Mibefradil, a new class of compound to study TRPM7 channel function

Transient receptor potential (TRPM) is a family of non-selective cation channels that are widely expressed in mammalian cells. TRP channels are composed of six transmembrane domains and the family consists of eight different channels, TRPM1–TRPM8. TRPM7 is compromised of an ion channel moiety essential for the ion channel function, which serves to increase intracellular calcium levels and to help regulate magnesium ion homeostasis. The current hypothesis is that decreased cytosolic Mg2+ ion concentration activates the divalent cation-selective TRPM7 current 2.

Xiong and colleagues4 were the first to demonstrate TRPM7 mediated sensing of Ca2+ concentration. The permeability of TRPM7 channels to monovalent cations is decreased In the presence of divalent ions, promoting outward rectification in whole cell recording with a reversal potential of 0mV. When divalent ions were removed from the external buffer solution a large TRMP7 current was activated. The order of permeability of TRPM7 to divalent cations follows the order Zn2+> Ni2+> Ba2+> Co2+> Mg2+> Mn2+> Sr2+> Cd2+> Ca2+. Mutation of the mouse TRPM7 gene in vivo demonstrates that it is essential for early embryogenesis, organ development, cardiac automaticity, and systemic Mg2+ homeostasis. Deletion of the TRPM7 gene in cells in vitro showed that this bi-functional channel, not only regulates divalent cation homeostasis, but also cell motility, proliferation, mechanosensitivity and exocytosis. TRPM7 may also play a role in anoxic neuronal death, immune responses, hypertension, neurodegenerative disorders, tissue fibrosis and in tumour growth2,3, 4,5,. TPRM7 is clearly an important drug target.

Figure showing; Regulation of cellular Mg2+ homeostasis

Shamin 1

Andrea M.P. Romani Arch Biochem Biophys. 2011 Aug 1; 512(1): 1–23.

Schafer & colleagues1 have recently reported on mibefradil and naltriben, two new classes of compound (fig 1a) that act on 5-HT7 receptors. In the calcium imaging assay, mibefradil potentiated TRPM7 current (fig 1b) in the absence of extracellular Mg2+ and blocked the TRPM7 current in it is presence (1C). Mibefradil also induced TRPM7 mediated Ca2+ entry in a concentration dependent manner with an EC50 of 53mM (fig 1d). The Influence of intracellular Mg2+ concentration was also investigated in a whole cell e-phys study which shown that in the absence of intracellular Mg2+ the addition of mibefradil failed to increase the current further (fig 2a). When cells were perfused with the physiological concentration of free Mg2+ (0.9mM) there was a pronounced increase in the whole cell current (fig 2b). These results suggest that mibefradil preferentially activates TRPM7 responses at physiological or low intracellular Mg2+. Extracellular divalent cations are known to block the TRPM7 channel, resulting in a small divalent cation selective inward current at the physiological membrane potential. Mibefradil elicited a moderate increase of the inward current for the TMRP7 (E1047Q) mutant compared to WT channel at physiological Mg2+ concentration. Application of mibefradil, stimulate both inward as well as outward monovalent currents in this mutant (fig 3a). The authors conclude that mibefradil acts as a specific agonist of the TRPM7 channel, and proposed that mibefradil specifically affects TRPM7 channel gating in a Mg2+ – dependent manner.

Shamin 2

Sebastian Schafer et.al. (2016) Eur J Physiol 2016; 468; 623-634

Mibefradil is a calcium channel antagonist of both L and T type voltage-dependent calcium channels and has been used clinically for the treatment of angina and hypertension7. Unfortunately, side effects arising from inhibition of cytochrome p450 enzymes led to the withdrawal of mibefradil withdrawn from clinical use. Currently, mibefradil is only used experimentally as a tool compound to identify voltage gated calcium channels. Mibefradil shows moderate potency for TRPM7 activation when compared to P/L type voltage gated calcium channel, the EC50 53mM, compared to 2.7mM for T-type, and 18.6mM for L-type Ca2+ channels. Published data are consistent with mibefradil action at the TRPM7 channel (reference?). Specifically, mibefradil induces the TRPM7 –mediated influx of divalent cations such Ca2+, Zn2+ and Mg2+ which may alter cellular process.

