Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5884_Библиотеки_им_академика_М_И_Перельмана.pdf
16 Computational Study of Conformational Changes in Nuclear Receptors... 471
enzalutamide is an AR competitive antagonist approved drug in 2009, patient
resistance happens after months of treatment [53].
As mentioned earlier, EDCs are xenobiotic compounds interacting with NRs and
by mimicking endogenous, causing a broad range of diseases. Epidemiological
studies reported that exposure to xenoestrogens such as diethylstilbestrol (DES)
during fetal development and exposure to dichlorodiphenyltrichloroethane (DDT)
during puberty increases the risk of breast cancer [54]. Another study offers initial
insight into the neural effects of human exposure to bisphenol (BPA). The results
propose that when expectant mothers are exposed to BPA during prenatal stages, it
may cause modifications in the microstructure of white matter in preschool-aged
children and these changes in white matter can mediate the connection between
early-life exposure to BPA and the emergence of internalizing problems [55]. Similarly, a rising number of cancerous testicular or malformations of the male genital
tract also could be attributed to exposure to EDCs [56]). A study has revealed the
relationship between the level of plasma polychlorinated biphenyls (PCBs), a persistent and lipophilic aromatic chemical, and reduced semen quality, particularly
reduced sperm motility [57].
Class II of NR has been also the target of therapeutics. Among them, PXR, CAR,
and FXR and their heterodimerization partner RXR are of substantial interest and,
therefore, introduced shortly in the next sections.
3 Computational Approaches to Evaluate NRs Protein
Dynamics
This chapter focuses on the protein dynamics induced by ligand binding and are not
discussing purely ligand-based approaches used to understand NR-binding, such as
machine learning and classical QSAR studies. Starting from the most classical
structure-based approaches, molecular docking has been large ly used to study NRs
(as reviewed by [45]). For more information about molecular docking approaches
and their limitations, readers are referred to chapter (see Chap. 7). Despite the several
advantages of docking, as a fast and rational method, this approach is unable to
capture NRs’ characteristic flexibility and the role of explicit solvent [45, 58–60]. In
addition, multiple binding modes for congener ligands are not unheard of for NRs.
Some examples, such as the nonanoic acid binding to PPARγ, even displ ay multiple
binding modes for the same ligand [61]. This could be even further illustrated by
PXR’s or CAR’s large ligand binding pocket, which allows such freedom for ligand
movement (see their respective sessions below).
These observations prompted us to hypothesize that small molecules or fragments
(such as EDs and lipids) might adopt multip le binding modes suggesting that a single
static model for the NR-ED interaction would be insufficient to accurately describe/
predict their outcomes. We identified this as an inherent limitation of the docking
and subsequently proceeded with MD simulations (please, see Chap. 8 for more
472 A. Rashidian et al.
methodological details) to assess the ligand stability within NRs’ LBP, besides
capturing the NRs conformational dynamic, in many of the illustrative examples
below.
In this sense, MD simulations are a relevant and underused tool to elucidate NR
conformational rearrangement upon ligand binding and can also be used to discover
the ligand entry/exit pathways [62, 63 ]. Historically, steered MD simulations were
used to identify two distinct pathways on the retinoic acid recept or (150 ns), one for
ligand binding and another for unbinding [64]. In addition, random expulsion MDs
suggested that the retinoic acid may exit the binding site through flexible regions
close to the H1–H3 loop and β-sheets, without displacing H12 from its agonist
position [65]. It is important to highlight that both studies applied forces to propel the
ligand outside the pocket, even if the selection of the expulsion was unbiased. The
lack of a significant number of replicas suggests that even more interesting conformational changes could be observed in a modern setup.
Despite that, their observation is consistent with recent crystal structures where
the end of H1 and H2 can assume a highly flexible loop-like folding in some
receptors. Consistently, studies performed on TR- and ER-LBDs indicated that
ligand unbinding does not require the H12 displacement [66, 67]. Up to four
different unbinding routes were identified on ER-LBD, with half relying on the
H12 dislocation and the other half on the H8 and H11 separation , with this last being
potentially influenced by dimerization [66, 67].
