Author: Shencheng Hu Ge
The protein arginine methyltransferase PRMT5 plays an important role in maintaining cell homeostasis.
By catalyzing the symmetric dimethylation of arginine in more than 100 nuclear proteins and cytoplasmic proteins, PRMT5 is involved in gene expression and nuclear protein bodies.
Biosynthesis, mRNA splicing, protein translation, DNA damage response and immune function regulation; PRMT5 is also an essential gene, its complete inactivation is incompatible with the survival of most cell lines
PRMT5 has been shown to have carcinogenic functions, and its expression level is dysregulated in many solid and hematological malignancies.
This event is related to advanced disease and poor clinical outcomes; therefore, PRMT5 is an attractive drug target in oncology , Potent and selective inhibitors of the catalytic pocket have been developed and are in clinical trials
In view of the important role of PRMT5 in normal tissue homeostasis, PRMT5 inhibition may have a limited therapeutic window, and there is currently no molecule on the market for this target
In a previous article, related reports on allosteric inhibitors, covalent inhibitors, PROTACS, and Amgen's strategy of choosing "synthetic lethality" were also discussed.
This article analyzes the other aspects of Merck's choice of PRMT5 development strategy.
One route-dual inhibitors
Merck's dual suppression strategyMerck's dual suppression strategy
In 2021, Merck reported in JMC magazine a class of 5,5-bicyclic nucleoside PRMT5 inhibitors for the treatment of cancer; activated PRMT5 and aptamer MEP50 form a heterooctamer, and use S-gland Glycosylmethionine (SAM) acts as a cofactor to transfer the methyl group to the substrate arginine (see Figure 1);
Figure 1 Combination mode of SAM (green, PDB 4X61) and PRMT5, the yellow dotted line shows the main effect
The active site of the enzyme consists of a conserved SAM binding pocket and a nearby protein substrate binding pocket.
The two binding pockets are connected by a short and narrow channel in the enzyme; in addition to SAM, the endogenous ligand methyl group Thioadenosine (MTA) also binds to and inhibits PRMT5 in a way that SAM competes.
In view of the structural characteristics of the catalytic site of PRMT5, there are three most common types of inhibitors: including SAM competition, substrate competition, and dual SAM/substrate Substance competition inhibitor; dual SAM/substrate competition inhibitors occupy the SAM pocket and extend to the narrow groove connecting the substrate protein pocket.
This mechanism of inhibiting the binding of SAM, substrate and PRMT5 enzyme can be used to design inhibitors, This strategy can improve the efficacy and extend the residence time of the compound
The goal of Merck's development is: potent, highly selective, high oral bioavailability and low predicted human administration dose of PRMT5 inhibitors of nucleoside derivatives
In Figure 1, the diol in the SAM structure forms hydrogen bonds with Tyr324 and Glu392, the purine C6-amino group is hydrogen bonded with Asp419, and the purine N1-nitrogen hydrogen bond forms a hydrogen bond with the main chain amide-NH of Met420
Based on these key binding information, Merck restructured the SAM mimetic compound JNJ64619178 previously developed.
It contains an aminoquinoline fragment.
Modeling shows that it can extend from the SAM binding pocket to the peptide binding site channel; aminoquinoline It is specially simulated to form a bidentate hydrogen bond with Glu444, similar to the arginine residue of the substrate, so it can prevent the binding of PRMT5 protein to SAM and substrate, achieving the purpose of dual inhibitor (SAM/substrate)
Merck's compound design aims to develop new nucleosides and substituents that can interact with glu444
A methyl group was introduced into the alpha position of the hydroxyl group on the five-membered ring to obtain analog 3 of JNJ64619178 (Figure 2).
The EC50 obtained by the PRMT5 target engagement (TE) assay was 4 nM, indicating that the hydroxyl position here can tolerate steric hindrance.
The compound obtained by further conformational constrained cyclization and release of a part of the tension has an EC50 of 1nM
According to the X-ray crystal structure of compound 4 (Figure 2B), the nucleoside diol and base retain the key hydrogen bond interaction that mimics the adenosine part of SAM (as a SAM inhibitor).
In addition, the aminoquinoline part can indeed be extended To the pocket connecting SAM and the substrate, it forms a face-to-face π-stacking interaction with Phe327, and forms a bidentate hydrogen bond with Glu444 (as a substrate inhibitor), all of which confirm that 4 is a double competitive inhibitor
Figure 2 (A) Designed 5,5 bicyclic skeleton by fusion of 3'and 4'; (B) several key nucleoside binding pharmacophores of 4
Figure 3 SAR of 5, 5-bicyclic ribose
5, 5-Bicyclic ribose SAR discussion: Merck mainly investigated the structure-activity relationship of bicyclic ribose when oxygen and methylene are used as the linking chain, and replaced the NH2 of purine with methyl, cyclopropyl, and H groups.
In the amino quinoline fragment, the ortho positions of the amino group were investigated for different halogen substitutions and methyl and H substitutions.
It can be seen from Figure 3 that compounds 37, 44, and 45 show excellent activity, and the amino quinoline fragments in the ortho position The influence of halogen electrical factors is greater than steric hindrance.
