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| Targets |
PRMT5 (IC50 = 1 nM)
PRT-543 targets protein arginine methyltransferase 5 (PRMT5), an enzyme that symmetrically dimethylates arginine residues (SDMA) on various protein substrates essential for spliceosome assembly and RNA processing. The compound binds the SAM-binding pocket of the PRMT5/MEP50 complex with high affinity, confirmed by co-crystal structure at 2.5 Angstrom resolution. A key structural feature is the dichloride phenyl ring that displaces F327 and forms a unique pi-pi interaction with Y324 in the alpha1 helix, conferring selectivity. PRT543 is highly selective against 37 other methyltransferases. The primary pharmacodynamic effect is reduction of symmetrically dimethylated SmD3, a core splicing factor and PRMT5 substrate. |
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| ln Vitro |
PRT543 is a potent, selective, oral PRMT5 inhibitor with robust preclinical efficacy. As an orally available small molecule inhibitor of protein arginine methyltransferase 5 (PRMT5), PRT543 shows potent antiproliferative and antineoplastic activities. Although the exact mechanism of action has not been completely determined, it is reported that PRMT5 inhibitor PRT543 selectively binds to the substrate recognition site of PRMT5 and inhibits its methyltransferase activity. This decreases the levels of both monomethylated and dimethylated arginine residues in histones H2A, H3 and H4 and modulates the expression of genes involved in several cellular processes, including cellular proliferation. As a result, PRT543 may increase the expression of antiproliferative genes and/or decrease the expression of genes that promote cell proliferation, which may lead to decreased growth of rapidly proliferating cells, including cancer cells. PRTM5, an arginine methyltransferase that catalyzes the formation of both omega-N monomethylarginine (MMA) and symmetric dimethylarginine (sDMA) on histones and a variety of other protein substrates, is overexpressed in several neoplasms[2].
PRT-543 potently reduces the methyltransferase activity of the PRMT5/MEP50 complex in cell-free enzymatic assays. It robustly inhibits cell proliferation in a broad panel of cancer cell lines, with particular activity in splicing factor-mutant leukemia models. The compound demonstrates synthetic lethality with cells bearing splicing factor mutations (SF3B1, U2AF1, SRSF2), which are preferentially sensitive to PRMT5 inhibition compared to wild-type cells. Treatment causes dose-dependent reduction of SDMA marks and induces extensive global changes in RNA splicing, including intron retention and exon skipping. Genes affected include those involved in interferon-alpha response, MYC targets, and DNA repair pathways. |
| ln Vivo |
As of 18 June 2021, 49 unselected pts with measurable disease refractory to established therapies had enrolled (17 pts BIW, 11 pts 5x/week [wk], 21 pts QD). Median number of prior lines of systemic therapies was 3. Six of 21 pts dosed at 25-50 mg QD experienced dose limiting toxicity (DLT) of thrombocytopenia, and 1 pt of 6 dosed at 45 mg 5x/wk had DLT of fatigue. The 45 mg 5x/wk regimen was selected as the expansion dose. Increased intron retention was seen in peripheral blood mononuclear cells. A complete response (CR) has been maintained for >1 yr in a pt with homologous recombination deficiency-positive (HRD+) ovarian cancer who remains on study therapy. An additional 5 pts exhibited stable disease ≥6 mo. Dose-dependent inhibition of target engagement and functional activity of PRMT5 were observed. PRT543 demonstrated encouraging clinical activity with a CR in HRD+ ovarian cancer and prolonged stable disease in multiple pts. An expansion phase in biomarker-selected solid tumor cohorts is ongoing [1].
In vivo, PRT-543 inhibits tumor growth in AML xenograft models. A Phase I dose-escalation/expansion trial enrolled 40 patients with R/R myeloid malignancies (18 lower-risk MDS, 11 higher-risk MDS, 7 AML, 4 MDS/MPN). The recommended Phase 2 dose (RP2D) was 35 mg daily 5x/week. Clinical responses included hematologic improvement (HI-erythroid) in lower-risk MDS, marrow CR (mCR) in higher-risk MDS, and CRi with platelet transfusion independence in AML. Responding patients included those with SRSF2 mutations. Pharmacodynamic analysis demonstrated mean reduction in serum SDMA by 41.9%, confirming target engagement. Limited efficacy was partly attributed to lower peak concentrations (1.48 uM) vs preclinical models. |
| Enzyme Assay |
For PRMT5 enzyme inhibition assays, a TR-FRET or radiometric methyltransferase assay is used. Recombinant PRMT5/MEP50 complex (1-10 nM) is incubated with PRT-543 (0.1-1000 nM) in assay buffer (50 mM Tris-HCl pH 8.5, 10 mM NaCl, 0.01% Tween-20, 1 mM DTT, 0.1% BSA) containing 0.6 microM S-adenosylmethionine (SAM) and 200 nM biotinylated histone H4 peptide (residues 1-21) as substrate. After 2 hours at room temperature, europium-labeled anti-SDMA antibody and streptavidin-XL665 are added. TR-FRET signal (excitation 340 nm, emission 620/665 nm) is measured. IC50 is determined from the 665/620 ratio. The assay demonstrates PRT543 as SAM-competitive and substrate-uncompetitive.