A large range of structures cocrystalized with (partial-) agonists and antagonists
are available for some NRs [68–71] highlighting changes in the H12 position.
However, other receptors lack such diversity. The work from Alvarez et al. [72]in
GR aimed to fill this structural gap by exploring the correlation between ligand
identity and GR’s H12 behavior using (steered and classical) MD simulations. They
generated relevant GR-LBD models in the so-called agonist and nonagonist states,
simulating those systems in the presence of an agonist (dexamethasone) or an
antagonist ligand (RU-486), in a combinatorial fashion. Their description of the
H12 as a dynamic ensemble of conformations is influenced by the interacting ligand.
On one hand, simulations of GR-dexamethasone display a deep minimum potential
energy surface favoring specific conformation. On the other hand, RU-486 bound
systems would favor a wider H12 conformational amplitude, consistent with a flatter
potential landscape.
Additionally, their steered dynamics trajectories on GR [72] oriented by the Cα’s
RMSD of H3, H11, and H12 as collective variables, showed conserved secondary
structure elements changing conformation among themselves without deformation.
Interestingly, the high-resolution structure of progesterone receptor (PR) complexed
with RU-486 shows high flexibility of the H12 [73], but not full displacement. Given
the crystallization procedure, starting from an agonist-bound starting structure that
was later soaked with RU-486, one can postulate that the antagonist binding is a
dynamic equilibrium process.
Of note, H12 of several LBDs are described as a stable but flexible helix in the
apostate, which could be located away from the LBD’s helix bundle [74, 75]. However, time-resolved fl
uorescence anisotropy experiments showed that, despite H12
16 Computational Study of Conformational Changes in Nuclear Receptors... 473
flexibility and conformation diversity, the LBD retained its globularity in the
apostate [76].
3.1 Retinoid X Receptor and the Role of Allosteric in the NR’s
Dynamics
Retinoid X receptor (RXR) is a type II NRs and forms heterodimers with approximately one-third of the other NRs [77]. In humans there are three isoforms of
RXR; α, mainly found in the liver, kidney, and intestine; β, found in most human
tissue; and γ, which mainly exists in the brain and muscles [78]. Malfunctioning of
these isoforms has been linked to various health issues [11, 79–81].
RXR activati on can be categorized into two groups: permissive and
nonpermissive heterodimer. In the permissive heterodimers [82], activation can be
induced by binding the agonist to either RXR or NR partner or both receptors.
Examples of this group are RXR/FXR, RXR/LXR and RXR/PPAR. In the case of
the nonpermissive heterodimer, only agonist binding to the RXR partn er triggers
activation, but RXR can still bind to the agonist, releasing the corepressor and
recruiting coactivator. Examples of this are RXR/TR and RXR/VDR. In this case,
RXR can also bind to the agonist and lead to synergistic action in the presence of a
heterodimer ligand [82, 83].
The main small molecule compound binding to RXR is 9-cis -Retinoic acid
(9cRA). It belongs to a retinoid family and has a critical role in cell growth,
development, differentiation, and apoptosis. Other RXR ligands are 9cRA-related
compounds and indenoisoquinolines. Remarkably, the work from Bexarotene and
Diarylamines [25] employed X-ray crystallography to describe the PPARγ and
RXRα structure. They explained how these two receptors interact with DNA,
highlighting the influence of DNA in governing the interaction between the two
receptor domains through specific rearrangements. They also revealed the cooperative nature of multiple domains of PPARs, which can modulate the properties of the
PPARγ-RXRα complex. In this complex, the LBD of PPARγ tightly couples with
RXRα domains. Accordingly, the conformational change induces a reposition of
receptor domains responsible for DNA binding and optimizing their contact with
DNA. They also examined the dynamic properties of structures using amide hydrogen/deuterium exchange mass spectrometry (H/D-Ex). The result revealed that the
helices H10/H11, involved in LBD-LBD heterodimerization, adopt a slightly
shifted/curved conformation when the protein is contacted with DNA, facilitating
optimal contact of the receptor with DNA [25].
