Molecular Dynamics Pinpoint the Global Fluorine Effect in Balanoids

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Molecular Dynamics Pinpoint the Global Fluorine Effect in Balanoids Binding to PKC# and PKA Ari Hardianto, Fei Liu, and Shoba Ranganathan J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.7b00504 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Journal of Chemical Information and Modeling

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Molecular Dynamics Pinpoint the Global Fluorine

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Effect in Balanoids Binding to PKCε and PKA

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Ari Hardianto, † Fei Liu, † Shoba Ranganathan *, †

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† Department of Chemistry & Biomolecular Sciences, Macquarie University, Sydney, NSW

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2109, Australia

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ABSTRACT: (-)-Balanol is an ATP mimic that inhibits protein kinase C (PKC) isozymes and

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cAMP-dependent protein kinase (PKA) with little selectivity. While PKA is known as a tumour

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promoter, PKC isozymes can be tumour promoters or suppressors. In particular, PKCε is

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frequently involved in tumorigenesis and a potential target for anticancer drugs. We recently

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reported that stereospecific fluorination of balanol yielded a balanoid with enhanced selectivity

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for PKCε over other PKC isozymes and PKA, although the global fluorine effect behind the

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selectivity enhancement is not fully understood. Interestingly, in contrast to PKA, PKCε is more

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sensitive to this fluorine effect. Here we investigate the global fluorine effect on the different

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binding responses of PKCε and PKA to balanoids using molecular dynamics (MD) simulations.

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For the first time to the best of our knowledge, we found that a structurally equivalent residue in

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each kinase, Thr184 in PKA and Ala549 in PKCε, is essential for the different binding

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responses. Furthermore, the study revealed that the invariant Lys, Lys73 in PKA and Lys437 in

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PKCε, already known to have a crucial role in the catalytic activity of kinases, serves as the main

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anchor for balanol binding. Overall, while Thr184 in PKA attenuates the effect of fluorination,

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Ala549 permits remote response of PKCε to fluorine substitution, with implications for rational

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design of future balanol-based PKCε inhibitors.

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Keywords: balanoids; fluorine effect, PKCε; PKA; binding response; Molecular dynamics

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simulations.

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INTRODUCTION

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(-)-Balanol (referred to as balanol) is a fungal natural product1 and a structural mimic of ATP.2

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It has a four ring structure that completely occupies the flexible ATP site of protein kinases

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(Figure 1).2, 3 The benzamide moiety (ring A) resides in the adenine subsite, whereas the azepane

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ring (ring B) occupies the ribose subsite. The benzophenone moiety, comprising rings C and D,

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fills the triphosphate subsite. As the central moiety,4 the azepane ring connects the benzamide

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and the benzophenone moieties, using amide and ester linkages, respectively.

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Balanol non-selectively inhibits cAMP-dependent protein kinase (PKA) and protein kinase C

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(PKC) isozymes at their ATP sites.5 While PKA consistently shows tumour promoting

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activities,6 most PKC isozymes can act as tumour promoters or suppressors, depending on the

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context.7 In breast cancer, for example, PKCβI is a tumour suppressor, whereas PKCα, PKCβII,

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and PKCδ are tumour promoters.7 On the contrary, PKCβI and PKCδ show promoter and

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suppressor activity in prostate cancer, respectively. Nonetheless, PKCε prominently displays

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oncogenic activities and is a potential target for anti-cancer drugs.7 Despite several decades of

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research, the development of isozyme-specific PKC regulators (both activators and inhibitors)

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has remained elusive.8 However, the ATP sites among PKC isozymes are highly homologous8

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and fine tuning ATP mimics, such as balanol, for specific inhibition of PKC isozymes and PKCε

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in particular, is essential for lead compound discovery in cancer therapy.


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Figure 1. (A) Balanol structure, divided into three moieties that occupy the different subsites

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based on structural overlay with ATP.3 (B) Balanol analogues which are fluorinated in the

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azepane moiety.4 1a and 1c are analogues monofluorinated at C6(S) and C5(S), whereas 1d is

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difluorinated at C6 while 1e is trifluorinated at C6 and C5(S). 1b (C6(R) – not shown) is the

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stereoisomer of 1a.

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To enhance selectivity for a specific PKC isozyme, balanol has been subjected to extensive

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structure and activity relationship (SAR) studies,9-13 with PKA also included for comparison.10,

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11, 13

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(ring B),10 and benzophenone moieties (rings C and D).11,

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moiety revealed the importance of C5′OH for PKC inhibition.9 Derivatisation studies on the

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benzophenone ring showed that acidic functional groups are necessary for balanol activity.10, 11

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In some SAR studies,9, 10 the azepane ring was replaced with a five-membered pyrrolidine ring,

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but this replacement did not substantially improve target selectivity of the balanol derivatives.

These SAR studies explored chemical diversifications on the benzamide (ring A),9 azepane


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The studies on the benzamide

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These studies showed that as the central moiety, the azepane ring could tolerate various

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derivatisation, but selectivity remains a challenge.11

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Table 1. Dissociation constant values (Kd) characterising balanoids interacting with PKA or

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PKCε4.

