Background: This article presents research that is based on the concept of using methylation and demethylation of histone (proteins in a cell’s chromatin that help condense long DNA) lysine which results in a gene being activated or being silences. KDMs are histone lysine demethylases that play important roles in many cancer models. Jumonji KDMs are a KDM family whose family members are involved in demethylation of histone-3-lysine-6 trimethyl and dimethyl (H3K9me3/H3K9me2) and also in histone-3-lysine-36 trimethyl and dimethyl (H3K36me3/H3K36me2) especially KDM4A-C. The former of the above is involved in gene transcription activation while the latter is involved in gene repression among others. The gene KDM4C overexpression is involved in esophageal and breast cancer while KDM4A in involves in chemotherapy resistance of ovarian cancer and multiple myeloma. Hence, the importance of synthesizing KDM4 inhibitors is very important.
Jumonji KDMs function in an enzymatic via the Fe and “2-oxoglutarate (2-OG)-dependent oxygenase mechanism”. The goal of the research was to develop series of KDM4 demethylase inhibitors that also possess cell antiproliferative capacity making the KDM4 inhibitors contain a carboxylic acid and also making the inhibitors perform 2-OG interactions of the demethylases.
Experimental Methods: The Experiment was set out so as to develop KDM4 inhibitors that are also antiproliferative and also inhibitors that can work pharmacologically and pharmacokinetically. To do this, the researchers first used the pyridine-2,4-dicarboxylic acid that was cocrystallized in KDM4A. With this as the base, and then considering its fragments, they came up with compound 1(the lead) which is 3-(methylamino)isonicotinic acid (Chart 1) while also exploring the potential of compound 1 in inhibiting KDM4C enzymatic action. The differences between these compounds will be talked in the results and analysis section.
Accomplishing this, the experiment proceeded to develop compound 2 by adding tetrahydronaphthalene to the methyl of 3-(methylamino)isonicotinic acid. Then, using KYSE-150 esophageal cell line for measuring antiproliferative qualities of 2, the test was performed so as to find which enantiomer of compound 2 (S or R) was the effective one1. When they found this out, the researchers then added methoxy group to the six positon of tetrahydronaphthalene to come up with 3 and then performed SAR (Structure-activity-relationship) development on it with 3 in a KDM4A site2. Then, the researchers used to intensity based Mass Spectrometry in order to analyze the engagement of compound 3 in a cell. This Mass Spectrometry was performed after treating KYSE-150 esophageal cells with compound 3, some at 2 μM for 24 hours, some at 300 nM for 96 h, and some were left untreated. These cells were then obtained and histones from these cells were purified, “subjected to in-gel chemical alkylation”, trypsin digested, and then set up for “high-resolution UHPLC-MSMS analysis”. Plotting the MS data, the researchers analyzed the histone residues H3K4, H3K9, H3K36. Obtaining the results, they then moved on to developing a mechanism of assay (MOA) in order to measure the histone mark changes. This they did by making 150 cells to overexpress KDM4C using lentiviral transfection. Then, they treated these cells at various concentrations with compound 3 and measured H3K36me3 histone at 24h using a HTRF assay. When the researchers had completed the H3K36me3 MOA, they designed three other compounds just by changing the R group at the end of tetrahydronaphthalene 6-position inorder to improve the hydrophobic interactions of the inhibitor and to have more cell permeability. They also tested the inhibiting capacity, the antiproliferative capacity, H3K36me3 MOA, and cell permeability (Table 1). When the researchers saw that compound 6 as a good inhibitor have good antiproliferative capacity, they checked its selectivity with the KDM family in general. Compound 6’s SAR was also developed
The synthesis of the drug and the steps used are detailed in the Scheme 1. Reactions like hydroboration, reduction of alkenes were used. Furthermore, the researchers also checked the inhibiting capacity of compound 6 in breast and colon cancer samples. Pharmacokinetics of the drug have also been tested in female mice. Finally, Finally, compound 6 was tested on breast cancer modeled mice where 6 was given in various dosages and the tumor volume measured. 6 was also treated on breast cancer modeled xenograft cells, and the cells that were dissociated were taken from the highest dosage (50mg/kg) and control groups and put into NSG mice. This was to measure tumor initiating cell frequency.
Results and analysis: First, Compound 1 was made to compound 2. The difference between these two compounds is the addition of the tetrahydronaphthalene ring because the researchers thought that this would give better inhibiting qualities to the drug that 1 or even the 2,4-PDCA that they started with could give. The KDM4C IC50 of 2,4-PDCA (starting fragment) was 19nM while the IC50 of 1 was 1400nM3. Hence, 2 beat both of them (IC50=12nM, KYSE-150 EC50=6 μM)2. Analyzing the enantiomers of 2 showed that the R enantiomer is the effective enantiomer with better inhibiting capacity and better antiprolifertive capacity compared to the S enantiomer (Chart 2). Compound 3 was developed by molecular modeling of 2 and by adding a methoxy group to the 6-position of tetrahydronapthalene of compound 2. SAR development of 3 showed the 2-OG interactions, carboxylic acid’s hydrogen bond interactions, and the pyridine Nitrogen’s dative bond with Ni (Fe surrogate) among others. This SAR development of 3 should’ve helped the researchers to see that compound 3 could KDM4 demethylases and participate in their mechanism4.
