cells and inhibition of leukemic progenitor cell function in vitro. We find that potency and efficacy for antagonism at NF-κB requires effective inhibition of both IRAK1 and IRAK4. We also find that the relative potency of NF-κB inhibition correlates with suppression of leukemic progenitor cell function in vitro, providing pharmacological validation that inhibition of both IRAK1 and IRAK4 are necessary for optimal inhibition of NF-κB and effect on leukemia progenitor cell function. We utilized an NF-κB reporter system expressed in human AML cells (THP1) to measure NF-κB dependent activation in response to a variety of IRAK4, IRAK1, or IRAK1/4 antagonists. The cells are highly responsive to both TLR agonists as well as to IL-1b, which allows for measurement of IRAK-mediated antagonism of multiple receptor-mediated pathways. Using the IRAK4-selective antagonists PF-06650833 and BAY 1834845 we find that both compounds fully suppress signaling through TLR2 (IC50vs. Pam3CSK4 = 7.2 and 150 nM, respectively). The IRAK4 antagonists inhibit signaling through the IL-1R with similar relative potency (IC50vs. IL-1b = 5.7 and 81 nM), but do not fully suppress signaling through this receptor (Span = 75% and 59%, respectively). Interestingly, the IRAK1 selective covalent inhibitor JH-X-119-01 does not inhibit signaling against either the TLR or the IL-1 receptor agonist. Together these data imply that neither IRAK4 nor IRAK1 inhibition alone is sufficient to fully inhibit NF-κB-mediated signaling through multiple receptor mediated pathways. For this reason, we then examined the activity of a series of IRAK4/IRAK1 inhibitors in AML cells. This allowed us to study the effect of adding in additional inhibitory activity at IRAK1 on the background of high potency IRAK4 inhibitors. Using two reference compounds with varying IRAK1:IRAK4 potency, we then compared the relative ability to inhibit TLR-agonist or IL-1b-agonist stimulated NF-κB activity in AML. KME-2780 has an IRAK1:IRAK4 potency ratio of 36 whereas KME-3859 has an IRAK1:IRAK4 potency ratio of 100. Unlike what we observed with the IRAK4-selective antagonists, both compounds can completely suppress NF-κB signaling through IL-1b, with relative potencies apparently determined by their activity at IRAK1: (IC50vs. Pam3CSK4 = 6.3 and 73.8 nM for KME- 2780 and KME-3859, respectively); (IC50vs. IL-1b = 9.3 and 227 nM for KME- 2780 and KME-3859, respectively). Finally, we examined the correlation between potency in the biochemical kinase assay at IRAK1 or IRAK4, activity in the NF-κB reporter assay, and leukemic progenitor cell activity in the colony forming assay for a series of KME compounds in AML and MDS cells. We find a correlation between kinase activity for both IRAK1 and IRAK4 and NF-κB activity that extends to the leukemia colony forming assays. This suggests that NF-κB signaling contributes to leukemia progenitor cell function and that optimal inhibition requires potent antagonism of both IRAK1 and IRAK4 in the setting of MDS and AML.

Potent Pre-Clinical and Early Phase Clinical Activity of EP300/CBP Bromodomain Inhibitor CCS1477 in Multiple Myeloma

