Allosteric Binding Sites On Nuclear Receptors: Focus On Drug Efficacy and Selectivity

Abstract

Nuclear receptors (NRs) are highly relevant drug targets in major indications such as oncologic, metabolic, reproductive, and immunologic diseases. However, currently, marketed drugs designed towards the orthosteric binding site of NRs often suffer from resistance mechanisms and poor selectivity. The identification of two superficial allosteric sites, activation function-2 (AF-2) and binding function-3 (BF-3), as novel drug targets sparked the development of inhibitors, while selectivity concerns due to a high conservation degree remained. To determine important pharmacophores and hydration sites among AF-2 and BF-3 of eight hormonal NRs, we systematically analyzed over 10 µs of molecular dynamics simulations including simulations in explicit water and solvent mixtures. In addition, a library of over 300 allosteric inhibitors was evaluated by molecular docking. Based on our results, we suggest the BF-3 site to offer a higher potential for drug selectivity as opposed to the AF-2 site that is more conserved among the selected receptors. Detected similarities among the AF-2 sites of various NRs urge for a broader selectivity assessment in future studies. In combination with the Supplementary Material, this work provides a foundation to improve both selectivity and potency of allosteric inhibitors in a rational manner and increase the therapeutic applicability of this promising compound class. , glucocorticoid receptor (GR), progesterone receptor (PR), and minearlocorticoid receptor (MR) share a common domain architecture as well as a similar fold regarding their ligand binding domain (LBD), the selectivity concern for allosteric NR inhibitors remains ( Figure 1B). For example, it has been reported that the AF-2 and BF-3 sites of AR and GR have a sequence identity of approximately 50% [9,28]. Further on, drug-like mimetics of coactivator peptides at the AF-2 site have the potential to disrupt protein-protein interactions for multiple NRs and, ultimately, promote off-target toxicity [16,29].While several structures with cocrystallized ligands have been determined for the AR, targeting superficial binding sites such as AF-2 and BF-3 of other receptors remains a challenge due to their comparably large size, shallowness, and high flexibility [30,31]. Knowledge regarding binding hotspots and distinct pharmacophores among structurally similar NRs is crucial for the design of effective and selective inhibitory compounds [31][32][33][34]. In this regard, cosolvent molecular dynamics (MD) simulations are a suitable computational tool to determine hotspots and assess their druggability, as well as to obtain detailed information on potentially useful pharmacophores to improve drug potency and selectivity for the site of interest. In this simulation protocol, which was inspired by crystallographic observations of small fragments binding to protein surfaces, organic solvent molecules mimicking drug fragments are added to the aqueous phase to monitor and quantify their interaction with a protein. Compared to similar methods for bi...

Computational Selectivity Assessment of Protease Inhibitors against SARS-CoV-2

“…In the next step, we further characterized and compared the selected proteases according to their hydration and small-molecule hot-spots by using different molecular probes including water, acetonitrile, isopropanol, and pyridine. The use of specific functional groups represented by the different organic probes associating with the active sites can be used to fine-tune the selectivity profile of protease inhibitors [ 18 , 28 ]. Similarly, selectively targeting hydration sites occurring in one protein, but not in an anti-target, offers potential to be exploited in structure-based design.…”

“…Binding modes obtained from molecular docking have been widely used to establish selectivity factors toward different targets [ 13 , 18 , 31 ]. Here, we compiled compound sets comprising experimentally verified ligands of nine proteases to assess the selectivity of recently reported SARS-CoV-2 M pro inhibitors ( Figure 3 ) by cross-docking them into the respective protein active sites.…”

“…Computational methods such as molecular docking and specialized molecular dynamics (MD) protocols can be exploited to explore the selectivity of small-molecule compounds, as it was evidenced for various targets. For example, we previously applied docking combined with cosolvent MD simulations and determination of hydration hot-spots to investigate the selectivity of allosteric inhibitors against eight nuclear receptors to support targeted experimental profiling of novel compounds [ 18 ]. In general, it was discussed that cosolvent MD simulations can provide valuable insights for the development of potent and selective compounds [ 19 , 20 ].…”

Advances in Molecular Dynamics Simulations and Enhanced Sampling Methods for the Study of Protein Systems

“…As the name suggests, cosolvent MD simulation uses solvent mixtures that usually consist of organic molecules or fragments that possessed key pharmacophore features crucial for ligand binding such as hydrophobic centers, aromatic rings, hydrogen bond donors and hydrogen bond acceptors [ 81 ]. There are various successful protocols utilizing cosolvent MD simulation for binding ‘hotspot’ prediction, namely, SWISH, MixMD, MDMix, SILCS, mCSM and many others [ 93 , 95 , 96 , 97 , 98 , 99 , 100 , 101 ]. A recent development of SWISH—a Hamiltonian replica-exchange-based method developed to survey hydrophobic patches on proteins for druggability—demonstrated proficiency of using cosolvent probe in precisely identifying cryptic pockets [ 93 , 95 ].…”

Mechanistic Insights into the Allosteric Inhibition of Androgen Receptors by Binding Function 3 Antagonists from an Integrated Molecular Modeling Study

“…This finding, together with the discovery of several BF3 inhibitors (e.g., flufenamic acid and Triac), suggests an alternative AR-directed PCa therapeutic strategy through targeting BF3 . Moreover, the BF3 site is potentially more selective than the AF2 site as the former is less conserved than the latter among the nuclear receptors (NRs) . Extensive effort has been devoted to developing BF3-directed agents with improved potency and specificity, ,,, of which several BF3 antagonists bearing an indole scaffold demonstrated nanomolar anti-AR activity and could effectively abrogate the androgen-induced proliferation of androgen-sensitive (LNCaP) and enzalutamide-resistant (MR49F) PCa cell lines. − …”