3
3
474 A. Rashidian et al.
3.2 Pregnane X Receptor’s Dynamic Pocket Allows Binding
of Diverse Ligand Sets
Pregnane X receptor (PXR), also known as the steroid and xenobiotic sensing
nuclear receptor (SXR) [84], is encoded by the NR1I2 (nuclear receptor subfamily1,
group1, member 2) gene on chromosome 3 [85, 86]. PXR heter odimerizes with
RXRα, β and γ (NR2B1–3) [87–89] at H10/H11 region. Additionally, the PXR-LBD
demonstrates a distinctive characteristic by homodimerizing at its β1′ interface.
Homodimerization occurs through the conserved Trp223 and Tyr225 residues in
each monomer (Fig. 16.3)[90]. These amino acid residues involved in the interface
exhibit high conservation among various species, including humans, rhesus monkeys, rabbits, mice, rats, pigs, and dogs. However, in the canine PXR, Trp223 is
replaced by Gln223 [90]. Noble et al. (2006) showed mutation of Trp223 and
Tyr225 does not interfere with DNA, RXR, or ligand binding, rather it disrupts the
homodimerization, reducing the recruitment of the coactivator SRC-1 and transcriptional activity [90].
When an agonist ligand binds to the PXR-RXRα heterodimer in the nucleus, it
promotes coactivator binding and release of corepressor from AF-2 [92]. Subsequently, this activated PXR complex induces the expression of the target gene. PXR
structure, like other NRs, has a large hydrophobic LBD. The primary PXR isoform is
composed of 434 amino acids, featuring a notable hydrophobic triad consisting of
F288, W299, and Y306. Unlike other nuclear receptors (NRs), the PXR-LBD lacks
the typical stable H2′ and H6 helices. Crystallographic structural data clearly
illustrate the lack of stability in the H2′ region, which appears disordered in all
publicly accessible PXR structures. These characteristics result in a more expansive
and flexible LBD for PXR, distinguishing it from other NRs [93–95]. Consequently,
the PXR-LBD can accommodate a diverse range of ligands. Presently, the Protein
Data Bank (https://www.ebi.ac.uk/pdbe/, accessed on November 2023) repository
contains 52 cryst al structures of human PXR in its active mode, complexed with the
αAF-2
α6
sheets
W22
Y225
Fig. 16.3 Crystal structure of PXR homodimer. The amino acids Trp223 and Tyr225, located on
β1′, mediate the homodimerization. The interface is shown in pink. PDB ID: 1NRL [91]
coactivator
Y225
W22
β sheets
α11
α10/
α3
16 Computational Study of Conformational Changes in Nuclear Receptors... 475
coactivator protein SRC1. Among these structures, 44 exist in a homodimer assembly, while 8 are available in heterodimer form.
PXR is a ligand-dependent transcriptional factor involved in small molecule
metabolism and regulation of diverse cellular processes including bile acid metabolism, glucose homeostasis, cell proliferation as well and inflamma tion. PXR mostly
exist in the liver and intestine. It regulates the gene expression of enzymes
and transporters that are responsible for the different pathways of endogenous and
xenobiotic pharmacokinetics including absorption, distribution, metabolism, and
excretion (ADME). Gene targets of PXR are cytochrome P450 genes (CYP2B,
CYP2C, CYP3A) and efflux and uptake transporters of the ATP-binding cassette
[96–99]. Besides endogeno us ligands, PXR is activated by a broad number of
diverse small molecules, including drugs, environmental pollutants, and natural
products. These various functions make PXR a potential therapeutic candidate.