CCS1477 (also known as inobrodib) is a potent and selective inhibitor of the bromodomains of EP300 and CBP, two homologous histone acetyltransferases with critical roles in cellular growth and differentiation. In preclinical analyses we find that CCS1477 induces potent growth inhibition though inducing cell cycle arrest at low nanomolar concentrations (GI50<100nM) across a panel of 12 human myeloma cell lines, including lenalidomide refractory lines. In vivo, in an OPM-2 human myeloma cell line NOD-SCID xenograft model, there was dose dependent inhibition of tumour growth, with 20mg/kg daily eliciting tumour regression and sustained inhibition of tumour growth for two weeks beyond the end of treatment. RNA sequencing of tumours recovered from mice after five days of CCS1477 treatment revealed substantial down regulation of transcription factor genes including MYB, MYC, IRF4, E2F1 and FOXM1; and GSEA showed significant enrichment among down regulated genes of gene sets associated with both MYC and E2F targets, consistent with drug-induced proliferative arrest. In the OPM-2 model, ChIP sequencing showed that EP300 binding peaks were predominantly distributed over transcription factor binding motifs for IRF, Runt, bHLH, bZIP and MYB/SANT factor classes. Within six hours of 100nM CCS1477 treatment there was a localized but significant reduction of EP300 recruitment to intronic FGFR3 sites: FGFR3 is placed under the control of the active IGH promoter in the intergenic t(4;14) in OPM-2 cells. By 48 hours, in keeping with CCS1477-induced inactivation of IRF4 (a critical regulator of plasma cell differentiation required for myeloma cell growth) there was extensive genome-wide redistribution of EP300 binding away from sites with IRF4 motifs, while sites with other motifs remained occupied. In our ongoing phase I/IIa clinical trial investigating the safety and efficacy of CCS1477 in advanced haematological malignancies (NCT04068597), we have treated four heavily pre-treated relapsed/refractory myeloma patients at the recommended phase 2 dose (35mg PO twice daily, four days a week). Of these, two exhibited objective responses with substantial reductions in serum or urinary M-protein, with one patient achieving a PR and the other exhibiting a complete remission after four months. Both patients remain on treatment as of the submission of this abstract. Treatment was well tolerated, with moderate thrombocytopaenia being an anticipated on-target side effect. Finally, we evaluated the activity of CCS1477 in an OPM-2 xenograft model in combination with existing standard of care agents in myeloma and observed additive or synergistic growth inhibitory activity with each of bortezomib, lenalidomide and vorinostat in combination with CCS1477. These encouraging data suggest strong promise for the clinical utility of CCS1477 in the treatment of multiple myeloma as monotherapy and/or in combination with standard of care agents.

Three-Dimensional Proteome-Wide Scale Screening for the 5-Alpha Reductase Inhibitor Finasteride: Identification of a Novel Off-Target.

Finasteride, a 5-alpha reductase (5α-R) inhibitor, is a widely used drug for treating androgen-dependent conditions. However, its use is associated with sexual, psychological, and physical complaints, suggesting that other mechanisms, in addition to 5α-R inhibition, may be involved. Here, a multidisciplinary approach has been used to identify potential finasteride off-target proteins. SPILLO-PBSS software suggests an additional inhibitory activity of finasteride on phenylethanolamine N-methyltransferase (PNMT), the limiting enzyme in formation of the stress hormone epinephrine. The interaction of finasteride with PNMT was supported by docking and molecular dynamics analysis and by in vitro assay, confirming the inhibitory nature of the binding. Finally, this inhibition was also confirmed in an in vivo rat model. Literature data indicate that PNMT activity perturbation may be correlated with sexual and psychological side effects. Therefore, results here obtained suggest that the binding of finasteride to PNMT might have a role in producing the side effects exerted by finasteride treatment.

Effects of Modified Magnetite Nanoparticles on Bacterial Cells and Enzyme Reactions.

Current paper presents biological effects of magnetite nanoparticles (MNPs). Relations of MNP’ characteristics (zeta-potential and hydrodynamic diameters) with effects on bacteria and their enzymatic reactions were the main focus. Photobacterium phosphoreum and bacterial enzymatic reactions were chosen as bioassays. Three types of MNPs were under study: bare Fe3O4, Fe3O4 modified with 3-aminopropyltriethoxysilane (Fe3O4/APTES), and humic acids (Fe3O4/HA). Effects of the MNPs were studied at a low concentration range (< 2 mg/L) and attributed to availability and oxidative activity of Fe3+, high negative surface charge, and low hydrodynamic diameter of Fe3O4/HA, as well as higher Fe3+ content in suspensions of Fe3O4/HA. Low-concentration suspensions of bare Fe3O4 provided inhibitory effects in both bacterial and enzymatic bioassays, whereas the MNPs with modified surface (Fe3O4/APTES and Fe3O4/HA) did not affect the enzymatic activity. Under oxidative stress (i.e., in the solutions of model oxidizer, 1,4-benzoquinone), MNPs did not reveal antioxidant activity, moreover, Fe3O4/HA demonstrated additional inhibitory activity. The study contributes to the deeper understanding of a role of humic substances and silica in biogeochemical cycling of iron. Bioluminescence assays, cellular and enzymatic, can serve as convenient tools to evaluate bioavailability of Fe3+ in natural dispersions of iron-containing nanoparticles, e.g., magnetite, ferrihydrite, etc.