Schafer & colleagues1 also identified naltriben as an activator of the TRPM7 channel at high-level intracellular Mg2+ levels. The authors have suggest that that two types of TRPM7 agonist act in different ways: type 1 naltribune, induces TRPM7 activity independently of Mg2+ concentration whereas type 2 Mibefradil acts in a Mg2+ -dependent manner2. Mg2+ is the most abundant intracellular cation (~15-18mM) and is mostly complexed with phosphormetabolites such that physiological levels of free Mg2+ are only in the range of 0.5-1mM. Under certain conditions, the free intracellular concentration may reduce further e.g. as a result of increasing phosphormetabolite synthesis during cell division. The authors propose that the two different types of TRPM7 activator could be directed to two different types of target; Type 1 to stimulate TRPM7 irrespective of Mg2+ concentration and Type 2 to act preferentially on cells with reduced Mg2+ levels.

Blog written by Shamim Choudhury


    1. Sebastian Schafer et.al. (2016) Mibefradil represents a new class of benzoimidazole TRPM7 channel agonists. Eur J Physiol 2016;468;623-634
    2. Andrea M.P. Romani (2011) Cellular Magnesium Homeostasis. Arch Biochem Biophys. 2011 august 1; 512(1): 1–23. Doi:10.1016/j.abb.2011.05.010.
    3. Valdimir Chubnav, silvia Ferioli, Thomas Gudermann (2017) Assessments of TRPM7 functions by drug like molecules. Call Calcium 2017;67;166-173
    4. Xiong Z, Zu W and Macdonald JF (1997) Extracellular calcium sensed by a novel cation channel in hippocampal neurons. Proc Natl Acad Sci USA 1997;94;7012-7017
    5. Yugang Sun et.al. (2015) TRPM7 and its role in neurodegenerative diseases Channels 2015; 9:5,253-261
    6. Yosuke Kaneko and Arpad Szallasi (2014) Transient receptor potential (TRP) channels : a clinical perspective British J Pharmacology 2014; 171 2474-2507
    7. S. Aczel, B. kurka, & S. Hering (1998) Mechanism of voltage and use dependent block of class Ca2+ channels by mibefradil. British J of Pharmacology 1998;125;447-45



Conference Report: RSC BMCS Mastering MedChem IV, 14th March 2018


On Wednesday 14th March 2018 I attended the RSC Biomedical and Medicinal Chemistry Sector’s 4th Mastering MedChem Symposium. The opening session chair, Professor Joe Sweeney (University of Lancaster) described the event as the sector’s “most successful new conference”. This year it was being held at my almer mater, University of Strathclyde in Glasgow.

The day was split into three sessions where a series of talks from a mixture of speakers across academia and industry shared stories of good practice in drug discovery. A variety of disease areas were covered. Prof. Stuart Conway (University of Oxford) gave a useful overview of epigenetics and bromodomain ligands. Malaria was covered by Prof. Ian Gilbert of the University of Dundee’s Drug Discovery Unit, although I was surprised to learn the project had progressed as far as it had given no mode of action for their compounds had been elucidated due to the complex nature of the malaria disease pathway.

During the lunchbreak and afternoon coffee session I was able to chat to attendees about the poster I had brought which summarised some of my PhD project involving the development of kinase inhibitors to target cancer via a synthetic lethality strategy. It was interesting discussing with other students attending with posters how they had prepared similar benzimidazole compounds to mine but for entirely different disease targets.

While I was a tad concerned there would be no conversation around kinases given the general nature of a number of the talk titles, Dr. Iain Simpson of AstraZeneca shared the story of the optimisation of a compound for a kinase project that, serendipitously, had the same core as one of my own series. It was very useful to speak to him afterwards to compare notes on dialling in selectivity for our respective targets.

There were also more generic talks about the industry and techniques for improving the efficiency of the laborious drug discovery process. Prof. Adam Nelson (University of Leeds) commented on the small pool of reactions most medicinal chemists use for preparing libraries (amide formation, Pd-catalysed couplings, alkylations etc.). He also shared a way of finding bioactive compounds faster without having to purify every single reaction run in the lab by assaying crude reaction mixtures and then only scaling up and purifying reactions with bioactive components, which he termed “activity-directed synthesis”, a rather intriguing method but must have limitations given the toxicity of many chemical reagents!

Dr. Craig Johnstone (Evotec) gave examples of case studies where focussing on multiple drug parameters at once had increased the productivity of a number of his company’s projects, instead of the scenic route most projects take to optimise individual physiochemical properties. He spoke about the potential of artificial intelligence to predict what reactions would work before heading into the lab and he also took an opportunity to justify his theory that every good drug candidate has a logD of around 2.