Protein (-)-balanol, 1 kinase

(6S)-F

(5S)-F

(6)-diF

(5S)-(6R,S)-triF

1a

1c

1d

1e

9.2 ± 0.8

43 ± 4

Kd (nM) PKA

5.9 ± 0.5

7.9 ± 0.5

PKCε

0.73 ± 0.06

19 ± 8

6.4 ± 0.1

0.4 ± 0.02 110 ± 19

38 ± 9.5

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Fluorine is an important substituent in medicinal chemistry and widely utilised in drug

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development due to its distinctive features.14, 15 It has a small atomic size of 1.47 Å which is

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close to 1.20 Å for hydrogen (Supporting information: Note 1 and Table S1). Thus, hydrogen

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atom substitution by fluorine does not significantly increase the size of a molecule.16, 17 As the

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most electronegative element with Pauling electronegativity of 4.00, fluorine forms a highly

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polarized C–F bond. The attraction of partial positive and partial negative charges on carbon and

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fluorine atoms, respectively, makes the C-F bond short and strong with significant ionic

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character.17 Due to its unique characters, the C–F bond can cause a perturbation to the

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conformation of a molecule via dipole–dipole interactions, charge–dipole interactions, and

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hyperconjugation effects (referred to as fluorine perturbation4; see Supporting information: Note

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1 and Figure S1), which can control a molecule shape.15,

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offers productive effects on conformation, intrinsic potency, pKa, metabolic pathways,

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membrane permeability, and pharmacokinetic properties.15 Thus, fluorination may improve

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balanol selectivity to a specific protein kinase.

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Proper fluorination of a molecule

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In our recent study,4 we showed that stereospecific fluorination provides a tool to achieve

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balanol-kinase selectivity in highly homologous ATP sites. We stereospecifically incorporated

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single fluorine (Figure 1.B, Table 1: 1a and 1c) and multiple fluorine atoms (Figure 1.B, Table 1:

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1d and 1e) on the azepane moiety of balanol for the first time. 1b (C6(R) – not shown in Figure

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1) is the stereoisomer of 1a. Binding affinity measurements of balanol and its fluorinated

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analogues (referred to as balanoids) to PKA and PKC isozymes were assayed to assess the effect

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of azepane fluorination on protein selectivity.4 We found that fluorination results in different

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responses of the kinases to balanoids. As 1b showed very similar binding affinity values to 1a,4

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this balanoid has not been investigated further. In particular, the C5(S)-fluorinated balanol

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analogue, 1c, enhanced the binding affinity and selectivity for PKCε (see Table 1). This study

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also revealed an interesting result that, in contrast to PKA, PKCε is more sensitive to the effect

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of fluorination on balanoids.4

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Computational analysis to understand the basis of varied binding responses of PKA and PKCε

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to balanoids will provide useful information for further development of balanol-based ATP-

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mimicking inhibitors. Our previous molecular docking study, which complements binding

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affinity measurements, suggests that stereospecific fluorination provides conformational

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adjustment of balanol in a kinase-dependent manner.4 Here, we present molecular dynamics

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(MD) simulations, for a more detailed and insightful interaction analysis18 of balanoids to the

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ATP sites of PKA and PKCε, such as hydrogen bond (H-bond) conservation analysis. The

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analysis can then be used to study protein flexibility and the fluorine effect or fluorination

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perturbation.4 This method also offers an investigation of the ligand conformational change

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during binding and an estimate of the binding energy from ensemble conformations. Moreover,


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since protein kinases are known to provide induce-fit binding interactions,2 MD simulations give

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a chance to explore this plasticity in ligand binding19 using PKCε and PKA.

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In order to carry out MD situations, the charge state (the net charge derived from the formal

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charges on ionisable functional groups) of balanoids bound to PKA and PKCε need to be

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correctly assigned. In our previous study,20 we computationally investigated the charge state

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characteristics of balanoids in the ATP sites of PKA and PKCε. Our results identified the

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charged state of different functional groups on balanoids (1-1e) that correlated with experimental

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binding energy values (Table 1). All balanoids have consistent charge states in both PKA and

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PKCε, except 1a (Supporting information: Figure S2). Briefly, the natural balanol 1 and the most

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potent analogue 1c bear charges on the azepane ring (N1), the phenolic group (C6′′OH) and the

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carboxylate (C15′′O2H) on the benzophenone moiety. The multiple fluorinated analogues (1d

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and 1e) have charges on the C6′′OH and the C15′′O2H. Balanoid 1a can exit in two charge states.

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One species of 1a (referred to as 1a.1) only carries a charge on the C15′′O2H, whereas the other

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(1a.2) bears charges on the N1, C′6′OH, and C15′′OOH. In PKA, 1a.1 is the preferred state while

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in PKCε, both charge states correlate well with the experimental binding affinity value.20 This

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charge state information is important for conducting the MD analysis on balanoids bound to the

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ATP sites of PKA as well as PKCε.

