Potent Dihydroorotate Dehydrogenase Inhibitor Research Articles

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Discovery of JNJ-74856665: A Novel Isoquinolinone DHODH Inhibitor for the Treatment of AML.

Acute myelogenous leukemia (AML), a heterogeneous disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes. As a result of virtual screening and structure-based drug design approaches, a novel series of isoquinolinone DHODH inhibitors was identified. Further lead optimization afforded JNJ-74856665 as an orally bioavailable, potent, and selective DHODH inhibitor with favorable physicochemical properties selected for clinical development in patients with AML and myelodysplastic syndromes (MDS).

Discovery of Alternative Binding Poses through Fragment-Based Identification of DHODH Inhibitors.

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival. Recently, DHODH has been identified as a potential target for acute myeloid leukemia therapy. Herein, we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode. Additionally, these compounds had low P-gp efflux ratios, allowing for applications where exposure to the brain would be required.

The Potent Dihydroorotate Dehydrogenase Inhibitor, Hosu-53, Exhibits Compelling Monotherapy Efficacy in Multiple Myeloma and Augments CD47 Targeted Therapy

Worldwide, multiple myeloma (MM) is the second most common hematological malignancy characterized by the expansion of aberrant mature plasma cells in the bone marrow. Despite the FDA approval of several drugs with different mechanisms of action such as immunomodulatory drugs (IMiDs) and proteosome inhibitors (PIs), MM remains a challenging incurable disease where most patients ultimately relapse and become refractory to available . The resistant nature of MM underscores the need for novel therapeutic strategies that can directly affect MM cells while enhancing the outcome with MM current standard of care (SOC). Among the most promising MM SOC agents is monoclonal antibodies (mAb) such as the CD38 antibody daratumumab, which significantly improved the management of newly diagnosed and relapsed/refractory MM. Therefore, we aimed to develop novel therapies that can enhance mAb therapy in MM. Dihydroorotate dehydrogenase (DHODH) is an enzyme which mediates the fourth and rate-limiting step in the de novo pyrimidine synthesis pathway converting dihydroorotate to orotate, the precursor of uridine. Pyrimidine starvation using DHODH inhibitors has been shown to have potent antiproliferative activity in several malignancies including acute myeloid leukemia (AML) and MM. Thus, we developed a novel potent DHODH inhibitor, HOSU-53 for the treatment of hematological malignancies. We previously discovered the ability of HOSU-53 to modulate surface CD38 expression and found that HOSU-53 had potent monotherapy activity in MM and provided impressive synergy in combination with daratumumab using the NCI-H929 MM cell line derived xenograft (CDX) model (2022 AACR annual meeting abstracts). Herein, we further expanded our studies to pre-clinically develop HOSU-53 as a new potential MM therapy and explore additional synergistic immunotherapy combinations that could maintain efficacy in the case of anti-CD38 therapy resistance. We evaluated the in vitro antiproliferative efficacy of HOSU-53 against a panel of MM cell lines and found nanomolar potency validating that HOSU-53 would have monotherapy efficacy. Indeed, we conducted in vivo studies using two additional MM CDX subcutaneous (s.c) models, OPM-2 and RPMI-8226, and found significant tumor delay and survival advantage. In the OPM-2 model, HOSU-53 has a median survival of 54-days compared to vehicle at 28-days, while the median survival for HOSU-53 was 60-days compared to vehicle at 26-days in the RPMI-8226 model. We further verified HOSU-53 efficacy using the disseminated MM1.S luciferase CDX model and found a significant prolonged survival in the HOSU-53 cohort (median survival53-days) compared to vehicle (median survival28-days) that was further enhanced with isatuximab combination resulting in superior survival benefit (median survival 69-days). Given the continuous challenge in maintaining a durable response with anti-CD38 therapies due to resistance, we sought to explore additional combination regimens to maximize HOSU-53 efficacy. Currently there is significant clinical interest in CD47 antibody therapy such as magrolimab for both solid tumors and hematological malignancies. Our previous work suggested a strong synergy between CD47 antibodies and HOSU-53 in AML with curative potential (2022 ASH annual meeting abstracts), and hypothesized that we would observe similar synergy with HOSU-53 in combination with anti-CD47 in MM. Indeed, we found complete tumor regression in all mice treated with HOSU-53 + B6.H12 anti-CD47 therapy in the NCI-H929 s.c CDX model and significant prolonged survival and reduced bioluminescence in the NCI-H929 luciferase disseminated CDX model. Together, these two studies validate a potential clinical benefit to explore HOSU-53 + anti-CD47 regimen in MM patients. Furthermore, we observed that calreticulin (pro-phagocytosis signal) was modulated post HOSU-53 in vitro treatment suggesting its role in the observed synergy between HOSU-53 and CD47 blockade therapy. In summary, we show compelling survival benefit for HOSU-53 as a monotherapy which is further enhanced when combined with anti-CD38 or anti-CD47 therapies. HOSU-53 is expected to enter phase 1 clinical trials in 2024 and our data is supportive for its expansion into MM.

