Isoform-Specific Inhibition of Cyclophilins

Abstract

Cyclophilins belong to the enzyme class of peptidyl prolyl cis/trans isomerases which catalyze the cis/trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family CypA and CypB exhibit specific cellular functions in several inflammatory diseases, cancer development and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4+ T cells by the inhibitor provided biological proof of discrimination in vivo.

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“…Our FEP/MC simulations have indicated that 2 takes advantage of these subtle differences via different binding motifs that fine-tune their selectivity for CypA by exclusively weakening their nonbonded interactions with the catalytic Arg63 and Asn110 residues in CypB. Relative free energies of binding, ΔΔG bind , given in Table 2 for the 2a-2c inhibitors were computed in both CypA and CypB using the MC/FEP transformation sequence given in Figure 9 and were found to yield excellent agreement with the experimental free-energy differences derived from the K i protease-free PPIase assay at pH 7.8 and 283K [113,189]. Figure 10 shows two binding modes for 2a in the overlaid CypA and CypB active sites where the lighter colored structure is the preferred binding conformation in CypA and the darker structure is favored in CypB.…”

“…This isomerization has been identified as the rate-limiting step in protein folding [106,107]. Review of the biochemistry of the PPIases is well-represented in the literature with an emphasis on overall biological relevance [108,109], foundational biochemistry [110,111], mechanism [112], inhibitors [113][114][115][116], and possible therapeutic utility [117]. Eight human Cyps with molecular masses ranging from 18 to 150 kDa [107] and an additional 12 multidomain Cyps (masses up to 352 kDa) have been reported that contain a highly conserved active site, making specific inhibition of a particular family member difficult [118].…”

“…It has only been shown recently that small molecules can differentiate between isoforms of cyclophilins, particularly CypA and CypB. These results have emanated primarily from the laboratories of Fisher [113,183,184] and Li [185][186][187]. Recent structural analysis of known human cyclophilins utilizing both X-ray crystal structures and homology modeling assuming an invariant Arg (Arg55 and Arg63 for CypA and CypB, respectively) have identified two specific locations near the catalytic site likely to confer isoform selectivity: the S2 pocket and the S1′ pocket.…”

“…Our recent computational study utilizing FEP/MC simulations [189] elucidated the origin of the experimentally observed nanomolar inhibition of CypA by acylurea-based compounds, 1a-1j, featuring variations of R 1 and R 2 λ where the 2-Cl,6-F-phenyl ring derivative (1a) was the best reported inhibitor with an IC 50 of 2.6 nM for CypA inhibition. As a point of comparison CsA exhibits a 2.9 nM inhibition of CypA [113]. Table 1 provides the computed ΔΔG bind values with the correct binding affinity trend predicted when compared to the IC 50 enzyme inhibition assay results.…”

“…Compound 2a (R 1 = NO 2 , R 2 = OH, and R 3 = NO 2 ) is particularly interesting because its K i value, as determined by a fluorescence quench assay, is reported to be 0.52 ± 0.15 μM in CypA and >100 μM in CypB implying a >200-fold selectivity between the cyclophilins from in vitro and in vivo experiments despite a completely conversed active site [113].…”