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Here, we present MD analysis of balanoid binding in the ATP sites of PKA and PKCε, using

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the charge states determined in our earlier study.20 The results suggest that stereospecific

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fluorination influences the shape, conformational bias, and bound position of the azepane ring in

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the ribose subsite that contribute to differential binding responses of PKA and PKCε to

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balanoids. We also find that an invariant Lys, which is essential for the kinase activity, is the

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major contributor in the binding energy of balanoids in both kinases. More importantly, to the


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best of our knowledge, we find, for the first time, that a single amino acid residue residing in the

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ribose subsite and close to the adenine subsites, Thr184 in PKA and Ala549 in PKCε, is essential

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for global fluorine effect in distinct binding responses of the two kinases. This residue plays a

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unique role in modulating the remote effect of fluorine onto other moieties of balanoids and thus

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prescribe the different levels of sensitivities from PKA and PKCε to different fluorine

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substitutions. Finally, this study provides pertinent information for future rational design of

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balanol analogues to achieve improved PKC isozyme selectivity.

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MATERIALS AND METHODS

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Homology modelling

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We built kinase domains of human PKA (UniProt ID: P17612) and PKCε (UniProt ID:

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Q02156) which bind balanol using a homology modelling approach as described previously.20

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The X-ray crystal structure of mouse PKA-balanol (PDB ID: 1BX6)3 was used as the template

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for homology modelling of human PKA. In modelling human PKCε, we utilised two X-ray

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crystal structure templates, the mouse PKA (PDB ID: 1BX6) and a human PKCη (PDB ID:

139

3TXO). To examine the structural conservation between both templates, we carried out structural

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alignment using jCE algorithm.21 Subsequently, we used CLUSTALX 2.122 to align the sequence

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of human PKCε to 1BX6 and 3TXO sequences. Additionally, the resulting sequence alignment

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was manually edited to map the ‘open’ conformation23 of the Gly-rich loop (GXGXXG) from

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1BX6 to PKCε model. Calculation of the percentage identity and similarity between human PKA

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and PKCε sequences was carried using MatGAT.24

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Homology modelling was performed using MODELLER 9.14.25 The modelling process

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included the balanol transfer, from mouse PKA-balanol (PDB ID: 1BX6) to the generated query

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structure, to maintain the three dimensional (3D) features of the ATP binding site in the resulting


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model. The resulting models were assessed using the Discrete Optimized Protein Energy

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(DOPE) score26 which reflects the quality of the model. Furthermore, we superimposed models

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from multiple runs of homology modelling and selected the structure which possessed residues

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adopting consensus conformations. The selected model was also evaluated using Ramachandran

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plot on PROCHECK webserver.27

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Molecular dynamics simulation preparation and protocol

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As fully activated human PKA and PKCε are phosphorylated at specific sites,28 we added

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phosphate groups on PKA (Thr198 and Ser339) and PKCε (Thr566, Thr710, and Ser729) using

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Discovery Studio Visualizer.29 The starting conformations of balanoids were adopted from the

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ligand in 1BX6 (mouse PKA with bound balanol). Atomic charges of balanoids were computed

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using the Austin Model 1 - Bond Charge Corrections (AM1-BCC)30 protocol in

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AmberTools16.31 To determine parameters for balanoids, we used the General Amber Force-

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fields (GAFF)32 and the parmchk program in AmberTools16. For protein force-fields, we

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assigned regular amino acid residues and their side chains with ff14SB33 and phosphorylated

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residues with phosaa10.34

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In the MD system preparation, we solvated each complex of either PKA- or PKCε-balanoid

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with explicit water molecules using TIP3P,35 with a bounding box of 10 Å. We also added

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sufficient Na+ and Cl- ions to achieve neutral charge and a salt concentration of 0.15 M, which is

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the equivalent of physiological salt concentration, using the tleap utility in AmberTools16.

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We utilised GPU-accelerated Particle-Mesh Ewald Molecular Dynamics (PMEMD), as

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implemented in Amber16,31 throughout simulations where periodic boundary conditions were

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applied. For every simulation, two consecutive steps of energy minimisation were performed

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with restraints of 25 and 5 kcal.mol-1.Å-2 to the solute, the protein-ligand complex. The system


8

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temperature was then increased to 300 K under NVT condition for 50 ps, followed by

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equilibration steps. In the following NPT simulation over 50 ps, the system density was

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equilibrated to 1 g.cm-1. Subsequently the system was switched to NVT. In the NVT simulation,

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the restraint on solute was gradually decreased from 5 kcal.mol-1.Å-2 by 1 kcal.mol-1.Å-2 every 50

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ps where in the last 50 ps simulation, the restraint was totally removed.

