Diverse dynamics features of novel protein kinase C (PKC) isozymes determine the selectivity of a fluorinated balanol analogue for PKCε

Background (−)-Balanol is an ATP-mimicking inhibitor that non-selectively targets protein kinase C (PKC) isozymes and cAMP-dependent protein kinase (PKA). While PKA constantly shows tumor promoting activities, PKC isozymes can ambiguously be tumor promoters or suppressors. In particular, PKCε is frequently implicated in tumorigenesis and a potential target for anticancer drugs. We recently reported that the C5(S)-fluorinated balanol analogue (balanoid 1c) had improved binding affinity and selectivity for PKCε but not to the other novel PKC isozymes, which share a highly similar ATP site. The underlying basis for this fluorine-based selectivity is not entirely comprehended and needs to be investigated further for the development of ATP mimic inhibitors specific for PKCε. Results Using molecular dynamics (MD) simulations assisted by homology modelling and sequence analysis, we have studied the fluorine-based selectivity in the highly similar ATP sites of novel PKC (nPKC) isozymes. The study suggests that every nPKC isozyme has different dynamics behaviour in both apo and 1c-bound forms. Interestingly, the apo form of PKCε, where 1c binds strongly, shows the highest degree of flexibility which dramatically decreases after binding 1c. Conclusions For the first time to the best of our knowledge, we found that the origin of 1c selectivity for PKCε comes from the unique dynamics feature of each PKC isozyme. Fluorine conformational control in 1c can synergize with and lock down the dynamics of PKCε, which optimize binding interactions with the ATP site residues of the enzyme, particularly the invariant Lys437. This finding has implications for further rational design of balanol-based PKCε inhibitors for cancer drug development. Electronic supplementary material The online version of this article (10.1186/s12859-018-2373-1) contains supplementary material, which is available to authorized users.


Note 1: Charge state validation of 1c in novel PKC isozymes
Our previous investigation proposed that 1c has same charge state when it is bound to the ATP site of PKCε or PKA [1]. Balanoid 1c has a positive formal charge on the amine (N1) group on the azepane ring. Additionally, negative formal charges present on the phenol (C6′′OH) and carboxylic (C15′′OOH) groups of the benzophenone moiety. Thus, we assigned these charges to PKCδ-, PKCε-, PKCη-, and PKCθ-bound 1c (annotated as charge combination I). To increase the number of data points for charge state validation, we also included PKAbound 1c to charge combination I. Each kinase-1c complex was then subjected to molecular dynamics simulation. Binding energy value of each complex was calculated using MMGBSA approach within 10-ns-sliding-window every 10 ns. The resulting ∆ ° values were evaluated further by plotting them over the MD trajectory and calculating their correlation coefficients with experiment.
The ∆ ° profile ( Figure S1.A) shows that binding energy values of PKCδ-bound 1c are outliers, even though the r 2 plot depicts good correlations, above 0.70 from 40 to 90 ns ( Figure S1.B). The experimental binding affinity order for 1c to kinase is PKCε > PKCδ ≈ PKA > PKCη ≈ PKCθ. The ∆ ° values of 1c bound to PKCδ, however, are close to those of PKCη-and PKCθ-bound 1c ( Figure S1.A). We then revisited the charge state of PKCδ-bound 1c. Our preceding study on charge state exploration of fluorinated balanoids suggests that local environment of the ATP site may influence the charge state of the phenol (C6′′OH) group, where it can be charged or neutral [1]. According to this result, we assigned C6′′OH neutral for PKCδ-bound 1c, whereas charge states of the remaining isozyme-bound 1c molecules are unchanged. We labeled those charge states as charge state combination II and run another MD simulation. The resulting ∆ ° profile shows the calculated binding energy values of PKCδ-bound 1c moves closer to those of PKA-bound 1c ( Figure S2.A), which is expected.

