In the present study, we demonstrated that two novel CCR5 inhibitors, AK530, and AK317, are lodged in a hydrophobic cavity located between the upper transmembrane domain and ECL2.CCR5 is a member of GPCRs, the largest superfamily of proteins in the body. The understanding of the structure of human GPCRs would be invaluable in elucidating their roles in a number of biological processes and should also greatly aid designing therapeutics. In particular, the elucidation of detailed structure of human CCR5 and its interactions with HIV-1 envelope glycoprotein should help establish a strategy for HIV-1 intervention. However, no crystal structures of human CCR5 are yet available; bovine rhodopsin and β2
-adrenergic receptor are the only GPCR species, for which crystallographic data have been obtained.32; 37
Thus, we have previously explored an alternate approach: combination of site-directed mutagenesis-based saturation binding assay and molecular modeling based on the crystallographic data of bovine rhodopsin.22
The site-directed mutagenesis-based saturation binding assay alone can give certain insights as to which residues of the receptor are implicated in the binding with the inhibitor. However, it does not indicate which specific atoms of the residue are involved. Homology modeling and docking give insights to interactions between atoms, but these methods produce multiple possible solutions, and it is difficult to differentiate between distinct interactions that are within a small energy range. Combining these complementary methods of site-directed mutagenesis and computational protein structure determination have made it possible to conduct robust structural analyses.22
However, certain validation of the model is crucial. Thus, in the present study, we chose five amino acid residues of CCR5 that were judged to be critical for APL binding based on our previous analysis and additional mutagenesis studies were carried out. When an amino acid substitution, P84H, C101A, F109A, T195P, or T195S, was introduced to CCR5WT
and saturation binding assay was performed using 3
H-APL, all KD
values proved to be greater than 100 nM, while the KD
value of 3
H-APL with wild-type CCR5 was 3.6 nM. This corroborated the notion we previously made 22
that the alternate approach using saturation binding assay combined with our computational protein structure determination can provide reasonably robust structural insights.
In the present study, we attempted to obtain a refined structural CCR5 model through molecular dynamics computation. In an inhibitor-unbound CCR5, we identified key interactions between residues located in different transmembranes, and between transmembrane and ECL2 amino acid residues. Amino acids involved in such intramolecular interactions included Y37, H175, Y108, Y251, E283, and S180. The intramolecular hydrogen bond interactions observed among different transmembrane (TM) regions, and between TM and ECL2 loop presumably stabilize the unbound conformation of CCR5 (). In order to determine the structures of AK317, and AK530 complexed to CCR5, we utilized a novel algorithm to incorporate receptor flexibility and induced-fit effects.56
The optimized AK317-CCR5 and AK530-CCR5 complex structures indicated a rotation of Y108 (TM3) away from both Y251(TM6) and E283(TM7) to make room for the inhibitor within the transmembrane helices. As a result of inhibitor binding, the hydrogen bond between Y108 and E283 and that between E283 and S180 seen in the unliganded CCR5 were disrupted, and these residues form hydrogen bond or tight van der Waals interactions with the inhibitors instead (–). The disruption of inter-helix residue interactions may cause movement of the TM helices and changes in the conformation of ECL2. A similar mechanism, involving disruption of inter-helix hydrogen bond interactions, is thought to be responsible for changes in loop conformation in rhodopsin,27; 29
and in the activation of β2
It is of note that Y37(TM1) is involved in a hydrogen bond interaction with ECL2 via H175. We have observed that Y37 interacts with SCH-C, and TAK-779.22
These arrangements of hydrogen bond interactions between transmembrane helices and ECL2 should also provide an explanation of the reason these inhibitors exert potent antiviral activity against HIV-1. Thus, the disruption of hydrogen bond interactions between TM helices, and between TM helices and ECL2, should be a mechanism of allosteric inhibition observed by the binding of CCR5 inhibitors to a binding domain residing mostly within the transmembrane residues.