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Production-phase for each system was simulated at 300 K under the NPT to yield 100 ns

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trajectory, which was recorded every 10 ps. Long-range electrostatic interactions were treated by

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particle-mesh Ewald (PME) method. Meanwhile, a 10 Å cut-off was used for short-range non-

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bonded interactions which was also employed by others.36,

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implemented to constrain all bonds involving hydrogen atoms. To maintain constant pressure

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and temperature, we used algorithms of Berendsen barostat39 and Langevin thermostat,40

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respectively.

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A SHAKE algorithm38 was

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Binding energy calculation

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° Experimental binding energy values (∆ ) of balanoids to PKA or PKCε were calculated

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from dissociation constant (Kd) values (Table 1).4 At equilibrium and under standard conditions,

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the binding energy is directly related to the equilibrium constants and can be computed using the

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following formula (eq. 1):

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° ∆ = − =

(1)

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° where ∆ is experimental binding energy, Ka and Kd are association and dissociation constant

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respectively, R is the universal gas constant, and T is absolute temperature.


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Estimated binding energy values of balanoids to PKA or PKCε as well as their per-residue

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decomposition were determined using Molecular Mechanics Generalised Born Surface Area

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(MMGBSA) method41 as implemented in MMPBSA.py.42 The MMGBSA binding free energy

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° (∆ ) is calculated as follows in eq. 2:

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° ∆ = 〈 〉 − 〈 〉 − 〈 〉

(2)

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° where 〈 〉 , 〈 〉 , and 〈 〉 are the average value of ∆ for complex, enzyme,

201

and ligand, respectively, over snapshots i extracted from MD trajectories. can be broken

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down as shown in eq. 3:

203 204

= ! + #$ + #$

(3)

205 206

where ! is the gas phase energy, #$ the electrostatic portion of solvation energy

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determined using Generalised Born (GB) implicit solvent model, and #$ the hydrophobic

208

contribution to the solvation energy. The hydrophobic contribution is approximated by the

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Linear Combination of Pairwise Overlaps (LCPO) method.43 ! is estimated by the molecular

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mechanics energy of the molecule consisting of internal energy terms: bond (Ebond), angle

211

(Eangle), and torsion energies (Etorsion) as well as van der Waals (EvdW), and electrostatic

212

interactions (Eel) (eq. 4).

213 ! = % !&' + % !' &'#

' #

+

(#

(#

*+

*+

% !(#' + % !$) + % !(#((

(#'#

214

(4)


10

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Internal energy terms were omitted in this study, since single-trajectory MD simulations were

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used in the calculation.44

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MD trajectory analysis

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We utilised cpptraj program in AmberTools16 to analyse MD trajectories. The analysis

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includes computation of H-bond conservation, dihedral angles in the azepane ring, Solvent-

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Accessible Surface Area (SASA), and Root-Mean-Square Fluctuation (RMSF).

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Images and graphs generation

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Conformational ensembles of balanoids were generated in VMD 1.9.2.45 For analysis

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purposes, balanoid conformations were collated at intervals of 1250 frames, from the MD

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trajectories for 50-100 ns (5000 frames). Representative non-covalent interactions of kinase-

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balanoid, according to H-bond conservation analysis, were visualised using BIOVIA Discovery

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Studio Visualizer 2016.29 2D structures of balanoids were sketched in BIOVIA Draw 2016.46 All

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plots were generated using an R47 package (ggplot2)48 in RStudio 0.99.892.49 GNU Image

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Manipulation Program (GIMP) 2.8.1450 and Inkscape 0.48.551 were utilised for image editing.

231 232

RESULTS AND DISCUSSION

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Overall effect of balanoid binding on conformational freedom of PKA and PKCε

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The binding of balanoids to PKA and PKCε reduces the conformational freedom of both

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kinases, regardless of their binding affinity values (Supporting information: Figure S3). For

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instance, the binding of 1e has a similar effect to other balanoids in decreasing conformational

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freedom of PKA, although 1e binds relatively weakly to this kinase. Likewise, 1c and 1d, ligands


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with the strongest and the weakest affinity to PKCε respectively,4 reduce the conformational

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freedom of PKCε. Moreover, the Wilcoxon sum-rank test shows that the binding of 1c or 1d

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leads to a statistically significant reduction of conformational freedom of the Gly-rich loop, the

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ATP pocket, and even the entire kinase domain of PKCε (Supporting information: Table S2).

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To investigate whether the strength of balanoid binding to PKA or PKCε is related to their

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Solvent-Accessible Surface Area (SASA), we conducted accessible surface calculations. The

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results suggest that there is no notable effect on SASA on the binding affinity of balanoids to

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PKA or PKCε. For example, 1d- and 1c-bound PKCε have comparable SASA (Supporting

246

information: Figure S4). Likewise, 1e and other balanoid-bound PKA complexes show similar

247

SASA.