Absorption, metabolism and excretion of 14C-emvododstat following repeat daily oral dose administration in human volunteers using a combination of microtracer radioactivity and high radioactivity doses.

Emvododstat is a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of COVID-19 and acute myeloid leukemia. Since the metabolism and pharmacokinetics of emvododstat in humans is time‑dependent, a repeat dose study design using a combination of microtracer radioactivity and high radioactivity doses was employed to evaluate the metabolism and excretion of emvododstat near steady state. Seven healthy male subjects each received 16 mg/0.3 µCi 14C-emvododstat daily oral doses for 6 days followed by a 16 mg/100 µCi high radioactivity oral dose on Day 7. Following the last 16 mg/0.3 µCi 14C‑emvododstat dose on Day 6, total radioactivity in plasma peaked at 6 h post-dose. Following a high radioactivity oral dose (16 mg/100 µCi) of 14C-emvododstat on Day 7, both whole blood and plasma radioactivity peaked at 6 h, rapidly declined from 6 h to 36 h post-dose, and decreased slowly thereafter with measurable radioactivity at 240 h post-dose. The mean cumulative recovery of the administered dose was 6.0% in urine and 19.9% in feces by 240 h post-dose, and the mean extrapolated recovery to infinity was 37.3% in urine and 56.6% in feces. Similar metabolite profiles were observed after repeat daily microtracer radioactivity oral dosing on Day 6 and after a high radioactivity oral dose on Day 7. Emvododstat was the most abundant circulating component, M443 and O-desmethyl emvododstat glucuronide were the major circulating metabolites; M474 was the most abundant metabolite in urine, while O‑desmethyl emvododstat was the most abundant metabolite in feces. Significance Statement This study provides a complete set of the absorption, metabolism and excretion data of emvododstat, a potent inhibitor of dihydroorotate dehydrogenase, at close to steady state in healthy human subjects. Resolution of challenges due to slow metabolism and elimination of a lipophilic compound highlighted in this study can be achieved by repeat daily microtracer radioactivity oral dosing followed by a high radioactivity oral dosing at therapeutically relevant doses.

Targeting DHODH reveals therapeutic opportunities in ATRA-resistant acute promyelocytic leukemia

Although all-trans retinoic acid (ATRA)-induced differentiation has transformed acute promyelocytic leukemia (APL) from the most fatal to the most curable hematological disease, resistance to ATRA in high-risk APL patients remains a clinical challenge. In this paper, we discovered that dihydroorotate dehydrogenase (DHODH) inhibition overcame ATRA resistance. 416, a potent DHODH inhibitor previously obtained in our group, inhibited the occurrence of APL in cells and model mice. Excitingly, 416 effectively overcame ATRA resistance in vitro and in vivo by inducing apoptosis and differentiation. Further mechanistic studies showed that PML/RARα lost the regulation of Bcl-2 and c-Myc in NB4-R1 cells, which probably contributed to ATRA resistance. Notably, 416 maintained its Bcl-2 and c-Myc down-regulation effect in NB4-R1 cells and overcome ATRA resistance by inhibiting DHODH. In conclusion, our study highlights the potential of 416 for APL therapy and overcoming ATRA resistance, supporting the further development of DHODH inhibitors for clinical use in refractory and relapsed APL.

Identification of a dihydroorotate dehydrogenase inhibitor that inhibits cancer cell growth by proteomic profiling.