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| Cell Assay |
For cell-based assays, leukemia cell lines (e.g., K562, MV4-11, or patient-derived splicing factor-mutant cells) are cultured in RPMI-1640 with 10% FBS at 37degC in 5% CO2. For proliferation assays, cells are seeded in 96-well plates (10,000 cells/well) and treated with PRT-543 (0.001-10 microM) for 72-120 hours. Viability is assessed by CellTiter-Glo, and IC50 values are calculated. For pharmacodynamic assessment, cells are treated with 1-10 microM compound for 24-48 hours, then lysed for Western blot analysis of SDMA (symmetrically dimethylated proteins) and SmD3 methylation. RNA is extracted for splicing analysis by RT-PCR or RNA-seq to assess intron retention and exon skipping events.
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| Animal Protocol |
This study assesses safety, pharmacokinetics, pharmacodynamics, and preliminary tumor response (REClST v1.1) of PRT543 administered at varying schedules beginning with twice weekly (BIW) and increasing to once daily (QD) with doses ranging from 5-50 mg in 28-day cycles. Dose escalation followed a 3+3 design. Serum symmetric dimethylarginine (sDMA) and intron retention, a marker of PRMT5-mediated mRNA splicing fidelity, were assessed as measures of PRMT5 target engagement and function, respectively.[1]
In vivo efficacy is evaluated in mouse xenograft models using splicing factor-mutant AML cell lines. Female NSG or nude mice are injected intravenously or subcutaneously with 5 × 10⁶ leukemia cells. For subcutaneous models, when tumors reach ~150-200 mm3, mice are randomized and treated with PRT-543 orally at 35-100 mg/kg daily or 5x/week. Tumor volume is measured every 2-3 days. For disseminated models, survival is monitored. Pharmacodynamic assessment includes plasma SDMA levels, tumor tissue SDMA by immunohistochemistry, and splicing analysis by RNA-seq. The RP2D in humans (35 mg daily 5x/week) was established based on preclinical toxicology and Phase I data. Peak plasma concentration in patients reached 1.48 uM. |
| ADME/Pharmacokinetics |
Both Cmax (nM) and AUC (nM·hr) of PRT543 increased in a dose-dependent manner. At extended doses, with a T½ of 15 hours, plasma drug exposure (Cmax of 2142 nM; AUC of 26,293 nM·hr) exceeded that of the preclinical efficacy model. Serum sDMA levels decreased in a dose-dependent manner, decreasing by 77% at a once-daily 50 mg dose and by 69% at extended doses. [1]
Pharmacokinetics of PRT-543 have been characterized in preclinical species and humans. In mice and rats, oral bioavailability is moderate, with peak concentrations (Cmax) achieved 1-3 hours post-dose. The compound has a terminal half-life (t1/2) of 4-8 hours in rodents, supporting once-daily dosing. In patients with myeloid malignancies, the recommended Phase 2 dose is 35 mg administered orally 5 days per week. Peak plasma concentrations in patients reached 1.48 uM, lower than concentrations used in preclinical cell line studies (10 microM), which may explain limited clinical efficacy. Protein binding is high (>90%). Metabolism occurs primarily via CYP450 enzymes, with excretion in feces and urine. No significant accumulation is observed with repeated dosing. |
| Toxicity/Toxicokinetics |
PRT543 was well tolerated and had a high safety profile. The most common treatment-related adverse events (TRAEs) (of any grade) across all treatment regimens were fatigue (n=20, 41%), nausea (n=14, 29%), thrombocytopenia (n=13, 27%), and anemia (n=12, 24%). Grade ≥3 TRAEs occurring in ≥5 patients across all treatment regimens included thrombocytopenia (n=10, 20%) and anemia (n=6, 12%). Cytopenia was reversible and could be managed by dose adjustment. Treatment was discontinued in 44 patients, primarily due to disease progression, and in 2 patients due to adverse events (grade 3 thrombocytopenia and grade 4 cholangitis). [1]
In the Phase I clinical trial (NCT03886831), 40 patients with R/R myeloid malignancies were treated with PRT-543 monotherapy. The most common treatment-emergent adverse events of any grade were anemia (50.0%), thrombocytopenia (30.0%), nausea (27.5%), and diarrhea (27.5%). Grade ≥3 AEs occurred in 85.0% of patients. AEs leading to treatment interruption, dose reduction, and discontinuation occurred in 40.0%, 12.5%, and 20.0% of patients, respectively. Serious AEs were documented in 32.5% of patients, all unrelated to PRT-543. In preclinical toxicology, no significant organ toxicity was reported at therapeutic doses. Thrombocytopenia is a class effect of PRMT5 inhibitors due to the role of PRMT5 in megakaryocyte differentiation. |
| References |
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| Additional Infomation |
PRMT5 catalyzes the symmetric dimethylation of arginine residues in protein substrates that play crucial roles in cancer cell growth and survival. PRT543 is a potent and selective oral PRMT5 inhibitor with significant preclinical efficacy (Bhagwat AACR 2020). An open-label Phase I study of PRT543 (NCT03886831) in unselected patients with advanced solid tumors and hematologic malignancies is ongoing. This article will present dose-escalation results in patients with solid tumors and lymphomas.