However, the activation of PXR can induce intestinal and hepatic first-pass metabolism and drug efflux transport [100] which in turn may lead to drug–drug interactions (DDI), adverse drug reactions or therapeutic failure of drugs [101–103]. To
exemplify, one can consider the report about the reduced effect of rifampicin on
midazolam or contraceptives due to the increased expression of CYP3A4 when these
medicines are coadministrated [104], or isavuconazonium which activates the
expression of CYP2B6 through PXR-mediated induction and decrease the exposure
of bupropion [96 , 105]. These observations raised the interest in designing PXR
antagonists along with the attempt to limit the activation of PXR in the presence of
xenobiotics. As of 2002, several “azole” compounds have been identified as PXR
inhibitors, such as ketoconazole, enilconazole, FLB-12, and SPA70, although it is
less known about the structural trigger of PXR-bound antagonists.
For instance, SPA70 and SJB7 are close analogues [106] where SPA70 act as an
antagonist of PXR but SJB7 is a PXR agonist which highlights the promiscuity of
PXR-LBD. Several approaches from experiment al methods to computational techniques such as pharmacophore, quantitative structural-activity relationship (QSAR),
machine learning (please see Chaps. 4 and 6, respectively), and structure-based
methods have been utilized to investigate PXR activation upon ligand binding
[96]. Notably, due to the lack of crystal structures of PXR in complexes with
antagonist ligands, likely attributed to the complexity and high flexibility of the
system, computational studies play a crucial role in unraveling the conformational
dynamics of the PXR-antagonist complex.
For a more thorough dissuasion regarding the capability of MD simulations in
capturing the dynamic of NRs, we refer to the study conducted by Chandran et al
[107] They studied the dynamic behavior of PXR-LBD apo structure comparing it to
the agonist-bound state, through short MD simulations that last for 100 ns
[107]. Although their short simulations would not allow sidechain and loop reorganization, yet, it was able to identify several conformational states for apo PXR-LBD
showing different pocket’s volume, while with agonist binding (SR12813), the
compound restricted both LBD conformation and binding pocket’s size and shape.
Alternatively, Motta et al. employed a different strategy by sim ulating the entry of
SR12813 into PXR’s LBP utilizing the MD-binding method. Their result suggested
476 A. Rashidian et al.
that the ligand accessed the LBP through a channel between H2 and H6 helices
[62]. To enhance the sample of the SR12813-bond conformations, they utilized
scaled MD simulations with a total of 2 μs). Remarkably, their finding confirmed that
the SR12813 binding mode observed in the crystal structure (PDB ID: 1NRL [93]) is
indeed the most stable through simulations.
In a comparative study, Huber et al. performed 200 ns MD simulations of wildtype PXR-LBD and Trp299Ala mutant, without ligand and with TO90131713
(agonist), SPA70, and SJB7 [108]. They suggested that the extra space conferred
by the Trp299Ala is the reason for the observed antagonist-to-agonist switch with
this mutant for SPA70. This extra space lets SPA70 reside deeper in the pocket,
preventing the αAF-2 dislocation and maintaining PXR active. In our work [109],
we used MD simulations starting from a PXR-apo structure, which has a similar
conformation with an agonist complex (SR12813, PDB ID: 1NRL). The long MD
calculations could decipher ligand-specificinfluence on conformations of different
PXR-LBD regions and could also be useful to guide how to alleviate PXR agonism.
Interestingly, even if star ting with the same conformation, long MDs can discriminate the dynamic behavior of the agonist versus the antagonist.