General overview
Decomposition analysis of per-residue binding energy at the ATP site ( Figure S7) suggests that novel PKC isozymes contribute differently to 1c. The only exception is the contribution from the adenine subsite where the benzamide moiety shows similar docking position (main paper: Figure 2). The adenine subsite residues also show similar H-bond profiles with the benzamide moiety (Table S8), particularly among novel PKC isozymes.
In the ribose subsite, two residues among kinases studied exhibit different binding energy contributions to 1c binding ( Figure S7). The most dispersed contributions are ranging from -  (Table S8).
As the regions where the flexible Gly-rich loop is mainly located, the residues of the triphosphate subsite also show diverse binding energy contributions to 1c binding among the given kinases. In particular, the invariant Lys provides the most varied contributions (-6.34 to -16.83 kcal.mol -1 ). Additionally, this residue frequently provides the strongest binding energy contribution among the other residues in the ATP sites of the kinases, except in the ATP site of PKCδ. The other corresponding residues for Glu403 in PKCη and Glu428 in PKCθ, which cause repulsive binding to 1c ( Figure S7), also shows diverse binding energy values (-2.66 ± 3.60 kcal.mol -1 ).
As mentioned in the paragraph above, the invariant Lys residues among novel PKC isozymes provides the strongest binding energy contribution to 1c. This result is similar to our previous study [2] that the invariant Lys residue in PKA and PKCε contribute a major binding contribution to the binding of balanoids studied. The invariant Lys can provide four different ways to non-covalently bind 1c, which include H-bond, charge-charge, alkyl-π hydrophobic, and cation-π interactions. The following discussions are presenting the effect of the dynamics features of nPKC isozymes on the interactions between the invariant Lys and 1c as well as binding energy contributed by this residue.
The invariant Lys378 in PKCδ provides a relatively weaker binding contribution to 1c. This may be because the unique dynamics of PKCδ and the uncharged phenolic C6′′OH group which leads to fewer possibilities for interacting with Lys378. The group non-covalently binds Lys378 only through an alkyl-π hydrophobic interaction and very a transient H-bond (0.4% conservation, Table S8). As a result, 1c only obtains -8.79 kcal.mol -1 from Lys378 when bound to PKCδ. However, in the ribose subsite, Asp491 stabilizes the binding of 1c with -18.01 kcal.mol -1 . This binding energy contribution is associated with a conserved H-bond (99.3%) and a salt bridge between the negatively charged side chain of Asp491 and the protonated N1 amine group in the azepane ring (main paper: Figure 5.B).

Unfavorable binding energy
Diverse dynamics of nPKC isozymes affect 1c binding in terms of shape, conformational bias, docking mode (main paper: Figure 2), and also interactions with the residues in the ATP sites. These different effects may implicate in unfavorable binding energy contributions that weaken the affinity of 1c to novel PKC isozymes. For example, Glu403 in PKCη contributes a repulsive binding energy of 0.14 kcal.mol -1 to 1c, whereas Asp421 and Glu428 in PKCθ serves 0.01 and 0.06 kcal.mol -1 ( Figure S7), respectively. In PKCδ, the azepane ring of 1c involves in a repulsive binding energy (0.19 kcal.mol -1 ) with Lys475 in the ribose subsite. This unfavorable binding energy violates the strong binding energy contributed (-18.01 kcal.mol -1 ) by Asp491 ( Figure S7), reducing the binding affinity of 1c to PKCδ.
Overall decomposition analysis of per-residue binding energy suggests that although the ATP site residues among novel PKC isozymes are highly similar, the different dynamics of novel PKC isozymes result in dispersed profiles of binding energy contributions ( Figure S7) to 1c. Interestingly, while the binding of 1c to PKCδ, PKCη, and PKCθ cause unfavourable binding interactions, PKCε-bound 1c is free from such binding interaction. Additionally, the binding of 1c to PKCε optimize the interactions with invariant Lys437 which was also reported in our previous work [2]. Hence, C5(S)-fluorine perturbation in 1c sets up properly the azepane ring and remotely drives the conformation of the benzophenone moiety to optimally interact with residues in the triphosphate subsite, particularly invariant Lys, which is only possible with PKCε. As a result, balanoid 1c has a high affinity and is selective to PKCε over PKA, other novel PKC isozymes, and also classical as well as atypical PKC isozymes (as suggested by ∆ ° calculation in Table S8).      Homology modelling and MD simulations for PKCα, βI, βII, γ, ɩ, and ζ were conducted as described in sub-sections of Homology modelling and Molecular dynamics simulation preparation and protocol in Materials and Methods. Phosphoryl moiety was added to each kinase as listed in Table S1.     in binding selectivity to PKCε. As seen in Table S6, 1c has decreased binding affinity to all PKC isozymes, except PKCε.