Dihydroorotate dehydrogenase (DHODH) is a central enzyme of the de novo pyrimidine biosynthesis pathway and is a promising drug target for the treatment of cancer and autoimmune diseases. This study presents the identification of a potent DHODH inhibitor by proteomic profiling. Cell-based screening revealed that NPD723, which is reduced to H-006 in cells, strongly induces myeloid differentiation and inhibits cell growth in HL-60 cells. H-006 also suppressed the growth of various cancer cells. Proteomic profiling of NPD723-treated cells in ChemProteoBase showed that NPD723 was clustered with DHODH inhibitors. H-006 potently inhibited human DHODH activity in vitro, whereas NPD723 was approximately 400 times less active than H-006. H-006-induced cell death was rescued by the addition of the DHODH product orotic acid. Moreover, metabolome analysis revealed that H-006 treatment promotes marked accumulation of the DHODH substrate dihydroorotic acid. These results suggest that NPD723 is reduced in cells to its active metabolite H-006, which then targets DHODH and suppresses cancer cell growth. Thus, H-006-related drugs represent a potentially powerful treatment for cancer and other diseases.

Absorption, distribution, metabolism and excretion of 14C-Emvododstat following a single oral dose in rats and dogs

Emvododstat is a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of acute myeloid leukaemia and COVID-19. Following an oral dose administration in Long-Evans rats, 14C-emvododstat-derived radioactivity was widely distributed throughout the body, with the highest distribution in the endocrine, fatty, and secretory tissues and the lowest in central nervous system. Following a single oral dose of 14C-emvododstat in rats, 54.7% of the dose was recovered in faeces while less than 0.4% of dose was recovered in urine 7 days post-dose. Emvododstat was the dominant radioactive component in plasma and faeces. Following a single oral dose of 14C-emvododstat in dogs, 75.2% of the dose was recovered in faeces while 0.5% of dose was recovered in urine 8 days post-dose. Emvododstat was the dominant radioactive component in faeces, while emvododstat and its two metabolites (O-desmethyl emvododstat and emvododstat amide bond hydrolysis product) were the major circulating radioactivity in dog plasma.

Preclinical Development of Hosu-53, a Novel Potent Dihydroorotate Dehydrogenase Inhibitor with Impressive Single Agent Efficacy and Combination Approaches in Hematological Malignancy

Common FDA approved strategies for hematological malignancies include chemotherapy, targeted therapies, immune therapies, hypomethylating agents, and cell-based therapeutics. However, recurrence often occurs resulting in poor prognosis particularly in acute myeloid leukemia (AML) with first relapse and multiple myeloma (MM) following several relapses based upon more therapies being available. The emergence of dihydroorotate dehydrogenase as an appealing mechanistic target in AML has propelled the development of several clinical candidate DHODH inhibitors (DHODHi) such as brequinar. Here we describe HOSU-53, a novel DHODHi with favorable oral bioavailability (>50% in mouse, rat, and dog) and a pharmacodynamic (PD) biomarker for target engagement, enabling clinical in vivo monitoring of potency and on-target anti-tumor activity. Here we describe the development of a series of DHODHi through a collaboration between Hendrix College and The Ohio State University (OSU). Our initial screen of 18 novel compounds synthesized at Hendrix College identified HOSU-3 as a potent DHODHi with efficacy equivalent to brequinar, however in vivo efficacy in a MOLM-13 AML xenograft model revealed further optimization would be required to compete with leading clinical candidates. Further structure activity relationship studies and lead optimization performed in collaboration with the Drug Development Institute at OSU led to screening over 200 additional analogs and the development of three additional compounds (HOSU-28, -49 and -53). HOSU-53 has subnanomolar biochemical activity against human DHODH in a cell-free enzyme assay, with a cellular IC50 ranging from 2 - 45 nM across a panel of AML cell lines, and 12-42 nM in MM cell lines. While HOSU-53 is highly plasma protein bound, it exhibits a moderate serum shift in assays using human plasma proteins with an IC50 of 457 nM, well below the in vivo Cmax and AUC achieved in rodent and canine pharmacokinetic (PK) studies (Cmax = 79.4-210 μM and AUC = 997-7961 μM-h). Additional absorption, distribution, metabolic and excretion (ADME) and safety studies revealed that HOSU-53 displays very favorable drug-like properties sufficient for development as a pre-clinical candidate. A MOLM-13 xenograft study verified that HOSU-53 was a potential new best in class DHODHi, with efficacy superior to the Bayer clinical candidate. We performed a follow-up study to determine the efficacy of dosing daily at 4, 10, and 20 mg/kg versus 30 mg/kg twice a week. Correlative PK/PD studies were also conducted to measure plasma HOSU-53 and DHO levels at day 1 and 14. We observed a dose linear relationship between HOSU-53 plasma concentration and DHO accumulation. Collectively, our efficacy and PK/PD studies indicate that 10 mg/kg is a highly efficacious and tolerable daily regimen, while higher doses can be safely administered intermittently, and DHO accumulation serves as an important plasma biomarker to identify in vivo therapeutic responses and toxicity. Confirmatory studies in rat subcutaneous MOLM-13 and MV4-11 xenograft models further demonstrated the efficacy of HOSU-53. Our group has previously described synergy between HOSU-53 and decitabine in AML, as well as monoclonal antibodies directed to CD38 in AML and MM. Furthermore, a novel combination between HOSU-53 and CD47 antibody is described in a separate abstract at this current meeting. We expanded our combination studies by testing HOSU-53 with another standard of care, the FLT3 inhibitor gilteritinib, using the MOLM-13 FLT3-ITD mutant xenograft model. NCG mice were enrolled ten days post engraftment to receive daily 4 mg/kg HOSU-53, 30 mg/kg gilteritinib or the combination. Single agent HOSU-53 and gilteritinib prolonged survival (median survival of 37 and 47 days, respectively) while the combination significantly improved survival with 9/10 mice in the combination group remaining on study at day 80. Collectively, we provide a thorough preclinical assessment for HOSU-53 for treatment in hematological malignancy both as a monotherapy and combined with other effective approved therapies including decitabine, monoclonal antibodies and gilteritinib. We have initiated IND enabling studies including GLP toxicity studies planned for late 2022 and have initiated human dose projection models based on our multi-species PK/PD analysis. We anticipate filing an IND application in 2023 for a phase 1 clinical trial in AML and MM.