To round up the day, the memoriam McGuigan Lecture was given by Prof. Chris Schofield (University of Oxford) who took us on a whistle-stop tour of the development of inhibitors of serine and Zn(II) dependent beta-lactamases and their involvement in combating bacterial resistance. Due to the unfortunate absence of Dr. Nicole Hamblin from Charles River laboratories (the only female speaker in the programme I might add), an alternative Q&A was held with a panel of the speakers from the day. Attendees asked a number of questions around the feasibility of artificial intelligence in drug discovery, the importance of specialisation for career progression and a particularly useful discussion point in their careers often have to keep moving around the country, jumping from project to project, before a rare long-term lectureship or industry post arises, if at all. The panel sympathised with this view and mainly came to the conclusion that the way research is funded needs to change to give scientists more sustainable livelihoods.

I found the symposium to be a very useful experience, particularly as it was aimed at early career researchers such as myself. It was nice to be back in Scotland for a bit and I would hope to attend future Mastering MedChem meetings.

Blog written by Fiona Scott, PhD researcher, Sussex Drug Discovery Centre



This publication written by Dong-Mei Zhang et al 1 describes the investigation of serum homocysteine and its role in cognitive impairment.

The authors investigate hyperhomocysteinemia (increased levels of serum homocysteine) and its association with an increased risk of cognitive impairment. According to Sudha Seshadri et al 2, an increased plasma homocysteine level is a strong independent risk factor for the development of dementia and Alzheimer’s disease, where they say that the risk of developing Alzheimer disease doubles with a plasma homocysteine level greater than 14 μmol/L, as well as being at major risk of suffering from coronary artery disease. Homocysteine levels increase with age, with common adult levels in Western populations being 10 to 12 μmol/L.

The graph below is taken from Axis-Shield 5 who carry out in-vitro diagnostic testing. The graph shows that the higher the homocysteine level, the greater the risk of developing coronary artery disease/Alzheimer’s disease.

Kamlesh 1

Homocysteine is a homologue of the amino acid cysteine, with an extra methylene bridge. It is biosynthesised in the body through metabolism of methionine (S-demethylation). Metabolism of homocysteine is aided by vitamin B12, folic acid and vitamin B6. As well as other factors, deficiencies in these vitamins may increase serum homocysteine levels. Hyperhomocysteinemia can contribute to a greater risk of developing diseases such as cardiovascular diseases/retinal vascular disease as well as neuropsychiatric diseases. B vitamins have therefore been considered as a possible option to reduce the risk of Alzheimer’s and dementia.

The authors wanted to address the discrepancy in the existing literature on the therapeutic effect of vitamin B and folates in patients with significant cognitive deficits, secondary to Alzheimer’s disease or dementia. They carried out a meta-analysis of randomized controlled trials in elderly patients with poor cognitive ability secondary to Alzheimers or dementia, who received homocysteine lowering B vitamins supplements and had serum homocysteine levels reported.

Existing evidence on vitamin B supplement induced reduction of cognitive decline by lowering homocysteine levels is conflicting. The authors mention other studies where daily folic acid supplements were taken by people with folate deficency (Durga et al 3, 800μg/d for 3 years and Fioravanta et al 4 15mg/d for 60 days), showing an improved cognitive performance. These results were not included in the meta-analysis because the serum homocysteine levels were not reported.

The overall results suggested that folate in combination with vitamin B12 and/or B6 supplements failed to offer any significant advantage in slowing down or preventing the progression of cognitive decline, although Vitamin B supplements were shown to significantly reduce homocysteine levels.

Blog written by Kamlesh Bala

1 Dong-Mei Zhang et al, Journal of Geriatric Psychiatry and Neurology 2017, Vol 30 (1) 50-59

2 Seshadri S, Beiser A, Selhub J et al, N Engl J Med 2002; 346:476-483

3. Durga J et al, Lancet. 2007; 369 (9557): 208-216

4. Fioravanti M et al, Arch Gerontol Geriatr. 1998; 26 (1): 1-13

5. http://www.homocysteine.co.uk/measuring-homocysteine/

Keywords: Hyperhomocysteinemia, homocysteine, cognitive decline

Modular C(sp2)-C(sp3) radical cross-coupling with PT-sulfones

The construction of sp2-sp3 carbon-carbon bonds is sometimes not trivial with standard two-electron coupling reactions such as Suzuki, Heck and Negishi. To offer chemists alternative options to form these types of carbon-carbon bonds the Baran Lab have been working on metal-catalysed radical cross-coupling (RCC) reactions. I have previously written about some of their earlier work in this area. In their latest paper (Science 360, 75 –80 (2018)) they write about their discovery that a redox-active phenyl-tetrazole (PT) sulfone could be used in these RCC reactions (figure 1).