Combination Treatment with Imetelstat, a Telomerase Inhibitor, and Ruxolitinib Depletes Myelofibrosis Hematopoietic Stem Cells and Progenitor Cells

Therapy with imetelstat (Ime), a telomerase inhibitor, has been shown to have disease-modifying effect in patients with myelofibrosis (MF) (Tefferi N Engl J Med 2015; Mascarenhas Blood 2018). This has been attributed to the ability of Ime to selectively deplete MF hematopoietic stem cells (HSC)/progenitor cells (HPC) (Wang Blood Adv 2018). As reported, the primary toxicities of Ime are cytopenias, however, they appear to be reversible, and most importantly, manageable without meaningful clinical consequences (Mascarenhas Blood 2018, Fenaux EHA 2019). Ruxolitinib (Rux) is the first and only JAK 1/2 inhibitor approved for use in patients with intermediate- or high-risk MF. Based on different mechanisms of action for Ime and Rux, we hypothesize that Ime in combination with Rux might create a regimen that would be more efficacious than single agent alone in depleting MF HSCs/HPCs. Using in vitro HPC assays and in vivo HSC assays, we evaluated the therapeutic potential of Rux and Ime. MF splenic (n=7) or cord blood (CB) (n=3) CD34+ cells (2.5×104/mL) were incubated in serum free expansion medium containing cytokines in the presence of Rux (50nM) alone, Ime alone (1.8uM), Rux+Ime (simultaneous treatment) or Rux→Ime (sequential treatment). Rux was dosed for 3 days and Ime for 14 days under treatment conditions containing either or both drugs. In addition, parallel cultures with mismatched oligonucleotides (MM) or vehicle alone were performed. Compared to either drug alone or to MM control, Rux followed by Ime sequential treatment resulted in significant reductions in the numbers of MF Lin-CD34+ cells (Rux→Ime vs. Rux alone, p=0.001; vs. Ime alone, p=0.059; vs. Rux→MM, p=0.052) and assayable HPCs [(CFUGM+BFU-E+CFU-GEMM), Rux→Ime vs. Rux alone, p=0.02; vs. Ime alone, p=0.05; vs. Rux→MM, p=0.04], while the simultaneous treatment with Rux+Ime did not significantly reduce MF Lin-CD34+ cells and assayable HPCs. By contrast, none of these treatment conditions affected the behavior of normal HSCs/HPCs. Genotyping of individual colonies showed that sequential drug treatment of CD34+ cells from 2 JAK2V617+ MF patients resulted in greater reductions in both the percentage and absolute numbers of JAK2V617F+ myeloid progenitors than that achieved with Ime alone or simultaneous combination treatment. These findings suggest that an additive inhibitory activity against MF HSCs and malignant HPCs can be achieved with sequential treatment of Rux followed by Ime. We next assessed if treatment with Ime (10mg/kg, 3 times/week for 4 weeks, I.P. injection) and Rux (45mg/kg, daily for 7 days, oral gavage) had additive effects on MF or normal HSCs by directly treating NSG mice transplanted with MF or CB CD34+ cells with these two drugs either alone, in combination simultaneously or sequentially. Mice were sacrificed 4 months after the transplantation. Both simultaneous and sequential drug treatment of mice transplanted with MF splenic CD34+ cells resulted in greater reductions in the degree of human CD45+ marrow cell chimerism (relative to vehicle alone: Rux→Ime: 54.0%; Rux+Ime: 72.4%; Rux alone: 76.6%, Ime alone: 80.0%) and human CD45+ splenic cell chimerism (relative to vehicle alone: Rux→Ime: 13.8%; Rux+Ime: 31.9%; Rux alone: 163.1%; Ime alone: 42.4%) as compared with either drug alone treatment. Similar to our findings in the above in vitro studies, a greater reduction in the absolute number of human CD34+ cells was observed with sequential but not simultaneous combination treatment as compared with either drug alone treatment in the marrow of the mice transplanted with MF CD34+ cells (relative to vehicle alone: Rux→Ime: 36.0%; Rux+Ime: 61.0%; Rux alone: 47.5%, Ime alone: 57.3%). Furthermore, similar results were also observed in depletion of MF long-term HSCs in mice receiving splenic CD34+ cells from an additional patient. However, this same sequential drug schema did not affect normal HSC function. Collectively, these data indicate that alterations of scheduling of the administration of Rux and Ime affect the efficacy of this drug combination in depleting MF HPC/HSCs. We propose that cycles of Rux followed by Ime represents a potentially effective therapeutic strategy that is capable of eliminating MF HSCs/HPCs with an acceptable toxicity profile. Disclosures Hoffman: Merus: Research Funding.

