Rigorous Computational Study Reveals What Docking Overlooks: Double Trouble from Membrane Association in Protein Kinase C Modulators

Abstract

Increasing protein kinase C (PKC) activity is of potential therapeutic value. Its activation involves an interaction between the C1 domain and diacylglycerol (DAG) at intracellular membrane surfaces; DAG mimetics hold promise as new drugs. We previously developed the isophthalate derivative HMI-1a3, an effective but highly lipophilic (clogP = 6.46) DAG mimetic. Although a less lipophilic pyrimidine analog, PYR-1gP (clogP = 3.30), gave positive results in computational docking, it unexpectedly presented greatly diminished binding to PKC in vitro. Through more rigorous computational molecular modeling, we reveal that, unlike HMI-1a3, PYR-1gP forms an intramolecular hydrogen bond, which both obstructs binding and reorients PYR-1gP in the membrane in a fashion that prevents it from correctly accessing the PKC C1 domain. Our results highlight the great value of molecular dynamics simulations as a key component for the drug design process of ligands targeting weakly membraneassociated proteins, where simulation in the relevant membrane environment is crucial for obtaining biologically applicable results.

Mechanistic Understanding from Molecular Dynamics in Pharmaceutical Research 2: Lipid Membrane in Drug Design

“…Neuroleptics Trifluoperazine, Haloperidol decanoate, Clozapine, Quetiapine, Olanzapine, Aripiprazole, Amisulpride [323] Alzheimer disease Pregnanolone sulfate, Pregnanolone glutamate [324], Carbazoles [325] Anticonvulsant and muscle relaxant Carbamazepine [326][327][328], Nordazepam [199], Lamotrigine [199], Chlorzoxazone [132] Cardiac arrhythmias Dronedarone [312] P2Y1 antagonist BPTU [329] Urea cycle disorders 4-phenylbutyrate Immunosuppressant Cyclosporine A and E [330] Cardiac Ca 2+ pump inhibitors CDN1163, CP-154526, Ro 41-0960 [331] Eye drops components Cetalkonium chloride, Poloxamer 188 [332] Vaccine adjuvant Cobalt porphyrin phospholipid [333], Lipidated nicotine [334] Other potential drugs Baicalin [282], Emodin [282], Siramesine [335], HMI and HMI-1a3 [336], Peptide mimicking GM1 [337], AMG3 [338], 1,8-naphthyridine derivatives [339], Protein kinase inhibitors [340], Bile salt export pump inhibitors [341] Pulmonary surfactants form a complex structure on the inner lung surfaces, the alveoli, including the only monolayer in the human body at the boundary between the pulmonary liquids and air. For drugs designed to treat lung conditions that are delivered through pulmonary administration, e.g., corticosteroid and salbutamol inhalers for the treatment of asthma, this monolayer is the first barrier that they encounter.…”

“…An analog of HMI-1a3 with a phenyl ring substituted with pyrimidine (PYR-1gP) was synthesized that, unlike HMI-1a3, had an acceptable solubility profile, but this was found in experimental assays not to be effective as a PKC activator. An MD simulation study was carried out by Lautala et al, to determine the cause of this; they found that one of the reasons for this observed low potency to be a drastic change in the orientation of the functional group involved in binding from exposed to the water phase to embedded into the bilayer; this results from the formation of an internal H-bond that increase its effective hydrophobicity [336].…”

Expanding the Paradigm of Structure-Based Drug Design: Molecular Dynamics Simulations Support the Development of New Pyridine-Based Protein Kinase C-Targeted Agonists

“…The current lack of knowledge concerning the precise behavior and detailed activation mechanism of PKC at the membrane interface likely compromised numerous attempts to target the enzyme via its C1 domain. As standard docking and biological experiments alone seemingly cannot provide a sufficient level of information, studying the behavior of the protein and both endogenous/exogenous C1 domain-targeted ligands through molecular dynamics (MD) simulations have been used to elucidate this phenomenon. ,,,− Most of these studies incorporated the PKCδ C1B domain (PDB ID: 1PTR ), the ligand of interest, and the relevant membrane. Unfortunately, these types of simulations can be extremely laborious due to their complex nature.…”

“…In our previous work, we observed that modifying the structure of active lead compounds (e.g., HMI-1a3) by switching the phenyl core with a pyrimidine (e.g., PYR-1gP) to improve the hydrophilicity of the scaffold leads to a precipitous decrease in binding affinity. We discovered a possible explanation by carrying out a set of MD simulations of the ligands in the relevant bilayer: an intramolecular H-bond between the hydroxy group and one nitrogen atom of the pyrimidine core, enhanced by the lipophilic membrane environment, changes the orientation of the compounds in the membrane . Further, Ryckbosch et al have determined that even the H-bonding pattern of the ligand at the lipid surface can differentiate ligands with tumor-promoting/suppressing behaviors …”

Exploring Novel Directions in Free Energy Calculations

“…The large-scale dynamics of the enzyme-ligand complex were probed with MD simulations, and QM/MM calculations were used to propose a reaction mechanism. The value of MD simulations as a key component to refine docking results was showcased with modulators of the membraneassociated protein kinase C. 18 The unexpected poor experimental activity of a docking hit from a previous study could be rationalized using MD simulations and thermodynamic integration (TI) in the relevant environment.…”