Before proceeding ahead to developing compound 4, the researchers conducted an MS. The MS results show the significant increase in concentrations of H3K9me3 and H3K36me3 in treated KYSE-150 cells. They also found that concentration of H3K27me3 increased and for they gave two reasons: H3K27me3 could be a KDM4 substrate or the cell could’ve become more adaptive. These results of MS show that cells were engaging with the compound as the increase in concentrations of the histones H3K9me3 and H3K36me3 suggest. An MOA was performed to check on these MS results, and with the results showing that the initial low levels of H3K36me3 by overexpression of KDM4C gene had actually increased when the transfected cells were treated, the researchers saw that MOA results were in accordance with the MS results.
Compounds 4-6 were made by changing the methoxy group at the 6th position of tetrahydonaphthalene of compound 3(Table 1). Comparing these compounds, one clearly sees that although Compound 6 was not as good as 3 in its inhibiting capacity, it certainly had most other numbers almost its favor. For example, the antiproliferative capacity of 6 was much better that 3, 4, and 5 with 6 having KYSE-150 EC50=3.5(+/-)1. In the same way, 6’s H3K36me3 MOA EC50 numbers were good and although a little lower than 5, 6’s cell permeability was good with a PAMPA permeability of 51.18 nm/s. Hence, we can clearly see why the researchers came up with 6 as their KMD4 inhibitor having a considerable anitproliferative capacity. Compound 6 was effective on the KDM4 family except KDM5B as also the other one on which 6 had significant inhibiting capacity (Table 2). Compound 6 did not have a significant inhibiting capacity on the other of KDMs and this makes compound 6 as a fairly good inhibitor targeting mainly the KDM4s. The SAR development of 6 was somewhat similar to that of 3 and this is what the researchers would’ve wanted- containing to interactions to inhibit KDM4 demethylases.
When 6 was tested on breast and colon cancer patient samples, the results were that 6 had low inhibiting concentrations of IC50 of 5 nM on breast cancer models and IC50 of 13 nM on colon cancer patients. This proves that compound 6 could prove to be an effective KDM4 inhibiting drug in breast cancer and colon cancer models. Furthermore, the experiment on pharmacokinetics of compound 6 six found that the drug has more distribution in the body when taken orally than when it was administered intravenously. When tested in vivo on breast cancer modeled mice, compound six showed that increasing dose levels suppressed tumor growth (figure 4) and 50mg/kg of 6 had more effect on tumor suppression than control or other lower doses. Finally, being tested on NSG mice, results showed that compound 6 decreased the number of cells that were needed for tumor development.
Conclusions: The authors’ conclusion was that they had developed inhibitors of KDM4 demethylases that are also antiproliferative in KYSE-150 cells. They also claim that one of their developed inhibitors -compound 6- proved to be efficacious and especially in in vivo PDX breast cancer models. This compound, they say, also reduces the tumor initiating cell frequency.
The authors’ conclusion lined up with their results. The KDM4 inhibitors they developed were effective on the histones H3K36me3 and H3K9me3 that are responsible for gene repression and transcriptional activation respectively. These results were gotten by the intensity based MS analysis they performed. Furthermore, their inhibitors, especially compound 6 had amazing antiproliferative capacity (KYSE-150 EC50=3.5(+/-)1) and cell permeability (PAMPA permeability =51.18 nm/s). They also showed in the research that compound 6 actually had an effect in vivo. This we know from the results of the experiment on breast cancer modeled mice where the increase in dosage of compound 6 resulted in less tumor volume than the control. Hence, their conclusion is verified by their results. In other words, they provided substantial evidence to their claim of developing KDM4 demethylase inhibitors with antiproliferative effects on KYSE-150 cells.
However, although the authors provide substantial evidence to their claim, they fail to clearly answer something that their results showed. Compound six showed an effect on KDM5B among the KDM family was responsive to compound 6. This was not supposed to be and yet was the case. Only KDM4s should have been responsive. Could this demethylase responsiveness which was not supposed to be something they must look into if it would help the cause of developing a more potent inhibitor? Could this be dangerous because the potential drug is having an effect on something it should not have an effect on?
Personal Takeaways: Firstly, to see the aspects of organic chemistry we’re learning is good. This article had intensity based mass spectrometry even though this mass spectrometry is different from what we learnt which plotted relative abundance against m/z ratio. Intensity based Mass spectrometry doesn’t seem to do that. Moreover, the article was bent on developing a drug that could be synthesized. This is similar to how some of us solve organic chemistry problems which is to look at the product and come to it step by step. The synthesis also included reagents and a reaction we know. It had BH3, THF in step c of synthesis scheme and also alkene reduction in step b of Scheme 1. I also saw the importance of stereochemistry because in this drug development only one enantiomer of the drug is effective and not the other. There were also some functional groups whose names only I know and not much about their function. How important is the knowledge organic chemistry in pharmaceutical company!
Finally, it is nice to read an article that so close relates to pharmaceutical industry. My mom has been part of that industry for years. Now, I got to see the some of the graphs and stats she would have to plot when working for developing a drug.