Biological evaluation of molecules of the azaBINOL class as antiviral agents: Inhibition of HIV-1 RNase H activity by 7-isopropoxy-8-(naphth-1-yl)quinoline

Inspired by bioactive biaryl-containing natural products found in plants and the marine environment, a series of synthetic compounds belonging to the azaBINOL chiral ligand family was evaluated for antiviral activity against HIV-1. Testing of 39 unique azaBINOLs and two BINOLs in a single-round infectivity assay resulted in the identification of three promising antiviral compounds, including 7-isopropoxy-8-(naphth-1-yl)quinoline (azaBINOL B#24), which exhibited low-micromolar activity without associated cytotoxicity. The active compounds and several close structural analogues were further tested against three different HIV-1 envelope pseudotyped viruses as well as in a full-virus replication system (EASY-HIT). The in vitro studies indicated that azaBINOL B#24 acts on early stages of viral replication before viral assembly and budding. Next we explored B#24’s activity against HIV-1 reverse transcriptase (RT) and individually tested for polymerase and RNase H activity. The azaBINOL B#24 inhibits RNase H activity and binds directly to the HIV-1 RT enzyme. Additionally, we observe additive inhibitory activity against pseudotyped viruses when B#24 is dosed in competition with the clinically used non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz. When tested against a multi-drug resistant HIV-1 isolate with drug resistance associated mutations in regions encoding for HIV-1 RT and protease, B#24 only exhibits a 5.1-fold net decrease in IC50 value, while efavirenz’ activity decreases by 7.6-fold. These results indicate that azaBINOL B#24 is a potentially viable, novel lead for the development of new HIV-1 RNase H inhibitors. Furthermore, this study demonstrates that the survey of libraries of synthetic compounds, designed purely with the goal of facilitating chemical synthesis in mind, may yield unexpected and selective drug leads for the development of new antiviral agents.

PS991 CA‐4948, AN IRAK4/FLT3 INHIBITOR, SHOWS ANTILEUKEMIC ACTIVITY IN MOUSE MODELS OF FLT3 WILD‐TYPE AND FLT3 MUTATED ACUTE MYELOID LEUKEMIA (AML)