Hosu-53, a Novel Potent Dihydroorotate Dehydrogenase Inhibitor Accentuates CD47 Blockade Therapy Resulting in Long Term Survival in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a complex disease characterized by leukemia stem cells in a state of differentiation arrest leading to suboptimal innate immune function and immunosuppression. The effectiveness of allogeneic hematopoietic stem cell transplantation, NK cell directed therapies, and recently CD47 blockade highlights the importance of immune surveillance as potentially curative therapies for AML. Dihydroorotate dehydrogenase inhibitors (DHODHi) have been reported to have both cytotoxic and differentiation properties which propelled their clinical development for AML. We developed HOSU-53, a novel DHODHi which is described in detail in another abstract at this meeting. HOSU-53 has (i) nanomolar potency against human DHODH biochemical activity, (ii) in vitro anti-proliferative activity toward AML with a potent tumor DNA damage response, (iii) favorable oral bioavailability, (iv) a monitorable on-target pharmacodynamic biomarker predictive of efficacy and toxicity, and (v) superior in vivo survival outcome in disseminated AML cell line derived xenograft models. Prior anti-viral studies using DHODHi have shown that deprivation of pyrimidine synthesis and associated DNA damage response can enhance innate immune activation. Therefore, we hypothesized that HOSU-53 could enhance phagocytosis and antibody dependent cellular phagocytosis (ADCP) and thereby synergize with myeloid immune checkpoint blockade therapies. Herein, we report compelling preclinical data demonstrating curative potential in AML xenograft models. Treating AML cells with HOSU-53 resulted in restored ability to phagocytose E.coli suggesting enhanced innate immunity. Given our previous reports that DHODHi could induce cell surface markers such as CD38, we next validated that HOSU-53 can increase expression of calreticulin (CALR) and CD47. In vitro treatment with HOSU-53 demonstrated increased tumor surface expression of the pro-phagocytic signal, CALR and a dose dependent increase in CD47 expression. We performed ADCP assays using murine bone marrow derived macrophages and observed enhanced phagocytosis when AML cells were pretreated with HOSU-53, prompting us to conduct in vivo validation using the MOLM-13 disseminated model. Mice were enrolled four days post-engraftment to receive single agent anti-CD47 monoclonal antibody at 0.5mg daily for 21 days and either daily 4 or 10 mg/kg HOSU-53, based on our prior dose optimization studies. We also investigated the combination of CD47 antibody with each dose level of HOSU-53. All monotherapy cohorts resulted in significant prolonged survival, but still eventually succumb to AML disease. In contrast, the combination cohorts resulted in disease-free survival as determined by the absence of measurable human CD45+ cells in spleen or bone marrow cells harvested from mice at the end of treatment on day 80, or on day 106 after remaining on study for 25 days without continued treatment. The curative potential in this model prompted us to repeat our study in a model with higher tumor burden. In this follow-up study, mice were enrolled ten days post engraftment to receive daily HOSU-53 (4 or 10 mg/kg) or B6H12 anti-CD47 (0.5mg daily or every three days for 21 days) or combinations of these treatments. We observed that anti-CD47 monotherapy was ineffective with this more established tumor model, but HOSU-53 monotherapy was comparable to our previous study and both 10 mg/kg combination cohorts significantly prolonged survival (Fig. 1, ongoing study). Further mechanistic studies of HOSU-53-mediated synergy of CD47 therapy are ongoing. Herein, using our DHODHi HOSU-53, we describe that pyrimidine depletion can enhance innate immunity to potentiate CD47 blockade therapy, representing a novel treatment approach. We show HOSU-53 increased tumor innate immune function, CALR expression and subsequently increased in vitro phagocytosis with CD47 blockade. Furthermore, in vivo combination of HOSU-53 with anti-CD47 antibody promoted dramatic synergy exhibiting a potentially curative approach in an otherwise very aggressive tumor model. HOSU-53 is currently in the IND-enabling studies phase with plans for filing an IND application in 2023 for phase 1 clinical trials in AML and MM. This data and our previously reported combination of HOSU-53 with anti-CD38 antibodies warrants the clinical translation of DHODHi in combination with immunotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SAR studies toward discovery of emvododstat (PTC299), a potent dihydroorotate dehydrogenase (DHODH) inhibitor