248 249 250

Comparison of conformational ensembles of balanoids in the ATP sites of PKA and PKCε

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Sequence analysis24 shows that PKA and PKCε share 43% identity and 68% similarity in their

252

kinase domains. At the ATP sites, however, their residue identity and similarity level are higher

253

(68% and 79%, respectively). Of the nine ATP-binding residues (indicated by green bars in

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Supporting Information: Figure S5) six are conservatively substituted and two are non-

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conservatively substituted. Visual inspection of overlaid PKA and PKCε homology models (data

256

not shown) reveals that two (occurring in the Gly-rich loop of the kinases, in Supporting

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Information: Figure S5, underlined by orange bars) of these nine non-identical residues

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participate in the ATP site only through their backbones. Other non-identical residues between

259

PKA and PKCε mainly reside outside of the ATP site.

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The natural product 1 binds stronger to PKCε than PKA (0.73 nM vs. 5.9 nM,4 respectively).

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Superimposition of conformational ensembles (Figure 2.A) revealed that 1 binds differently in


12

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the ATP sites of the two kinases. Overall, 1 in PKA is more flexible than that in PKCε

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(Supporting information: Note 2). The flexibility of each moiety of the ligand increases in the

264

following order: benzamide moiety < azepane ring < benzophenone moiety. The benzamide

265

moiety shows comparable conformation and binding mode in both kinases, with limited

266

flexibility, whereby the azepane ring in PKA is more mobile than in PKCε. The largest

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conformational variations occur on the benzophenone moiety in the ATP site of PKA and PKCε.

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In PKA, the benzophenone moiety of balanol (1) is closer to the ATP site entrance, whereas in

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PKCε, this moiety moves deeper inside the triphosphate subsite.


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Figure 2. Superimposition of conformational ensemble of balanol (1) and its fluorinated

273

analogues bound to PKCε and PKA. (A) depicts the superimposition of PKCε- (grey) and

274

PKA-bound 1 (white) where the right side figure displays a 90°-rotated view. Superimposition of

275

1 (shown in black) and its fluorinated analogues (1a-1e in green, pick, blue and orange,

276

respectively) bound to PKA is shown in (B) and to PKCε in (C). 1a.1 is shown, as a

277

representative for 1a. Each ATP subsite occupied by the respective balanol moiety is represented


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in dashed lines: blue for the adenine subsite, yellow for the ribose subsite, and orange for the

279

triphosphate subsite.

280

Stereospecific fluorination of 1 on its azepane ring results in large changes in binding affinity

281

to PKCε (Table 1). In one instance, C5(S)-fluorinated analogue (1c) shows improved binding

282

affinity, compared to 1. By contrast, such binding affinity variation is not observed in PKA

283

(Table 1). PKA is less sensitive to the effect of fluorination than PKCε,4 which is consistent with

284

superimposition of conformational ensembles of balanoids (Figure 2.B and C). During binding,

285

all fluorinated balanoids in the ATP site of PKA exhibit conformational changes on their azepane

286

moiety relative to 1, but these changes do not alter the binding conformations of the other

287

moieties (Figure 2.B). Slight alterations occur only on the benzophenone moiety. Balanoid 1e,

288

which is the ligand with the weakest affinity to PKA, appears to have its benzophenone moiety

289

closer to the entrance of the ATP site. In the ATP site of PKCε, conformational changes on the

290

azepane ring are accompanied by changing positions of both the benzamide and benzophenone

291

moieties (Figure 2.C). As the ligand with the strongest affinity to PKCε, 1c shows enhanced

292

interaction to the protein whereby its benzophenone moiety is deeper inside the triphosphate

293

subsite. This is contrasted by 1d, the weakest binding ligand to PKCε, in which case the

294

benzophenone moiety is found closer to the ATP site entrance. The residential position of the

295

benzophenone moiety seems to correlate with the binding affinity of balanoids, whereby the

296

deeper this moiety is buried in the triphosphate subsite, the stronger the binding affinity.

297 298 299

A single residue important for the local and remote effects of fluorine substitution in PKA and PKCε


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Thr184 in the ATP site of PKA and its corresponding residue in PKCε, Ala549, may be a

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critical amino acid residue for the varied response of both kinases to balanoids due to local and

302

remote effects of fluorine substitution. As previously reported,52 the mutation T184A expands

303

the ATP site of PKA and improves the binding of several kinase inhibitors, particularly JNJ-

304

7706621, VX-680, and H-89. However, no mutation study of Ala549 in PKCε has yet been

305

reported. The mutation study in PKCε addressed only the phosphorylation sites,53 rather than the

306

ATP-binding site as a potential target for kinase inhibition.

307 308

Figure 3. Interactions between representative balanoids and the invariant Lys and the

309

azepane ring residues of PKA and PKCε. 1c and 1d are respectively the best and the worst

310

ligands for PKCε (Table1). (A) and (B) respectively depict interactions of 1c in PKA and PKCε

311

while (C) and (D) show interactions of 1d in PKA as well as PKCε, respectively. The invariant

312

Lys and the representative azepane ring are highlighted in cyan and orange, respectively. The


16

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313

key determinant residue (Thr184 in PKA and Ala549 in PKCε) is highlighted in yellow. Water-

314

mediated intramolecular interactions are highlighted in green. H-bonds are indicated by green

315

dashed lines, whereas electrostatic or ion pair interactions are depicted by orange dashed lines.

