New Research Confirms Potency Of RAD-140

New cutting-edge research confirms the potency of RAD-140 in binding & activating the Androgen Receptor

Fig.1. In silico model of RAD-140 (center) binding in the pocket of Androgen Receptor (blue coils) confirms the specificity of binding.
Fig.1. In silico model of RAD-140 (center) binding in the pocket of Androgen Receptor (blue coils) confirms the specificity of binding.

A research group in Germany has recently published some exciting results in the Journal of Steroid Biochemistry and Molecular Biology, demonstrating for the first time the unique potency of RAD-140 for binding the Androgen Receptor (AR). RAD-140 activation of AR is known to activate a cellular signal cascade that results in substantial lean mass gains. With this report, we now know why RAD-140 is so prized for its ability to trigger anabolic growth in muscle.

Selective androgen receptor modulators (SARMs) comprise compounds of diverse chemical structures, which can be classified into steroidal or non-steroidal compounds. By definition, SARMs bind as ligands to the androgen receptor (AR) and possess tissue-selective activity.

Ideally, they show preferential agonistic effects in anabolic target tissues (muscle, bone), with weak agonistic or antagonistic effects in reproductive organs (prostate). While the molecular mechanisms explaining this tissue-selectivity are not fully understood yet, potential mechanisms contributing to these properties are the distinct expression of metabolizing enzymes, different conformational changes of the AR. after binding, differing co-activator/co-repressor recruitment or triggering of distinct intracellular signaling cascades after AR binding.

Therapeutic interest of SARMs is based on the prevention or treatment of muscle wasting conditions (sarcopenia, cachexia) as well as the treatment of osteoporosis. However, no SARM has sustained clinical approval up to now. A very different field of interest is sports, where compounds with anabolic properties might be consumed by athletes to enhance performance, increase muscle mass or accelerate muscle regeneration.

RAD-140 is a non-steroidal SARM whose synthesis was first described in 2011. Investigations in vitro showed osteoblast differentiation potential in myoblast cells, neuroprotective properties in primary rat neurons, as well as AR agonistic effects and growth suppression of AR+ breast cancer cells. Moreover, tissue selectivity was shown for the orally bioavailable RAD-140 using the Hershberger assay.

The orally bioavailable GSK-2881078 was first described in a first-in-human clinical study in 2017. This study investigated pharmacokinetics and pharmacodynamics and showed safety of this nonsteroidal SARM in males and females. A second phase-1b study showed anabolic effects in healthy males and females ≥ 50 years by a dose-dependent increase in lean body mass. GLPG0492 is a hydantoin analogue, which also has been shown to possess oral bioavailability in the rat. Moreover, tissue-selective effects have been observed in the classical Hershberger assay inducing anabolic effects in the muscle (levator ani) without effects in the prostate.

Two preclinical studies showed the potential of GLPG0492 to enhance functional performance in a mouse model of muscular dystrophy as well as being effective in reducing muscle loss in a mouse model of hind limb immobilization. The intention of the present study was to investigate and directly compare the androgenic and anti-androgenic properties of the three SARMs RAD-140, GLPG0492 and GSK-2881078 in two different in vitro bioassays, the yeast androgen screen and a luciferase reporter gene assay in human PC3(AR)2 cells. As a further comparison the molecular modeling was performed in silico to provide androgen receptor binding mode hypotheses for RAD-140, GLPG0492 and GSK-2881078.

In this recent update to our understanding of the molecular binding of RAD-140, scientists in the Department of Molecular Cell Physiology and Endocrinology at Technical University Dresden have now quantified the selectivity of the SARM RAD-140 for binding AR in its anabolic setting.

Molecular Modeling

For the sake of binding mode comparison, two x-ray structures of the androgen receptor (AR) in complex with its normal binding partner dihydrotestosterone and with SARM S21 were chosen due to the high average resolution of 1.50 Å and 1.60 Å repectively. The S21-androgen receptor structure was selected as input structure for docking experiments due to the high structural similarity of S21 with RAD-140.

Docking experiments were performed for the three SARMs of interest using the GOLD v.5.2.2 algorithm using standard settings with an increased search efficiency of 200%. The binding site was defined using a sphere with a radius of 12 Å and the amide carbon atom of S-21 as center. For each compound, ten generic algorithm runs were performed. The obtained ligand orientations of RAD-140 and GSK- 2881078 underwent energy minimization in LigandScout v4.09 using the MMFF94 force field algorithm.

For the selected GLPG0492 conformation and all binding site residues, energy minimization was performed in MOE using the MMFF94 force field algorithm due to steric clashes and necessary rotation of Thr877. Binding mode hypotheses were selected considering the similarity to the binding mode and pharmacophore model of S-21.

Fig. 2. AR-binding dose-response curves of DHT (■) as positive control and of GLPG0492 (◯), GSK-2881078 (▽) and RAD-140 (◇). RAD-140 shows superior binding compared to the other SARMs.
Fig. 2. AR-binding dose-response curves of DHT (■) as positive control and of GLPG0492 (◯), GSK-2881078 (▽) and RAD-140 (◇). RAD-140 shows superior binding compared to the other SARMs.

This is the first study showing that all three non-steroidal SARMs, namely RAD-140, GLPG0492 and GSK-2881078 possess transactivation potential in the high specificity androgen screen. Moreover, the authors investigated the transactivation potency of the three compounds in a human prostate cell line. In contrast, all three SARMs induced luciferase at concentrations already high in human cells.

This difference in potency is also apparent regarding the EC50 values calculated for both in vitro test systems. This discrepancy can be explained by cell biological differences, as S. cerevisiae cells possess not only a cell membrane, but also an additional cell wall, which might prevent compounds from diffusion into the cell. Besides, the co-factors involved in transcription differ between cell types. Hence, the observation of false-negative results is possible. The advantage of this approach is the detection of the biological activity of individual compounds as well as mixtures, independent of the knowledge of their chemical structure. 

The binding mode hypotheses obtained for the investigated SARMs RAD-140, GSK-2881078 and GLPG0492 suggest that the observed androgenic activity is due to the interaction pattern similarity to the x-ray binding mode of the reported SARM S-21. While Nique and colleagues described molecular modeling of GLPG0942, this is the first description of the in silico docking of RAD-140 and GSK-2881078 to the androgen receptor ligand binding domain. This GLPG0942-AR ligand binding domain interaction modes show the same pattern as that observed by Nique and colleagues although their description was only simplified. Accordingly, these results and those of the previous study observed the hydrogen bonding of the nitrile group to Arg752 in subpocket s1 as well as the hydrogen bonding to Asn705 in sub-pocket s3.

The results of the both in vitro transactivation assays confirm the functional binding of the three SARMs to the AR ligand binding domain modeled in silico. The comparison of AR binding values reveals that RAD-140 is the most potent in human cells. Although GSK-2881078 lacks the ability for hydrogen bond donation in sub-pocket 2 compared to the two other SARMs (Figs. 3B, 4 B), the transactivational activity of GSK-2881078 in the PC3(AR)2 cells is higher than that of GLPG0492.

In conclusion, the authors showed that the AR binding activities induced by the three non-steroidal SARMs RAD-140, GLPG0492 and GSK-2881078 were different comparing the non-mammalian androgen screen and a luciferase expression assay in human cells. In addition to the biological testing, these authors demonstrated for the first time molecular modeling of the interaction between the human androgen receptor ligand binding domain and RAD-140 or GSK-2881078 respectively.

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