Dihydroorotate dehydrogenase (DHODH) is the enzyme that catalyzes a rate-determining step during the de novo synthesis of uridine, an important source of cellular pyrimidine nucleotides. Ability to modulate the activity of this enzyme may be used to control diseases associated with rapid, out-of-control cell growth in oncology, immunology, and virology. Emvododstat (PTC299) is a tetrahydro-β-carboline DHODH inhibitor discovered through the GEMS technology (Gene Expression Modulation by Small-Molecules). Described in this paper is the lead optimization campaign that culminated in the discovery of this highly potent DHODH inhibitor.

CML-164 Dihydroorotate Dehydrogenase Inhibition Reveals Metabolic Vulnerability in Chronic Myeloid Leukemia

There are chronic myeloid leukemia (CML) patients who show resistance to TKI therapy and are prone to progress to more advanced phases of the disease, and new therapeutic approaches are needed. Our study shows that CML cells are vulnerable to dihydroorotate dehydrogenase (DHODH) inhibition mediated by Meds433, a potent DHODH inhibitor developed by our group. Pyrimidine supply in resting and differentiated cells primarily relies on the salvage pyrimidine pathway, which is energetically affordable. This level of nucleotide production for fast-proliferating leukemic cells is insufficient, and they have to fulfill their needs via the de novo pathway. In this study, we shed light on the role of DHODH inhibition in CML and how it can be a promising approach for targeting leukemic cells. Sixty bone marrow samples and peripheral blood of newly diagnosed CML patients and 4 TKI-resistant patients were collected. In-vivo, in-vitro, and ex-vivo experiments were performed on primary CML CD34+ and various CML cell lines. Our data showed that DHODH is highly active in CML stem/progenitor cells, which supports a high proliferation capacity. Meds433, by targeting DHODH enzyme activity, induced apoptosis, cell growth, and cell cycle arrest in leukemic cells. Meanwhile, the administration of Meds433 reduced tumor growth and tumor burden in treated mice. Interestingly, the addition of exogenous uridine rescued all of the biological effects caused by DHODH inhibition, demonstrating the selectivity of Meds433. Based on RNA-seq data, most upregulated gene sets were related to apoptosis and immune response and most downregulated gene sets were related to MYC targets and metabolism pathways, which was confirmed by metabolic profile analysis. Also, we found that glutamic pyruvic acid transaminase 1 (GPT1) is among the top downregulated genes after treating CML cells with Meds433, and overexpressing GPT1 in CML cells interfered with the effect of Meds433. These data show that GPT1 downregulation could be one of the possible mechanisms in which Meds433 acts in CML. Our study shows that DHODH inhibition is a promising approach for targeting CML stem/progenitor cells and may help more patients discontinue the therapy. This work was supported by Associazione Italiana per la Ricerca sul Cancro: IG-23344.