316

As 1c and 1d are respectively the best and the worst ligands for PKCε (Table1), the following

317

sections will focus on these two balanoids. The interactions of the other two balanoids, 1a and

318

1e, are discussed in Supporting Information: Note 3. In PKA, the side chain of Thr184 is a key

319

relay station for a hydrogen bond network between the C1'=O of the amide linkage, the azepane,

320

and the ribose subsite of the protein (Figure 3.A and C, highlighted in yellow). Such a hydrogen

321

bond network helps to anchor the benzamide moiety in the adenine subsite and dampens the

322

transmission of the fluorination effect in the azepane ring. As a result, the cascade effect of

323

fluorination to the benzamide and benzophenone moiety is attenuated (Figure 2.B). On the

324

contrary, the replacement of Thr184 to Ala549 in PKCε (Figure 3.B and D, highlighted in

325

yellow) abrogates the essential H-bonding that holds and minimises the fluorine effect in the

326

azepane ring. As a consequence, perturbations caused by fluorination not only significantly

327

affect the azepane ring locally but also remotely influence interactions of the other moieties,

328

particularly in the benzophenone moiety, with the ATP site residues of PKCε (Figure 2.C).

329

Balanoid 1c (C5(S)-fluorinated balanol analogue) is the best available example that the

330

fluorine effect improves affinity to PKCε but not PKA (Table 1). In PKCε, the presence of a

331

fluorine atom at C5(S) in the azepane ring of 1c allows the intramolecular interaction with NH of

332

the amide linkage that is facilitated by a water molecule (Figure 3.B, highlighted in green).

333

Furthermore, the non-bonding orbital of the fluorine atom (σ*C5(S)-F) appears to be in

334

hyperconjugation with σC6-H, a fluorine effect suggested by Gillis et al.15 and Hunter.17 This

335

may serve to stabilise the azepane ring conformation (Supporting Information: Figure S6, 1c in


17


Page 18 of 29

336

PKCε). Additionally, the ammonium group of 1c form an H-bond with the backbone of Asn537

337

and an ion pair interaction with the side chain of Asp550 (Figure 3.B; orange dashed line).

338

Altogether, these interactions shape the conformation ensemble and bound position of the

339

azepane ring and further position the benzophenone moiety to interact better with residues in the

340

triphosphate subsite. In PKA, the azepane ring of 1c possesses a similar conformation

341

(Supporting Information: Figures S6, S7 and S8) to that of PKCε-bound 1c. Moreover, the same

342

water-mediated intramolecular interaction also occurs (Figure 3.A). Also, the ammonium group

343

of 1c form an H-bond with the side chain of Glu171 and an ion pair interaction with the side

344

chain of Asp185 (Figure 3.A; orange dashed line). Nevertheless, Thr184 anchors the azepane

345

ring and deters the remote effect of C5(S)-fluorination to the benzophenone moiety.

346

The fluorine effect on balanol may lead to a dramatic fall in binding affinity to PKCε as shown

347

by 1d (doubly fluorinated at C6 of the azepane ring, which leads to a zero formal charge on

348

N1)20 (Table 1). Without Thr184 that anchors C1'=O, the uncharged N1 group20 cannot maintain

349

H-bonds with Asp550 (Supporting information: Table S3). Meanwhile, one fluorine atom in 1d

350

interacts with the backbone of Lys416 and slightly moves the azepane moiety (Figure 3.D). Such

351

a change in the position of the azepane ring remotely interferes with the interactions between the

352

benzophenone moiety and residues in the triphosphate subsite. As seen in Table S3 (Supporting

353

information), the benzophenone moiety is missing H-bonds with Phe419 and Lys437, residues at

354

the triphosphate subsite. On the other hand in PKA, the interaction of Thr52 (which corresponds

355

to Lys416 in PKCε) with the azepane ring is counterbalanced by Thr184. The residue Thr184

356

also allows water-mediated intramolecular interactions that involve fluorine substituents and the

357

N1 group (Figure 3.C). These interactions with the azepane ring modify and restrain its


18

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358

conformation. Therefore, unlike the case with PKCε-bound 1d, PKA minimises the remote effect

359

of fluorine substitution on the binding of the benzophenone moiety.

360

Overall, balanoids 1c and 1d together demonstrate the malleability of PKCε to cooperatively

361

accommodate perturbations due to fluorine substitutions, that is absent in PKA. The structurally

362

equivalent residue in each kinase, Thr184 in PKA and Ala549 in PKCε, seems to be crucial for

363

the different responses of PKCε and PKA to the fluorine effect. Details of this important residue

364

and the malleability of PKCε are valuable for designing the next generation of balanoids for

365

improved affinity and specificity to PKCε and not to PKA.

366 367

Invariant Lys as the major contributor residue in balanoid binding

368

The invariant Lys in the triphosphate subsite is the most conserved residue of protein kinases.