When fluorine is substituted (aminoquinoline nitrogen has strong basicity), it is beneficial to form bidentate hydrogen bonds with Glu444
However, the PK and solubility tests of the C6 amino substitution series show (Figure 4) that the rat bioavailability of compounds 4 and 29~33 is relatively poor, and the better compound 33 is only 28%; when the oxygen atom is a linking chain, The solubility is poor, and when the ortho position of the aminoquinoline is F substituted and the methylene group is used as the bicyclic linker, the solubility is greatly improved
Figure 4 PK and solubility of C6 -amino base analogues
In the C6-methyl base substitution series (Figure 5), the bioavailability of rats has been greatly improved, and the bioavailability of compound 35 has increased to 71%
However, there is still room for improvement in MRT (Mean Residence Time), solubility, RI (reversible inhibition) and TDI (time-dependent inhibition) of CYP3A4
Figure 5 PK and solubility of C6-methyl base analogues
The current modification has not yet obtained a compound with high affinity, good PK (high F% and long MRT> 2h), suitable solubility (≥50 μM) and low risk of CYP3A4 mediated DDI (drug-drug interaction)
Therefore, Merck has developed an all-carbon 5,5 bicyclic analogue
Figure 6 lists the data of some all-carbon 5,5 bicyclic analogs.
Comparing 66 to 68, when C6 is methyl substituted instead of amino, the bioavailability can be improved.
The solubility of this kind of all-carbon inhibitors is also very good.
In line with the trend of −F> −Cl> −Br (especially when the pH is 7); the clearance rate and MRT value of most compounds also remain within a reasonable range, although %F remains moderate, 71 and 72 show this series The potential of MRT is ≥2.
5 h (not seen in bicyclic ribose compounds).
The fluorine substitution on the quinoline ring does not help increase %F.
It may block the metabolic sites in the aminoquinoline fragments, resulting in lower Clearance and higher MRT value
Figure 6 PK and solubility of all-carbon 5,5-bicyclic analogues
Comprehensive considerations, single-digit nanomolar activity, high F%, excellent solubility, and suitable PK make this all-carbon bicyclic ring a more promising framework
The representative compound 68 also showed good off-target selectivity in off-target selectivity experiments.
All compounds in this series also have a reduced risk of CYP3A4-mediated DDI drugs because of their reversible or time-dependent inhibition Tendency to decrease
Among them, compound 72 is a very prominent compound (TDI ratio = 1.
Considering its low nanomolar activity, excellent PK (low clearance rate, >2h MRT) and consistent data in multiple species, compound 72 has balanced physical and chemical properties, and low QD (once a day) human dose Predict and minimize the risk of DDI (drug-drug interaction)
Figure 7 Human dose prediction of all-carbon bicyclic PRMT5 inhibitor (Compound 72)
Merck finally discovered compound 72, a bicyclic nucleoside analog with good PK, high solubility, low DDI risk and low predicted human dose.
It is currently in preclinical evaluation as a lead
(The JMC was received in December 2020).
Since then, Merck has still applied for at least 4 WO patents (different structure types)
Merck's related patent layoutMerck's related patent layout
Search for keywords of PRMT5 in the database of the European Patent Office, query Merck’s patents on PRMT5 inhibitors, and find 10 compound patents and one Biomarker related patent (Table 1).
Among them, patent WO2020033288 has entered China, and examples The compound in 24 is the compound 72 in the JMC.
After that, Merck still disclosed other structural types of PRMT5 inhibitors, such as imidazo[1,2-a]pyridine (4 patents in total, including two special Compound patents)
Merck's PRMT5 inhibitor related patents
The compound 1b in patent WO2021126731 is modified according to GSK-3326595 (in phase II clinical).
The introduction of two deuterium atoms may be used to block the metabolic site (the alpha position of N has benzyl hydrogen); due to the possible inhibition of PRMT5 There is a limited therapeutic window.
The author speculates that compound 72 may have a limited therapeutic window and insufficient safety.
Therefore, the new structure is still being optimized.
According to the Medical Rubik’s Cube database, currently Merck has no molecular reports for clinical trials.
There are two special compounds.
The molecules in the patent are worthy of follow-up research.
Since only the activity data is disclosed in the patent, PK data, in vivo data, and safety data are not disclosed.
The author speculates that the molecule in Example 3 in WO2021126732 is worthy of attention.
Summary and outlookSummary and outlook
The synthetic lethal inhibitors of Amgen (AMG193) and Mirati (MRTX1719) are also expected directions.
The advantages of this strategy include: highly selective targeting of cancer cells with missing MTAP genes, with little impact on normal cells , While improving the therapeutic index (TI); the dual SAM/substrate inhibitor developed by Merck is aimed at improving the efficacy and prolonging the residence time of the compound, thereby reducing the dose and increasing the TI
From this point of view, the two strategies have reached the same goal by different routes
One of the main shortcomings of the first generation PRMT5 inhibitors is the narrow therapeutic index
However, the indications for synthetic lethal inhibitors are cancers with missing MTAP genes, which are different from the indications for SAM/substrate dual inhibitors.
The differentiation of indications is also one of the key factors affecting the success of new drugs on the market.
It is expected that PRMT5 inhibitors can be marketed as soon as possible to benefit patients
Note: The original text has been deleted
Machacek etal; The Discovery of Two Novel Classes of 5,5-Bicyclic NucleosideDerived PRMT5 Inhibitors for the Treatment of Cancer; J.
2021, 64, 3911−3939;
Alessandra Ianari etal; Discovery of a First-in-Class Inhibitor of the PRMT5−Substrate Adaptor Interaction; J.
2021, 64, 11148−11168;
 Merck & Co; PRMT5 inhibitor; CN112805006 A;
 European Patent Office database;
 Medical Rubik's Cube Database;
AACR-NCI-EORTC 2021: MRTX1719: A First-in-class MTA-cooperative PRMT5 Inhibitor that Selectively Elicits Antitumor Activity in MTAP/CDKN2A Deleted Cancer Models;