Lewis 1

Figure 1

Some PT-sulfone reagents are commercially available but others such as (3f) can be easily made from phenyl-tetrazole (6) by sulfur-carbon bond formation (alkyl halide displacement or Mitsunobu) and then oxidation of the resulting sulphide (mCPBA or ammonium molybdate/hydrogen peroxide). These sulfones are useful building blocks and can be used in RCC reactions as they are or further functionalised such as α-alkylation or α-fluorination (figure 2).

Lewis 2

Figure 2

A small set of fluorinated sulfone building blocks (8-11) were used to introduce mono/di-fluoromethyl and mono/di-fluoroethyl moieties onto a selection of aromatics (figure 3). Unfortunately, these reaction conditions are not able to install a trifluoromethyl group. Sulfones (7-11) are not currently commercially available but 100-500 mg quantities can be requested directly from the Baran lab via this link.

Lewis 3

Figure 3

Baran ran a series a competition experiments and under these reaction conditions observed the following reactivity trend Cl/Br < SO2PT < NHPI/TCNHPI (figure 4). This observation was tested with sequential chemoselective RCC reactions. Firstly, a decarboxylative cross-coupling (DCC) was performed followed by a desulfonylative cross-coupling (SCC) (figure 5).

Lewis 4

Figure 4

Lewis 5

Figure 5

These new reagents and chemoselective reaction conditions offer a simple and general method to add to the tool box of sp2-sp3 carbon-carbon bond forming reactions. The ability to diversify and fluorinate a common building block will increase the interest from medicinal chemists as will the ability to introduce simple alkyl fluorides without the use of harsh reaction conditions or toxic reagents.

Blog written by Lewis Pennicott


A Brief Comparison of Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC)

  1.  MST

MST assay is based on thermophoresis, the directed movement of molecules in a temperature gradient induced by an infrared laser. Thermophoresis is highly sensitive to all types of binding-induced changes, such as size, charge, hydration shell or conformation, which allows for a precise quantification of molecular events. (Jerabek-Willemsen, André et al. 2014) Initial, the molecules are distributed homogenously with an initial fluorescence signal. When the IR laser is activated, the fluorescent signal is decreased as a ‘T-Jump’ form. With the turnoff of IR-laser, the molecule diffusion is back, solely driven by mass diffusion. The trace difference between a fluorescent molecule binding with or without non-fluorescent ligands indicates a binding signal. (Figure 1)

MST can handle weak conformation change on binding of two molecules in different buffer system, such as biological liquids. Another advantage is low sample consumption and MST can measure dissociation constants from pM to mM. (Wienken, Baaske et al. 2010, Jerabek-Willemsen, André et al. 2014) However, in most of cases, the sample should be labeled with hydrophobic fluorophores which would probably cause non-specific binding effects. (Table 1)

 Tina 1

Figure 1. MST setup and experiments. A. The machine Monilith NT. 115 from NanoTemper Technologies GmbH. B. Schematic representation of MST optics. C. Typical signal of a MST experiment. D. Typical binding experiment.(Jerabek-Willemsen, André et al. 2014)

  1. ITC

ITC is a biophysical technique to measure the heat exchange associated with molecular interactions at a constant temperature. (Duff Jr, Grubbs et al. 2011, Milev 2013) It directly determines the binding affinity (Ka), enthalpy changes (ΔH), and binding stoichiometry (n) of the interaction between two or more molecules in solution. The experimental methodology involves performing several titrant injections from a syringe (usually the ligand) into the solution (usually the macromolecule) in the cell, while maintaining the system at isobaric, quasi-isothermal conditions. When the ligands are injected to the cells, the ligands bind to macromolecules and the machine detects the heat upon binding. With several injections, ligands bound to protein continually. However, when the target protein becomes saturated with the ligand, less binding occurs and the heat change starts to decrease. If the macromolecule is saturated with ligand, no more binding occurs, and only heat of dilution is observed.