369

It participates in the phosphotransfer reaction and orients ATP for catalysis by interacting with

370

the α- and β-phosphate groups of the nucleotide. Furthermore, this residue is also involved in

371

stabilising the conformation of other catalytically active kinases.54 Interestingly, our results

372

suggest that the invariant Lys (Figure 3, highlighted in blue) also frequently acts as a major

373

contributor to balanoid binding (Supporting information: Figure S9). This may indicate that

374

future inhibitor design may need to preserve and optimise interactions to the invariant lysine

375

residue.

376

In PKCε, this invariant residue is Lys437, which contributes higher binding energy (mean = -

377

16.33 ± 3.62 kcal.mol-1, Supporting information: Figure S9) than the corresponding Lys73 in

378

PKA (mean = -11.16 ± 2.06 kcal.mol-1). This higher binding energy could be attributed to

379

Lys437 providing more conserved H-bonding networks than Lys73 (Supporting information:

380

Table S3). Nevertheless, Lys73 in PKA remains the most prominent contributor to binding than


19


Page 20 of 29

381

the other residues in PKA to balanoids, except to 1a. We found that the invariant Lys can non-

382

covalently bind balanoids in four ways: H-bonding, charge-charge, alkyl-π hydrophobic, and

383

cation-π interactions (Supporting information: Figure S10). The last two interactions are

384

conserved in binding of all balanoids (1 to 1e) to PKA as well as PKCε. Such interactions may

385

be responsible for the stronger binding contribution of this invariant Lys (Supporting

386

information: Figure S9) more so than other kinase residues, even with transient H-bonding

387

interaction observed (Supporting information: Table S3).

388

The natural balanol 1 builds an H-bonding network with Lys437 in PKCε via its C1'=O of the

389

amide linkage. The hydrogen bonding network may persist or dissipate due to the effect of

390

fluorine substitutions that occur in the azepane ring of balanoids (Supporting information: Note

391

4). C5(S)-Fluorine substitution in 1c, however, eliminates the H-bonding between the C1'=O and

392

Lys437 and directs its phenolate C6''-O- group to interact intensively with the invariant Lys. The

393

interaction between the phenolate group C6''-O- and the positively charged side chain of Lys437

394

provides not only H-bonding but also a charge-charge interaction. Moreover, Lys437 forms

395

alkyl-π hydrophobic and cation-π interactions with the ring C of 1c which make this balanoid

396

exhibit all possible noncovalent binding interactions with the invariant Lys. As a result, the

397

binding of 1c to Lys437 in PKCε is the strongest among the balanoids studied here.

398

In PKA, the presence of Thr184 restrains the azepane rings of all balanoids. These restrained

399

azepane rings limit the chance of the invariant Lys73 to form H-bonds with balanoids, as shown

400

in Table S3 (Supporting information). Hence, the binding of balanoids to PKA is unable to

401

completely interact with Lys73, particularly in terms of H-bonding. As a consequence, the

402

binding energy contribution of the invariant Lys73 is within a narrow range for all balanoids (-

403

8.54 to -13.76 kcal.mol-1; Supporting information: Figure S9) and weaker than the contributions


20

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404

of Lys437 to PKCε-balanoid interaction (-12.86 to -21.39 kcal.mol-1). In particular, the

405

restraining effect of Thr184 on the triply fluorinated balanoid, 1e, diminishes its binding affinity

406

to both PKA and PKCε (Supporting Information Note 5 and Figure S11).

407

As we discussed above, the invariant Lys residues in both PKA and PKCε provide the main

408

contribution to balanoid binding over the other ATP site residues. While in PKA, the invariant

409

Lys73 cannot optimise the interactions with balanoids due to the restraining effect from Thr184,

410

Lys437 in PKCε is not under any restraint in its binding to 1c and, thus, provides a substantial

411

binding energy contribution to this balanoid. This finding suggests that optimising interaction

412

with Lys437 is an important requirement for designing PKCε inhibitors with greater affinity than

413

1c.

414

Interactions with the invariant Lys may also explain the correlation of the residential position

415

of the benzophenone moiety, where the deeper this moiety is buried in the triphosphate subsite,

416

the stronger the binding affinity (Figure 2). The invariant Lys is located inside the triphosphate

417

subsite. Thus, intensive interactions of a balanoid to this residue lead the benzophenone moiety

418

residing deeper inside the subsite, as observed in 1c bound to PKCε (Figure 2.C).