Brequinar and dipyridamole in combination exhibits synergistic antiviral activity against SARS-CoV-2 in vitro: Rationale for a host-acting antiviral treatment strategy for COVID-19

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and the associated global pandemic resulting in >400 million infections worldwide and several million deaths. The continued evolution of SARS-CoV-2 to potentially evade vaccines and monoclonal antibody (mAb)-based therapies and the limited number of authorized small-molecule antivirals necessitates the need for development of new drug treatments. There remains an unmet medical need for effective and convenient treatment options for SARS-CoV-2 infection. SARS-CoV-2 is an RNA virus that depends on host intracellular ribonucleotide pools for its replication. Dihydroorotate dehydrogenase (DHODH) is a ubiquitous host enzyme that is required for de novo pyrimidine synthesis. The inhibition of DHODH leads to a depletion of intracellular pyrimidines, thereby impacting viral replication in vitro. Brequinar (BRQ) is an orally available, selective, and potent low nanomolar inhibitor of human DHODH that has been shown to exhibit broad spectrum inhibition of RNA virus replication. However, host cell nucleotide salvage pathways can maintain intracellular pyrimidine levels and compensate for BRQ-mediated DHODH inhibition. In this report, we show that the combination of BRQ and the salvage pathway inhibitor dipyridamole (DPY) exhibits strong synergistic antiviral activity in vitro against SARS-CoV-2 by enhanced depletion of the cellular pyrimidine nucleotide pool. The combination of BRQ and DPY showed antiviral activity against the prototype SARS-CoV-2 as well as the Beta (B.1.351) and Delta (B.1.617.2) variants. These data support the continued evaluation of the combination of BRQ and DPY as a broad-spectrum, host-acting antiviral strategy to treat SARS-CoV-2 and potentially other RNA virus infections.

Identification of second generation benzylidene chromanone analogues as novel, potent DHODH inhibitors in acute myeloid leukemia cells

Dihydroorotate dehydrogenase (DHODH) remains as an active target at the preclinical level against acute myeloid leukemia (AML). Herein we report potent second generation benzylidene chromanone (SBL-105) analogues to inhibit DHODH in AML cells. Virtual docking and molecular dynamic simulations were performed. Human-recombinant (rh)DHODH, THP-1, TF-1 and HL-60 cell lines were used. MTT assay was used for cell viability. Flow cytometry was used for differentiation analysis. Computational modeling and simulations predict, SBL-105 analogs bind efficiently to DHODH with improved binding energies. While all tested analogues of SBL-105 inhibited rh DHODH enzyme, SBL-105-4 and SBL-105-6 more effectively inhibited rh DHODH with an IC50 value of 3.62 and 13.61 nM respectively. SBL-105-4 exhibited excellent anti proliferative effects against THP-1, TF-1 and HL-60 cells with GI50 values of 18.78, 38.11 and 63.83 nM respectively. A similar effect was also observed in SBL-105-6 treated AML cells with respective GI50 values of 34.56, 44.40 and 38.65 nM in THP-1, TF-1 and HL-60 cells. An increase in apoptotic populations were enumerated in all three AML cells. Both these compounds also increased the differentiation marker CD11b positive populations in all the three AML cells tested. In conclusion, SBL-105-4 and SBL-105-6 were identified as potent second generation DHODH inhibitors, which drives attention for further preclinical developments. Communicated by Ramaswamy H. Sarma

Abstract 5492: Activity of RP7214, a novel, selective, and potent small molecule inhibitor of DHODH, in AML