ITC has been widely applied as a major tool in drug discovery fields, validating and optimizing the hits (Leavitt and Freire 2001, Peters, Frasca et al. 2009) and also in binding studies, such as protein-protein, protein-DNA, small molecule-protein interactions(de Azevedo, Walter et al. 2008, Liang 2008). ITC is a fast and straight way to detect binding affinity of two molecules by the change of binding enthalpy. However, some complexes may exhibit rather small binding enthalpies that are not suitable for the ITC measurement. (Table 1)

Advantages ²   Small sample size

²   Immobilization free

²   Minimal contamination of the sample

²   Ability to measure complex mixtures

²   Wide size range for interactants (ions to MDa complexes)

²   Ability to determine thermodynamic binding parameters in a single experiment

²   Modification of binding partners are not required


Disadvantages ²   Hydrophobic fluorescent labelling required, may cause non-specific binding

²   No kinetic information

²   Highly sensitive to any change in molecular properties

²   Large sample quantity needed

²   Kinetics cannot be determined

²   Limited range for consistently measured binding affinities

²   Non-covalent complexes may exhibit rather small binding enthalpies since signal is proportional to the binding enthalpy

²   Not suitable for HTS

Table 1. Advantages and disadvantages of ITC and MST.

  1. Ligand-protein binding affinities detected by ITC and MST

One example is about comparing the biophysical data of small molecules with Protein kinase CK2 using both MST and ITC assays.(Winiewska, Bugajska et al. 2017) In this paper, the interactions of four halogenated benzotriazoles with the catalytic subunit of human protein kinase CK2 had been investigated. Among the four compounds, only one compound (5-BrBt) had a consistent binding affinity data in both MST and ITC assays, the solubility of which substantially exceeded the ligand concentration. For another three compounds, when the compounds titrated to the protein solution for ITC measurement, the binding affinities determined by ITC were around 10-folded weaker than by MST. The main problem was the limited titrant solubility that resulted in the formation of nano-aggregates. The issue was ignored by titrating the protein to the compound solution as the protein was soluble enough. (Figure 2) The protein-ligand affinities that derived from ITC may be underestimated because of the compound solubility problem, while the problem can be avoided by MST. Tina 2

Figure 2. Correlation between MST- and ITC-derived binding affinities determined for complexes of halogenated benzotriazoles with hCK2α. Kd(ITC), were obtained with ITC experiment, in which either inhibitor (red) or protein (blue) was used as a titrant. Vertical and horizontal bars represent standard deviation (MST) and 67% confidence intervals (ITC), respectively.(Winiewska, Bugajska et al. 2017)

Blog written by Xiangrong (Tina) Chen

de Azevedo, J., F. Walter and R. Dias (2008). “Experimental approaches to evaluate the thermodynamics of protein-drug interactions.” Current drug targets 9(12): 1071-1076.

Duff Jr, M. R., J. Grubbs and E. E. Howell (2011). “Isothermal titration calorimetry for measuring macromolecule-ligand affinity.” J Vis Exp 55: e2796.

Jerabek-Willemsen, M., T. André, R. Wanner, H. M. Roth, S. Duhr, P. Baaske and D. Breitsprecher (2014). “MicroScale Thermophoresis: Interaction analysis and beyond.” Journal of Molecular Structure 1077: 101-113.

Leavitt, S. and E. Freire (2001). “Direct measurement of protein binding energetics by isothermal titration calorimetry.” Current opinion in structural biology 11(5): 560-566.

Liang, Y. (2008). “Applications of isothermal titration calorimetry in protein science.” Acta biochimica et biophysica Sinica 40(7): 565-576.

Milev, S. (2013). “Isothermal titration calorimetry: Principles and experimental design.” General Electric 9.

Peters, W. B., V. Frasca and R. K. Brown (2009). “Recent developments in isothermal titration calorimetry label free screening.” Combinatorial chemistry & high throughput screening 12(8): 772-790.

Wienken, C. J., P. Baaske, U. Rothbauer, D. Braun and S. Duhr (2010). “Protein-binding assays in biological liquids using microscale thermophoresis.” Nature communications 1: ncomms1093.

Winiewska, M., E. Bugajska and J. Poznański (2017). “ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2.” PloS one 12(3): e0173260.