PRT-543 is an investigational drug that has completed Phase I clinical trials for splicing factor-mutant myeloid malignancies but has not received regulatory approval. The Phase I study (NCT03886831) demonstrated safety, target engagement (SDMA reduction), and preliminary efficacy including hematologic improvement, marrow CR, and CRi in a subset of heavily pretreated patients. However, clinical efficacy was modest, partly attributed to lower peak concentrations in patients vs preclinical models. Newer generation MTA-cooperative PRMT5 inhibitors are now in Phase I trials. PRT-543 has also been investigated in solid tumors. The compound demonstrated that PRMT5 inhibition disrupts RNA splicing, validating PRMT5 as a therapeutic target in splicing factor-mutant cancers. PRT-543 is available for research use only. |
| Molecular Formula |
C17H17CLN4O4
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|---|---|
| Molecular Weight |
376.79
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| Exact Mass |
376.09383
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| Elemental Analysis |
C, 54.19; H, 4.55; Cl, 9.41; N, 14.87; O, 16.98
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| CAS # |
1989620-03-2
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| PubChem CID |
122497020
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| Appearance |
White to off-white solid powder
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| Density |
1.73±0.1 g/cm3(Temp: 20 °C; Press: 760 Torr)(Predicted)
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| Boiling Point |
713.3±60.0 °C(Predicted)
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| LogP |
0.8
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
26
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| Complexity |
495
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| Defined Atom Stereocenter Count |
5
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| SMILES |
O[C@@H]1[C@@H]([C@@]([H])([C@@H](C2C=CC(Cl)=CC=2)O)O[C@H]1N1C=CC2=C(N=CN=C12)N)O
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| InChi Key |
ITEKIFMGFZAFPM-QFRSUPTLSA-N
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| InChi Code |
InChI=1S/C17H17ClN4O4/c18-9-3-1-8(2-4-9)11(23)14-12(24)13(25)17(26-14)22-6-5-10-15(19)20-7-21-16(10)22/h1-7,11-14,17,23-25H,(H2,19,20,21)/t11-,12+,13-,14-,17-/m1/s1
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| Chemical Name |
(2R,3R,4S,5R)-2-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-5-[(R)-(4-chlorophenyl)-hydroxymethyl]oxolane-3,4-diol
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| Synonyms |
PRT543; CHEMBL4585781; SCHEMBL18026293; SCHEMBL22508993; PRT-543; BDBM415556;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6540 mL | 13.2700 mL | 26.5400 mL | |
| 5 mM | 0.5308 mL | 2.6540 mL | 5.3080 mL | |
| 10 mM | 0.2654 mL | 1.3270 mL | 2.6540 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Link: https://clinicaltrials.gov/ct2/show/NCT03886831
Conditions:Relapsed/Refractory Advanced Solid Tumors|Relapsed/Refractory Diffuse Large B-cell Lymphoma|Relapsed/Refractory Myelodysplasia|Relapsed/Refractory Myelofibrosis|Adenoid Cystic Carcinoma|Relapsed/Refractory Mantle Cell Lymphoma|Relapsed/Refractory Acute Myeloid Leukemia|Refractory Chronic Myelomonocytic LeukemiaProducts are for research use only; We do not sell to patients
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