Investigation of Basolateral Targeting Micelles for Drug Delivery Applications in Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a complex disorder characterized by uncontrolled renal cyst growth, leading to kidney function decline. The multifaceted nature of ADPKD suggests that single-pathway interventions using individual small molecule drugs may not be optimally effective. As such, a strategy encompassing combination therapy that addresses multiple ADPKD-associated signaling pathways could offer synergistic therapeutic results. However, severe off-targeting side effects of small molecule drugs pose a major hurdle to their clinical transition. To address this, we identified four drug candidates from ADPKD clinical trials, bardoxolone methyl (Bar), octreotide (Oct), salsalate (Sal), and pravastatin (Pra), and incorporated them into peptide amphiphile micelles containing the RGD peptide (GRGDSP), which binds to the basolateral surface of renal tubules via integrin receptors on the extracellular matrix. We hypothesized that encapsulating drug combinations into RGD micelles would enable targeting to the basolateral side of renal tubules, which is the site of disease, via renal secretion, leading to superior therapeutic benefits compared to free drugs. To test this, we first evaluated the synergistic effect of drug combinations using the 20% inhibitory concentration for each drug (IC20) on renal proximal tubule cells derived from Pkd1flox/-:TSLargeT mice. Next, we synthesized and characterized the RGD micelles encapsulated with drug combinations and measured their in vitro therapeutic effects via a 3D PKD growth model. Upon both IV and IP injections in vivo, RGD micelles showed a significantly higher accumulation in the kidneys compared to NT micelles, and the renal access of RGD micelles was significantly reduced after the inhibition of renal secretion. Specifically, both Bar+Oct and Bar+Sal in the RGD micelle treatment showed enhanced therapeutic efficacy in ADPKD mice (Pkd1fl/fl;Pax8-rtTA;Tet-O-Cre) with a significantly lower KW/BW ratio and cyst index as compared to PBS and free drug-treated controls, while other combinations did not show a significant difference. Hence, we demonstrate that renal targeting through basolateral targeting micelles enhances the therapeutic potential of combination therapy in genetic kidney disease.

MARK4 protein can explore the active-like conformations in its non-phosphorylated state.

Microtubule affinity-regulating kinase 4 (MARK4) is a Ser/Thr protein kinase, best known for its role in phosphorylating microtubule associated proteins, causing their detachment from microtubules. In the current study, the non-phosphorylated conformation of the activation loop was modeled in a structure representing the enzymatically inactive form of this protein, and its dynamics were evaluated through a 100 ns initial all-atom simulation, which was prolonged by another 2 µs. Although the activation loop was folding on itself and was leaning toward ATP site in the initial modeled structure, soon after the initiating the simulation, this loop stretched away from the ATP binding site and stably settled in its new position for the rest of simulation time. A network of hydrogen bonds, mainly between the activation segment residues, αC-helix and the catalytic loop reinforced this conformation. Interestingly, several features of active kinase conformation such as formation of R-spine, Glu106-Lys88 salt-bridge, and DFG-In motif were observed during a considerable number of trajectory frames. However, they were not sustainably established during the simulation time, except for the DFG-In motif. Consequently, this study introduces a stable conformation of the non-phosphorylated form of MARK4 protein with a partially stretched activation loop conformation as well as partial formation of R-spine, closely resembling the active kinase.