We also examined the interplays of ECL2 and selected amino acid residues that consist of the largest hydrophobic cavity within CCR5, which accommodate small molecule CCR5 inhibitors. Our previously published data 22
and data by others 19; 23
showed that two CCR5 inhibitors (SCH-C, TAK-779) have no direct interactions with amino acid residues in extracellular domain including ECL2. However, all three SDP-based CCR5 inhibitors we examined (AK530, AK317, and APL) had substantial interactions with amino acids in ECL2, in particular with C178 located in the antiparallel β-hairpin structural motif of ECL2 and K191 located at the interface of ECL2 and TM5 (– ). The C178A substitution virtually abrogated the binding of the three inhibitors to CCR5. C178 is presumed to form a disulfide bond with C101 of TM3 and seems to be critical for the conformation of ECL2. The disruption of the disulfide bond with C178A mutation may result in decreasing the binding of the three SDP-based inhibitors. Our previous observation,22
and reports from others,19; 23
that the same C178A substitution did not affect the CCR5 binding of two other CCR5 inhibitors, SCH-C and TAK-779, suggest that C178 plays a unique but critical role in the binding of AK530, AK317, and APL. In the present study, K191A substitution also virtually nullified CCR5 binding of AK317 and APL (), although it did not significantly affect the binding of AK530 probably due to the absence of hydrogen bonding between AK530 and K191 (). Taken together, these data suggest that unlike the cases of SCH-C and TAK-779, at least a part of the hydrophobic cavity, where AK530, AK317, and APL are lodged within CCR5 involves ECL2. The disruption of ECL2’s β-sheet structure by the removal of disulfide bond through C101A and C178A substitutions virtually nullified both the binding of all three CCR5 inhibitors and the HIV-1 gp120-elicited fusion. This strongly suggests that ECL2 plays a crucial role not only in the binding of the three CCR5 inhibitors but also in the interaction of HIV-1 envelope glycoproteins with CCR5. The data also suggest that at least two amino acids in ECL2, C178 and K191, can be potential targets for the design of CCR5 inhibitors.
Several studies have shown that the resistance against a CCR5 inhibitor emerged without the change of coreceptor usage.38–43
Resistant R5-HIV-1 variants were reportedly obtained by passage 22 for AD101,39
passage 43 for TAK-652,40
passage 20 for VVC,42
and passages 12–18 for MVC. 41
However, we have failed in selecting R5-HIV-1 variants resistant to APL even after 60 passages in vitro
(over ~1.5 years) (Nakata et al.
unpublished), although the possibility of the emergence of HIV-1 variants resistant to APL cannot be ruled out in other settings.44
Pugach et al.
demonstrated that one of the mechanisms by which HIV-1 becomes resistant to CCR5 inhibitors such as VVC, is by "noncompetitive resistance", a process in which a resistant virus continues to enter target cells regardless of the concentration of the inhibitor, once HIV-1 acquires an ability to use the inhibitor-bound CCR5 for entry.43
Of note, Westby et al.
reported that a resistant virus against maraviroc (MVC) retained susceptibility to APL, suggesting that the virus can use MVC-bound CCR5 for entry but can not use APL-bound CCR5.41
Thus, APL is likely to have such a profile that does not allow or delay HIV-1 acquisition of an ability to utilize the "APL-bound" CCR5 for its cellular entry. This potentially favorable property of APL may be related to the direct interactions of APL with amino acids in ECL2, producing “substantially distorted” ECL2, with which HIV-1 gp120 cannot get engaged for its cellular entry, while certain unique allosteric changes of ECL2 conformation following the binding of APL might also explain the substantial delay or lack of the emergence of APL-resistant HIV-1.
The present data, taken together, demonstrate that structural modeling analysis coupled with CCR5 binding affinity data should help understand structural/molecular mechanism of the inhibition of HIV-1 infection by CCR5 inhibitors. The data should not only help delineate the structural dynamics of CCR5 following ligand binding but also aid in the design of therapeutic inhibitors. The data, in particular, demonstrate that through studying the properties of inhibitor-unbound and -bound CCR5, transmembrane residues such as Y108, Y251, and E283 are important for both gp120 fusion, HIV infectivity,22
and inhibitor binding. The loss of hydrogen bond interactions among these key transmembrane residues and the interactions between E283 and S180, which are essential for the formation and maintenance of the binding pocket for CCR5 inhibitors, might be responsible for changes in ECL2 conformation, providing insights to the mechanism of gp120 inhibition.