Background:AML is an aggressive hematopoietic malignancy that arises from a population of aberrant hematopoietic stem cells in the bone marrow (BM). Advances in understanding the molecular basis of AML has led to the development of new targeted therapies. CA‐4948 is a novel, oral IRAK4 kinase inhibitor with additional inhibitory activity against wild‐type (wt) and mutated FLT3 kinase. IRAK4 (Interleukin‐1 Receptor Associated Kinase 4) is required for interleukin 1 receptor (IL‐1R) and toll‐like receptor (TLR) innate immune pathway signaling, pathways that are frequently over activated in AML and myelodysplastic syndromes (MDS). For example, AML patients have increased IL‐1R agonist (IL‐1ß) levels that promote the survival of AML cells and IL‐1R KO represses AML cell growth in vitro and in vivo (Carey et al 2017). Dysregulation of the FLT3 signaling pathway is a well validated driver of AML. Constitutively activating mutations in FLT3 that comprise the ITD or the tyrosine kinase domain (KD) are frequently acquired late in AML disease and are poor prognostic factors with high relapse rates. FLT3 kinase inhibitors targeting FLT3‐ITD or ITD/KD double mutations show high remission rates; however, multiple resistance mechanisms have been reported in both nonclinical models and AML patients. CA‐4948 has both IRAK4 and FLT3 inhibitory activity, which may impart benefit to FLT3‐wt and FLT3‐mutant AML patients.Aims:Evaluate the ability of a novel IRAK4/FLT3 inhibitor, CA‐4948, to block IRAK4 and FLT3 in FLT3‐wt and FLT3‐mutant AML in vitro and in vivo.Methods:CA‐4948's kinome profile was assessed against 378 kinases and 9 FLT3 mutant variants (DiscoverX). CA‐4948's ability to inhibit TLR/IL‐1R or FLT3 signaling pathways was evaluated using NF‐kB reporter, cytokine production, or western blot. The growth inhibitory and pro‐apoptotic activity of CA‐4948 was tested in vitro against AML cell lines by viability assay or flow cytometry. For AML FLT3‐wt in vivo efficacy, THP‐1 cells were tail‐vein injected into mice and animal survival and degree of AML cell engraftment in BM were monitored in CA‐4948, FLT3i, or vehicle treated mice. For AML FLT3 mutant in vivo efficacy, subcutaneous MV4–11 and MOLM‐14 FLT3‐ITD and MOLM‐14 double FLT3‐ITD/KD tumor models were treated with CA‐4948 or other FLT3i.Results:CA‐4948 exhibited 23 nM (Kd) binding affinity for IRAK4, &gt;500‐fold selectivity against IRAK1, and high binding affinity for FLT3‐wt, ‐ITD, ‐ITD/D835 V, and ‐ITD/F691L at 8–31 nM (Kd). CA‐4948 blocked cellular TLR and IL‐1R‐stimulated signaling from 150–220 nM (IC50). Consistent with CA‐4948's additional FLT3 inhibitory activity, CA‐4948 induced apoptosis and exhibited cytotoxic activity against AML FLT3 mutant lines in vitro at 58–200 nM (IC50). In FLT3‐wt in vivo studies, CA‐4948 blocked THP‐1 bone marrow engraftment, while FLT3i quizartinib (Q) and midostaurin (M) had no inhibitory effect. In AML FLT3‐ITD tumors, CA‐4948 induced tumor regression equivalent to Q and M. CA‐4948 also induced tumor regression in FLT3‐ITD/D835Y tumors similar to M and showed greater efficacy in FLT3‐ITD/F691L tumors as compared to Q.Summary/Conclusion:These results demonstrate that targeting the IL‐1R/TLR signaling pathway with IRAK4 inhibitor CA‐4849 may be an effective therapeutic strategy in FLT3‐wt AML. Furthermore, CA‐4948's additional FLT3 inhibitory activity may repress the emergence of FLT3‐mutant AML clones. CA‐4948's dual IRAK4/FLT3 inhibitory activity merits further clinical investigation in FLT3‐wt and FLT3‐mutant AML/MDS.

The SERM/SERD bazedoxifene disrupts ESR1 helix 12 to overcome acquired hormone resistance in breast cancer cells.

Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ERα)) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER +breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We found that BZA possesses improved inhibitory potency against the Y537S and D538G ERα mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show BZA's selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ERα mutations.

Structure-Activity Relationships of Thiazolyl Resorcinols, Potent and Selective Inhibitors of Human Tyrosinase.