More recently, our group employed an in silico screen and experimental cellular
reporter assay to identify small molecule kinase inhibitors from an in-house compound library, the Tübingen kinase inhibitor collection (TüKIC) compound library,
which act also as a PXR inhibitor. In the experimental work [110] we describe the
identification of the C-100 compound and the biochemical binding and cellular
protein interacti on assays which categorize the novel compounds as mixed competitive/noncompetitive, passive antagonists by disrupting PXR coregulatory binding
[110]. This work was supported by structure-based virtual screening and molecular
dynamics (MD) simulations which reveal the ligand-specific conformational
rearrangements of PXR-LBD including H6 region, αAF-2, H1–H2′, β1′–H3 and
β1–β1′ loop [109] (Fig. 16.4).
PXR-LBD’s flexibility and promiscuity enable it to bind to a wide range of
ligands with different sizes and shapes. However, no considerable changes in
interaction patterns are associ ated with PXR conformational rearrangement. Ngan
et al. (2009) investigated the structural foundations of PXR’s promiscuity using
computational solvent mapping [111], specifically, they used servers such as FTMap
to dock small molecule probe fragments, which were followed by binding energy
calculation. This technique is designed to identify and characterize hot spot regions
within protein binding sites, those regions represent relevant residues/surface
regions that play a pivotal role in determining the binding free energy. Their result
shows that one of the most important regions for binding the different PXR ligands is
the hydrophobic cage (Fig. 16.5b). This subpocket is formed by a triad of Phe288,
Trp299 and Tyr306 where pi–pi interaction and edge-to-face interaction are
observed for ligand stability. In addition, the polar residues His407, Gln285,
Ser247, and Thr248 form hydrogen to bind with ligands either directly or mediated
with a water molecule [109]. Phe429, which is located on H11, and αAF-2 known as
the hydrophobic side participating in ligand binding [111]. However, the study
conducted by Delfosse et al. (2021) on the synergistic activation of the endocrine
t
αAαA
F-F-22
16 Computational Study of Conformational Changes in Nuclear Receptors... 477
A
α6 region
β1-β1'
α1-α2' loop
αAF-2
PXR agonistbinding conformation
B
α1-α2' loop
Shift in conformational
dynamics
β1-β1' loop
AntagonistAgonist
β1-β1'
loop
PXR antagonistbinding conformation
α6 region
α2'
α6
αAF-2
α1-α2' loop
αAF-2
PXR-Ag
PXR-AntAg
-An
Fig. 16.4 Ligand binding effect on PXR-LBD conformation. (a) Agonist-bound and antagonistbound PXR-LBD conformation. MD simulations revealed the shifts in PXR-LBD conformational
dynamics. The PXR-LBD regions undergoing the most extensive conformation are labeled. (b)
Markov state modeling result for the PXR-LBD bound to compound 100. The subregions with
extensive motions are shown individually, with a reference conformation from an agonist-bound
crystal structure (PDB ID: 1NRL) in transparent illustrated as follows: H1–H2′ loop, grey dashed
line; β–β1′ loop, white; H6 region, transparent light green located in the vicinity of H2′ (white
helix); β1′–H3 loop, transparent dark green; αAF-2, transparent dark brown located in the vicinity
of H3-helix (cyan helix). (For interpretation of the references to color in this figure legend, refer to
Fig. 16.3, modified from [109])
PXR-Ag
PXR-AntAg
PXR-AntAg
PXR-Ag
PXRAntAg
α3
PXR-Ag
disruptor mixtures on PXR-RXR heterodimer revealed that the PXR activation does
not necessarily occur due to the engagem ent of AF-2 region in ligand binding
[87]. Moreover, our in silico study also provided insight into the interaction profile
of PXR-LBD bound to full agonist and our identified competitive antagonist consistent with the already known interacting subregion of PXR-LBD for agonis t.
However, in the presence of an antagonist our MD simulations revealed that
His407 is not directly involved with Ligand binding rather it preferred to be engaged
with His404 through H-bond which might be one of the local triggers for inducing
PXR-LBD conformational rearrangement [109].
Our studies provided insight into which conformational behavior of PXR-LBD
can promote PXR antagonism. The discovery of drugs that can simultaneously
inhibit both PXR and protein kinases could offer new possibilities in cancer treatment and the possibility of dual PXR, and kinase inhibitors could be beneficial in
cancer treatment. and h elp overcome drug resistance.