Abstract Background: Dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme in pyrimidine synthesis, is overexpressed in certain cancers and impedes tumor cell proliferation via cell cycle arrest. RP7214 is a novel and potent DHODH inhibitor that inhibited DHODH activity in enzyme and PHA induced HWB/PBMC proliferation with IC50 and EC50 values of 7.8 & 2.5/0.60 nM respectively. Herein, we describe the efficacy of RP7214 as a single agent and in combination with standard of care drugs in preclinical models of AML Methods: Cell viability of AML cell lines (U937, HL-60, THP-1, and KG-1) following incubation with RP7214 as a single agent or in combination with Azacytidine or Venetolcax for 72 h was determined in an MTT-based assay. Cell surface expression of CD11b in THP-1 and human primary bone marrow AML mononuclear cells in presence of RP7214 was evaluated by flow cytometry. Apoptosis was evaluated following incubation of cell lines with compound for 48 or 72 h, subsequent staining with Annexin-V-PE and 7-AAD, and analysis by flow cytometry. Anti-tumor potential of RP7214 as single agent was tested in subcutaneous MV-4-11 human leukemia xenograft model. Results: RP7214 inhibited AML cell proliferation in all the four cell lines tested with GI50 ranging from 2.4 to 7.6 µM. Expression of the CD11b marker on THP-1 cell surface was enhanced by 40% following incubation with 5 µM compound; effect of which was reduced to 15% in the presence of 300 µM Uridine. Incubation of primary human bone marrow AML mononuclear cells with 10 µM of RP7214 increased the CD11b+ cells by > 15 %. Combination of 3 µM-RP7214 and 1 µM-Azacytidine inhibited cell proliferation by > 25% (P < 0.05) compared to single agents in MV-4-11 cells. Similarly, combination of 3 µM-RP7214 with 3 nM Venetoclax demonstrated 15% (P < 0.05) inhibition of cell growth compared to single agents in MV-4-11 cell line. RP7214 at 3, 10, and 30 mg/kg BID demonstrated significant anti-tumor activities in both tumor size and tumor weight in subcutaneous MV-4-11 human leukemia xenograft model. Conclusions: Results demonstrate the utility of RP7214 as a single agent and in combination with standard of care drugs in inducing apoptosis and promoting differentiation to fully mature cells in AML. A Phase 2a trial in AML patients is planned. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Swaroop Vakkalanka. Activity of RP7214, a novel, selective, and potent small molecule inhibitor of DHODH, in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5492.

Abstract 4049: Preclinical pharmacology profile of GTX-0196: A novel, potent and highly selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of hematologic malignancies

Abstract Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the de novo pyrimidine synthesis [Löffler et al., 1997]. Initial clinical studies with the DHODH inhibitor brequinar in solid tumors showed limited efficacy due to dose limiting adverse effects [Peters et al, 2018]. Recently, there is renewed interest in the use of more potent and selective DHODH inhibitors for the treatment of myeloid and lymphoid malignancies [Sykes et al, 2016]. Here we report the identification of the novel potent and highly selective DHODH inhibitor GTX-0196, with excellent physicochemical properties. GTX-0196 exhibited a biochemical IC50 of 3.7 nM in a DHODH biochemical assay and an antiproliferative IC50 of 1.1 nM in the MOLM-13 cell line, with brequinar showing IC50 values of 6.9 and 50 nM, respectively. The activity in the MOLM-13 proliferation assay was uridine-dependent, indicating absence of general toxicity of the compound and confirming DHODH-mediated efficacy. Profiling of GTX-0196 in the BioPrint panel (Eurofins/CEREP, at 10 µM), showed <50% inhibition for all 133 target proteins tested (GPCR’s, NR´s, kinases and other enzymes), illustrating the high selectivity for DHODH (> 2000-fold selectivity versus other targets tested). Furthermore, no inhibition was observed in a human CYP panel (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4, at 30 µM) and in a panel of transporters (at 10 µM). High metabolic stability in human liver microsomes and hepatocytes was observed with t½ > 6h and 10h, respectively. Compared to an earlier lead compound, GTX-0196 demonstrated enhanced potency and metabolic stability (particularly in preclinical species). Potent and efficacious inhibition of proliferation through DHODH was confirmed in a large number of solid tumor and hematologic malignancy cell lines, illustrating the broad potential use of this chemical series. Furthermore, tumor growth inhibition was observed in the MOLM-13 xenograft mouse model. Due to its favorable properties, GTX-0196 has progressed towards IND-enabling studies. In parallel, we are exploring mono and combination therapy opportunities in hematological malignancies as well as combination therapy opportunities in solid tumors to investigate the full therapeutic potential in oncology for this optimized DHODH inhibitor. Löffler et al. Mol. Cell. Biochem. 1997Peters GJ et al, Nucleosides, Nucleotides and Nucleic Acids 2018Sykes DB et al. Cell 2016 Citation Format: Allard Kaptein, Sonia Lain, Diede Brunen, Edward van Wezel, Tjeerd Barf. Preclinical pharmacology profile of GTX-0196: A novel, potent and highly selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4049.

