Mutations in the KCNA1 gene, which encodes voltage-gated Kv1.1 potassium channel α-subunits, cause a variety of human diseases, complicating simple genotype–phenotype correlations in patients. KCNA1 mutations are primarily associated with a rare neurological movement disorder known as episodic ataxia type 1 (EA1). However, some patients have EA1 in combination with epilepsy, whereas others have epilepsy alone. KCNA1 mutations can also cause hypomagnesemia and paroxysmal dyskinesia in rare cases. Why KCNA1 variants are associated with such phenotypic heterogeneity in patients is not yet understood. In this review, literature databases (PubMed) and public genetic archives (dbSNP and ClinVar) were mined for known pathogenic or likely pathogenic mutations in KCNA1 to examine whether patterns exist between mutation type and disease manifestation. Analyses of the 47 deleterious KCNA1 mutations that were identified revealed that epilepsy or seizure-related variants tend to cluster in the S1/S2 transmembrane domains and in the pore region of Kv1.1, whereas EA1-associated variants occur along the whole length of the protein. In addition, insights from animal models of KCNA1 channelopathy were considered, as well as the possible influence of genetic modifiers on disease expressivity and severity. Elucidation of the complex relationship between KCNA1 variants and disease will enable better diagnostic risk assessment and more personalized therapeutic strategies for KCNA1 channelopathy.
Microtubule polymerization dynamics result from the biochemical interactions of αβ-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αβ-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently 'poisoned' growth, yielding a wider range of growth rates compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP tubulin at the growing microtubule end may be an important early event determining catastrophe.
Overexpression of ABC transporters like P-glycoprotein (P-gp) has been correlated with resistances in cancer chemotherapy. Intensive efforts to identify P-gp inhibitors for use in combination therapy have not led to clinically approved inhibitors to date. Here, we describe computational approaches combined with structure-based design to improve the characteristics of a P-gp inhibitor previously identified by us. This hit compound represents a novel class of P-gp inhibitors that specifically targets and inhibits P-gp ATP hydrolysis while not being transported by the pump. We describe here a new program for virtual chemical synthesis and computational assessment, ChemGen, to produce hit compound variants with improved binding characteristics. The chemical syntheses of several variants, efficacy in reversing multidrug resistance in cell culture, and biochemical assessment of the inhibition mechanism are described. The usefulness of the computational predictions of binding characteristics of the inhibitor variants is discussed and compared to more traditional structure-based approaches.
P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer’s disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer’s associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.
P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer's disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer's associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.
The MtrCDE system confers multidrug resistance to Neisseria gonorrhoeae, the causative agent of gonorrhea. Using free and directed molecular dynamics (MD) simulations, we analyzed the interactions between MtrD and azithromycin, a transport substrate of MtrD, and a last-resort clinical treatment for multidrug-resistant gonorrhea. We then simulated the interactions between MtrD and streptomycin, an apparent nonsubstrate of MtrD. Using known conformations of MtrD homologues, we simulated a potential dynamic transport cycle of MtrD using targeted MD techniques (TMD), and we noted that forces were not applied to ligands of interest. In these TMD simulations, we observed the transport of azithromycin and the rejection of streptomycin. In an unbiased, long-time scale simulation of AZY-bound MtrD, we observed the spontaneous diffusion of azithromycin through the periplasmic cleft. Our simulations show how the peristaltic motions of the periplasmic cleft facilitate the transport of substrates by MtrD. Our data also suggest that multiple transport pathways for macrolides may exist within the periplasmic cleft of MtrD.
Antibiotic-resistant gonorrheal infections are an urgent health concern. The MtrCDE system confers multidrug resistance to Neisseria gonorrhoeae, an obligate human pathogen, and the causative agent of the sexually-transmitted infection gonorrhea. The inner membrane pump MtrD effluxes a variety of hydrophobic and amphiphilic substrates and thereby confers resistance to a multitude of antibiotics. Using a combination of free and directed Molecular Dynamics (MD) simulations, we analyzed the interactions of MtrD with Azithromycin, an MtrD substrate and one of the last remaining courses of treatment for multidrug resistant gonorrhea. We also simulated the interactions between MtrD and Streptomycin, a non-substrate of MtrD. Using targeted MD (TMD) techniques and known conformations of MtrD homologues, we guided MtrD through the conformational changes of a putative transport cycle by applying small forces to α-carbons of the protein backbone; forces were not applied to Azithromycin or to Streptomycin. In our TMD experiments, we observed the transport of Azithromycin (in three possible protonation states) and the rejection of Streptomycin. To supplement our findings, we then demonstrate the spontaneous diffusion of Azithromycin through the periplasmic cleft in long time-scale, unbiased MD simulations. Our findings support the hypothesis that the transition from Binding to Extrusion is an energy requiring step in the transport process. Our data also suggest that multiple binding modes, and potentially multiple residue contact pathways, exist within the periplasmic cleft of MtrD, even for bulky substrates. To our knowledge, this is the first computational demonstration of substrate transport, and non-substrate rejection, by MtrD.
Multi‐drug resistance (MDR) occurs when cancer cells become resistant to a diverse array of chemotherapeutics and xenobiotics. Among the many mechanisms of MDR, one of the most prominent is the overexpression of ATP‐binding cassette (ABC) transporters. ABC transporters harness the energy from ATP hydrolysis to transport xenobiotics, drugs, and other toxic compounds out of the cell. When ABC transporters are overexpressed in cancer cells, their efflux activity can lower the intracellular concentration of medicinal compounds to sub‐therapeutic levels. One member of the ABC transporter family, the Breast Cancer Resistance Protein (BCRP, or ABCG2), confers MDR to a variety of cancers. Transient inhibition of BCRP should therefore restore sensitivity of resistant cells to chemotherapeutics. In previous work by our lab, several potential BCRP inhibitors were identified via computational methods. Using cell viability assays, we tested the potential BCRP inhibitors using a BCRP overexpressing cell line. Here we compare the effectiveness of the inhibitors at re‐sensitizing the cells to Mitoxantrone relative to the parental cell line which does not overexpress BCRP. The potential toxicities of the experimental compounds were also assessed using the non‐cancerous HFL1 cell line.
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