478 A. Rashidian et al.
PXR-LBD
6
β1’
β1
Homodimerization
interface
α
1
α
α
3
α
10/11
α
5
α2’
αAF-2
4
α
PXR-LBP
H407
H327
W299
8
α
Y306
F288
M243
Q285
S247
T248
L411
F281
M425
F251
T422
F429
coactivator
Fig. 16.5 Representative snapshot of the PXR-LBD displaying binding mode. The binding pocket
is enclosed in a black rectangular box. Residues participating in binding are labeled. Hydrophobic
subpacket surrounded by dashed circle. The ligand is shown in an obscured yellow surface. H3,
H10/11, α-AF2 and Homodimerization interface colored in cyan, light brown, dark brown, and pink
color, respectively. (Modified from [109])
Most known ligands bind to orthosteric PXR-LBP; however, allosteric sites can
be an alternative region for the PXR modulator’s accommodation [112–114]. Allosteric sites are distant from orthosteric sites and accommodate structurally different
ligands. So far, 202 allosteric modulators have been reported for nuclear receptors
[115]. The proposed allosteric ligand binding sites are the AF-1 site, zinc fingers and
response elements, LBP (synergistic effect), the AF-2 site, and the binding function
3 (BF-3 site) [115, 116]. The BF-3 region was originally described in the androgen
receptor and is a hydrophobic cleft composed of the H1, the L:H3–H5 loop, and H9.
Despite their amino acid and conformation conser vation, no ligands were found to
bind in the PXR equivalent.
An example of an allosteric ligand binding pocket is reported by Delfosse et al.
(2021) where the simultaneous binding of 17-α -ethinylestradiol (EE2) and
transnanochlor (TNC) enhanced the CYP3A4 induction higher than the single
binding of either of compounds when compared to potent agonist SR12813
[87]. Ketoconazole [117] is an example of a modulator binding to AF-2 and acts
as a PXR inhibitor. To name, fluconazole, enilconazole, pazopanib [103], metformin
and leflunomide [118], FLB-12 [117, 119], coumestrol [120, 121], sulforaphane
[122], and campthotecin [123] are other AF-2 modulators.
In summary, while the adaptability of PXR-LBD has evolve d to safeguard
humans from environmental factors through its involvement in xenobiotic metabolism, this characteristic poses challenges in certa in treatments, contributing to issues
such as drug–drug interactions, and adverse and drug resistance. Advancements in
computational technology, coupled with in vitro validation, can enhance the study of
PXR and provide valuable insights.
16 Computational Study of Conformational Changes in Nuclear Receptors... 479
3.3 Constitutive Androstane Receptor and the Structural
Features for Constitutive Activity
Constitutive Androstane Receptor (CAR), encoded by the NR1I3 (nuclear receptor
subfamily1, group1, member 3) gene belongs to class II NRs. CAR is predominantly
expressed in the intestine and liver, [124, 125 ] heterodimerized with RXRα, β, γ
(NR2B1–3), and like PXR plays a critical role in regulating genes involved in
exogenous and endogenous metabolism. Alternative splicing generates multiple
CAR isoforms in humans and other primates, but not in rodents [126, 127]. This
mechanism, however, is not yet fully understood. Around 50% of transcripts encode
the wild-type CAR1 that displays high basal activity, CAR2 and CAR3 isoforms
(Fig. 16.6a–c) demonstrate ~10% and ~40%, respectively with low constitutive
activity, likely due to their reduced interaction with RXR which results in weaker
binding to DNA and coactivators [128, 129].
The unique feature of CAR is its constitutive activation, distinguishing it from
other nuclear receptors. Unlike other NRs, CAR does not require ligand binding for
its transcriptional activity although ligand binding can modulate CAR activity as an
agonist or inverse agonist. It can bind to a vast number of chemical compounds [130]
and regulates multiple genes involved in xenobiotic detoxification, which might
overlap with or be distinctive from PXR targe t genes.