419

Unfavourable binding interaction

420

The effect of fluorine substitution in the azepane ring may generate unfavourable binding

421

interactions in PKCε as well as PKA. For instance, in PKCε, the weakest binding ligand 1d

422

involves unfavourable binding contributions from Asp449 (0.02 kcal.mol-1) and Asn537 (0.28

423

kcal.mol-1). The unfavourable binding interaction from Asn537 is the result of water infiltration

424

that prevents the azepane moiety from interacting with the ribose subsite, whereas the one from

425

Asp449 is due to a remote fluorine effect in the azepane ring which interferes with the binding of

426

benzophenone moiety to the triphosphate subsite. Such unfavourable binding interactions explain


21


Page 22 of 29

427

why 1d is a poor PKCε ligand. Moreover, a relatively weaker binding energy contribution (-

428

13.82 kcal.mol-1) provided by invariant Lys (Lys437) may also contribute to the very low

429

binding affinity of 1d to PKCε (110 nM). On the contrary, fluorine substitution in 1c does not

430

result in any unfavourable binding interaction with the residues of the ATP site of PKCε

431

(Supporting information: Figure S9).

432

In PKA, an unfavorable binding interaction (+0.04 kcal.mol-1) is only found in the ATP site

433

with 1e, the weakest PKA ligand (Kd = 43 nM) among balanoids. The presence of such an

434

unfavorable binding energy contribution reveals that the fluorine substitution in 1e interferes

435

with the benzophenone moiety interacting optimally with residues of the ATP site of PKA.

436

In summary, we show that fluorine substitution can generate unfavourable binding interaction

437

between balanoids and the ATP sites. Nonetheless, judicious stereospecific fluorine

438

incorporation, such as in 1c, can produce a constructive effect which is free from any

439

unfavourable binding interaction. Thus, the design of the next balanol-based inhibitors needs to

440

consider substituents to avoid unfavourable binding interactions in the ATP site.

441 442

CONCLUSIONS

443

In this current study, using a molecular dynamics simulation approach, we unravelled the

444

perturbation effects of stereospecific fluorination on balanoid binding affinity in the ATP sites of

445

PKCε as well as PKA. Stereospecific fluorination affects balanoid binding in the ATP sites of

446

both kinases, by modifying the kinase conformation and ligand flexibility, especially the binding

447

mode of the azepane ring in the ribose subsite. The fluorination effects further remotely

448

influence the benzophenone moiety interactions in the triphosphate subsite. The effects are

449

receptor-dependent, which is also reflected in the experimental different binding affinity values


22

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450

of balanoids to PKA and PKCε. For instance, in PKCε-bound 1c, the azepane ring conformation

451

cooperatively set the benzophenone moiety to optimise interactions with the ATP site residues,

452

especially with the invariant Lys437. From our study, the invariant Lys, which has an essential

453

role in catalytic activity, is the major contributor for binding affinity of balanoids to PKA and

454

PKCε. On the other hand, in PKA, the azepane ring of 1c leads to an interaction decrease with

455

the invariant Lys73.

456

Here, for the first time to the best of our knowledge, we also found an interesting factor that

457

causes susceptibility of PKCε but insensitivity of PKA, to fluorination substitution. The

458

insensitivity of PKA is due to the presence of Thr184, which is situated in the ribose subsite,

459

close to the adenine subsite. Thr184 firmly holds the amide linkage through an H-bond and, thus,

460

dampens fluorine effect in the azepane ring. In PKCε, Ala549 replaces Thr184, preventing the

461

formation of an H-bond that protects the azepane ring from fluorine perturbation. As a result,

462

PKCε is sensitive to fluorine substitution, and our study showed that 1c, fluorinated at C5(S),

463

provides a fluorine effect which improves binding affinity. Overall, this study provides valuable

464

information which facilitates the further rational design of balanol-based inhibitor targeting

465

PKCε for cancer therapy.

466 467

ASSOCIATED CONTENT

468

Notes 1-5, Table S1-S3 and Figures S1-S11(PDF)

469

AUTHOR INFORMATION

470

Corresponding Author

471

SR: [email protected]


23


Page 24 of 29

472

Author Contributions

473

AH carried out the computational simulation study and drafted the manuscript. AH, FL, and SR

474

participated in the design of the study and interpretation of data. SR, FL and AH finalised the

475

manuscript. All authors have read and approved the final manuscript.

476

Notes

477

The authors declare that they have no competing interests.

478

ACKNOWLEDGMENT

479

We acknowledge the Indonesia Endowment Fund for Education scholarship to AH. This project

480

was partially supported by resources and services from the National Computational

481

Infrastructure (NCI), funded by the Australian Government.

482

ABBREVIATIONS

483

ATP: adenosine triphosphate; AM1-BCC: Austin Model 1 - Bond Charge Corrections; Balanol:

484

(-)-balanol; DOPE: Discrete Optimized Protein Energy; GAFF: General Amber Force-fields;

485

LCPO: Linear Combination of Pairwise Overlaps; MD: Molecular Dynamics; MMGBSA:

486

Molecular Mechanics Generalized Born Surface Area; PKA: cAMP-dependent protein kinase;

487

PKC: protein kinase C; PMEMD: Particle-Mesh Ewald Molecular Dynamics; RMSF: Root-

488

Mean-Square Fluctuation; SASA: Solvent-Accessible Surface Area.

489 490

REFERENCES

491 492 493

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Molecular Dynamics Pinpoint the Global Fluorine Effect in Balanoids

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