Tyrosinase inhibitors are of great clinical interest as agents for the treatment of hyperpigmentary disorders; however, most compounds described in the literature lack clinical efficiency due to insufficient inhibitory activity against human tyrosinase (hTyr). Recently, we reported that thiazolyl resorcinols (4-resorcinylthiazol-2-amines and -amides) are both selective and efficacious inhibitors of hTyr in vitro and in vivo. Here, we measured dose-activity profiles of a large number of thiazolyl resorcinols and analogous compounds to better understand the molecular basis of their interaction with hTyr. We show that both the resorcinyl moiety and the thiazole ring must be intact to allow efficient inhibition of hTyr, while the substituents at the thiazole 2-amino group confer additional inhibitory activity, depending on their size and polarity. The results of molecular docking simulations were in excellent agreement with the experimental data, affording a rationale for the structural importance of either ring. We further propose that a special type of interaction between the thiazole sulfur and a conserved asparagine residue is partially responsible for the superior inhibitory activity of thiazolyl resorcinols against hTyr.

Discovery and structure-activity relationship studies of 2-benzylidene-2,3-dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivatives as a novel class of potential therapeutics for inflammatory bowel disease

To develop effective therapeutics for inflammatory bowel disease (IBD), 2-benzylidene-2,3-dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivatives, were designed and synthesized and their structure-activity relationships (SAR) were investigated. Compounds 7, 25, 26, 32, 39, 41, 52, 54, and 55 showed potent inhibitory effect (>70%) on the TNF-α-induced adhesion of monocytes to colon epithelial cells, which is one of the hallmark events leading to IBD. Such inhibitory activity of the compounds correlated with their suppressive activities against the TNF-α-induced production of ROS; ICAM-1 and MCP-1 expression, critical molecules involved in monocyte-epithelial adhesion; and NF-κB transcriptional activity. In addition, compounds 41 and 55 significantly suppressed the lipopolysaccharide (LPS)-induced expression of the TNF-α gene, with compound 55 showing better efficacy. This inhibition of TNF-α expression by compounds 41 and 55 corresponded to their additional inhibitory activity against AP-1 transcriptional activity, which is another transcription factor required for high level TNF-α expression. The strong inhibitory activity of compound 55 against an in vivo colitis model was confirmed by its dose-dependent inhibitory activity in a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, demonstrating compound 55 as a new potential candidate for the development of therapeutics against IBD.

Possible role of P-glycoprotein in the neuroprotective mechanism of berberine in intracerebroventricular streptozotocin-induced cognitive dysfunction

The therapeutic potential of berberine has been well documented in various neurological problems. However, the neurological mechanism of berberine remains untapped in the light of its P-glycoprotein (P-gp)-mediated gut efflux properties responsible for reduced bioavailability. Verapamil, a well known L-type calcium channel blocker, has additional inhibitory activity against P-gp efflux pump. Thus, there is a strong scientific rationale to explore the interaction of berberine with verapamil as a possible neuroprotective strategy. The present study was designed to evaluate the effect of berberine, verapamil, and their combination on behavioral alterations, oxidative stress, mitochondrial dysfunction, neuroinflammation, and histopathological modifications in intracerebroventricular streptozocin (ICV-STZ)-induced sporadic dementia of Alzheimer's type in rats. Single bilateral ICV-STZ (3mg/kg) administration was used as an experimental model of sporadic dementia of Alzheimer's type. Berberine (25, 50, and 100mg/kg, oral gavage) or verapamil (2.5 and 5mg/kg, intraperitoneally) were used as treatment drugs, and memantine (5mg/kg, intraperitoneally) was used as a standard. Berberine and verapamil significantly attenuated behavioral, biochemical, cellular, and histological alterations, suggesting their neuroprotective potential. Further, treatment of berberine (25 and 50mg/kg) with verapamil (2.5 and 5.0mg/kg) combinations respectively significantly potentiated their neuroprotective effect which was significant as compared to their effect per se in ICV-STZ-treated animals. The augmentative outcome of verapamil on the neuroprotective effect of berberine can be speculated due to the inhibition of P-gp efflux mechanism and the prevention of calcium homeostasis alteration. Additionally, anti-inflammatory and antioxidant effects of both berberine and verapamil could also contribute in their protective effect.