CAR is primarily localized in the cytoplasm and forms a complex with heat shock
proteins [131]. The majority of CAR ligands act as direct activators, such as
6-(4Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O(3,4-dichlorobenzyl) oxime (CITCO) [132] in human and TCPOBOP [133]in
mouse. On the other hand, synthetic compounds like phenobarbital and acetaminophen, as well as endogenous compound bilirubin, are examples of indirect CAR
activators. It has been proposed that the Epidermal growth factor receptor (EGFR)
signaling pathway is inhibited by phenobarbital, therefore acting as a repressor,
leading to the dephosphoryl ation of CAR at Thr38 within the cytoplasm, which
enables its translocation to the nucleus [17, 18]. Flavonoids have been reported to
function as both direct and indirect activators depending on cellular context
[134]. Androstane metabolite, PK11195 [135], TO901317 [136] and S07662
[137, 138] are examples of CAR inverse agonists (Fig. 16.6d). Inverse agonists
can reduce the basal constitutive activity of CAR, acting as inhibitors/repressors.
Moreover, certain CAR activators, such as phthalates, antivirals, and artemisinin
derivatives display some isoform selectivity [128, 129, 139–141]. In the nucleus,
CAR plays a constitutive regulatory role in target genes, including
CAR-preferentially responsive gene CYP2B6. To achieve this, CAR interacts with
specific DNA motifs DR3, DR4, DR5, ER6, and ER8, located in the enhancer and
promotor region of target genes [142].
Another feature of CAR, as a member of NRs, is its permissive activity when
complexed with RXR. Several studies [137, 143, 144] have reported the synergistic
and additive effect of multiple xenobiotic compounds. Dauwe et al. (2023)
conducted their in vivo study with several pesticides—
recognized as ligands of
480 A. Rashidian et al.
AB
α6
α7
α3
α2
αX
α5
α1
CAR1 CAR2
α11
α10/
αAF-2
α8
α9
L:α6-α7
SPTV
αX
α7
α11
α10/
AF-2
α
α8
α4
α6
α3
α2
α5
α4
α1
D
Cl
Cl
Cl
CITCO
O
N
N
SN
S
H
N
O
S07662
H
N
F
F
F
FF
O
O
N
S
O
TO901317
F
OH
F
F
F
clotrimazole
C
α7
α2
N
N
α6
α3
αX
α5
α1
Cl
N
N
O
PK 11195
Cl
α11
α10/
L:α8-α9
αAF-2
APYLT
α8
α9
α4
Fig. 16.6 Structural overview of the CAR-ligand binding domains. (a) Featuring the CAR1-ligand
binding domain (CAR1-LBD) (b) in CAR2-LBD and (c) CAR3-LBD. The insertion loops are
highlighted within a grey circle (SPTV, L: H6–H7) for CAR2-LBD and in a blue circle (APYLT, L:
H8–H9) for CAR3-LBD. Various regions of interest, including H3, H5, β-sheets, H10/H11, αXhelix, and αAF-2 (α12) are visualized with distinct colors for clarity. (d) The small molecule CAR
ligands
CAR and Tri-butyl-tin (TBT) served the role of an RXR agonist. In mice subjects,
the concurrent administration of dieldrin (pesticide) and TBT prompted a synergistic
activation of CAR. Furthermore, combined effects were observed with
propiconazole, bisphenol, boscalid, and bupirimate [145].
An initial study [146] aimed to identify the conformational rearrangement of
CAR-LBP upon agonist binding by implementing short MD simulations (50 ns).
Their findings indicated that the activation helix retains its active conformation even
in the ligand’s absence, through van der Waals interactions and hydrogen bonds.
Additionally, their study revealed significant conformational changes within the