Dihydroorotate dehydrogenase inhibition reveals metabolic vulnerability in chronic myeloid leukemia

The development of different generations of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has led to the high overall survival of chronic myeloid leukemia (CML) patients. However, there are CML patients who show resistance to TKI therapy and are prone to progress to more advanced phases of the disease. So, implementing an alternative approach for targeting TKIs insensitive cells would be of the essence. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the de novo pyrimidine biosynthesis pathway that is located in the inner membrane of mitochondria. Here, we found that CML cells are vulnerable to DHODH inhibition mediated by Meds433, a new and potent DHODH inhibitor recently developed by our group. Meds433 significantly activates the apoptotic pathway and leads to the reduction of amino acids and induction of huge metabolic stress in CML CD34+ cells. Altogether, our study shows that DHODH inhibition is a promising approach for targeting CML stem/progenitor cells and may help more patients discontinue the therapy.

A novel and potent dihydroorotate dehydrogenase inhibitor suppresses the proliferation of colorectal cancer by inducing mitochondrial dysfunction and DNA damage

AbstractDihydroorotate dehydrogenase (DHODH) is a quite attractive target in cancer therapy. Nevertheless, the antitumor effects of DHODH inhibitors against colorectal cancer (CRC) and the underlying mechanism have seldom been studied. Here, we explored the anti‐CRC efficacy of M62, a novel and potent DHODH inhibitor. In the study, M62 significantly inhibited the proliferation of CRC cells and induced S phase arrest. The antiproliferative effects caused by M62 were rescued by uridine supplementation. Mechanistically, messenger RNA sequencing results showed that M62‐triggered gene changes related to mitochondrial dysfunction, DNA damage, and DNA damage repair. Further validation showed that M62 treatment induced the generation of reactive oxygen species and decreased ΔΨm and ATP production. Meanwhile, M62 induced the accumulation of γ‐H2AX and longer comet tail moment, which were both markers of DNA damage. The downregulated DNA repair proteins and PI3K/ATK pathway were observed after M62 treatment. Furthermore, M62 significantly inhibited CRC xenograft tumor growth without detectable toxicity. Therefore, we conclude that M62 inhibits CRC growth both in vitro and in vivo by causing mitochondrial dysfunction and DNA damage, suggesting that DHODH inhibitors are potential therapeutic strategies for treating CRC.

Emvododstat, a Potent Dihydroorotate Dehydrogenase Inhibitor, Is Effective in Preclinical Models of Acute Myeloid Leukemia.

Blocking the pyrimidine nucleotide de novo synthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH) results in the cell cycle arrest and/or differentiation of rapidly proliferating cells including activated lymphocytes, cancer cells, or virally infected cells. Emvododstat (PTC299) is an orally bioavailable small molecule that inhibits DHODH. We evaluated the potential for emvododstat to inhibit the progression of acute myeloid leukemia (AML) using several in vitro and in vivo models of the disease. Broad potent activity was demonstrated against multiple AML cell lines, AML blasts cultured ex vivo from patient blood samples, and AML tumor models including patient-derived xenograft models. Emvododstat induced differentiation, cytotoxicity, or both in primary AML patient blasts cultured ex vivo with 8 of 10 samples showing sensitivity. AML cells with diverse driver mutations were sensitive, suggesting the potential of emvododstat for broad therapeutic application. AML cell lines that are not sensitive to emvododstat are likely to be more reliant on the salvage pathway than on de novo synthesis of pyrimidine nucleotides. Pharmacokinetic experiments in rhesus monkeys demonstrated that emvododstat levels rose rapidly after oral administration, peaking about 2 hours post-dosing. This was associated with an increase in the levels of dihydroorotate (DHO), the substrate for DHODH, within 2 hours of dosing indicating that DHODH inhibition is rapid. DHO levels declined as drug levels declined, consistent with the reversibility of DHODH inhibition by emvododstat. These preclinical findings provide a rationale for clinical evaluation of emvododstat in an ongoing Phase 1 study of patients with relapsed/refractory acute leukemias.