Delocalization errors, such as charge-transfer and some self-interaction errors, plague computationally efficient and otherwise accurate density functional approximations (DFAs). Evaluating a semilocal DFA non-self-consistently on the Hartree–Fock (HF) density is often recommended as a computationally inexpensive remedy for delocalization errors. For sophisticated meta-GGAs like SCAN, this approach can achieve remarkable accuracy. This HF-DFT (also known as DFA@HF) is often presumed to work, when it significantly improves over the DFA, because the HF density is more accurate than the self-consistent DFA density in those cases. By applying the metrics of density-corrected density functional theory (DFT), we show that HF-DFT works for barrier heights by making a localizing charge-transfer error or density overcorrection, thereby producing a somewhat reliable cancellation of density- and functional-driven errors for the energy. A quantitative analysis of the charge-transfer errors in a few randomly selected transition states confirms this trend. We do not have the exact functional and electron densities that would be needed to evaluate the exact density- and functional-driven errors for the large BH76 database of barrier heights. Instead, we have identified and employed three fully nonlocal proxy functionals (SCAN 50% global hybrid, range-separated hybrid LC-ωPBE, and SCAN-FLOSIC) and their self-consistent proxy densities. These functionals are chosen because they yield reasonably accurate self-consistent barrier heights and because their self-consistent total energies are nearly piecewise linear in fractional electron numbertwo important points of similarity to the exact functional. We argue that density-driven errors of the energy in a self-consistent density functional calculation are second order in the density error and that large density-driven errors arise primarily from incorrect electron transfers over length scales larger than the diameter of an atom.
The current anticancer therapies are limited by their lack of controlled spatiotemporal release at the target site of action. We report a novel drug delivery platform that provides on-demand, real-time, organelle-specific drug release and monitoring upon photoactivation. The system is comprised of a model anticancer drug doxorubicin, an alkyltriphenylphosphonium moiety to target mitochondria in cancer cells, and a hydroxycinnamate photoactivatable linker that is covalently attached to the drug and mitochondria-targeting moieties such that it can be phototriggered by either UV (one-photon) or NIR (two-photon) light to form a fluorescent coumarin product and facilitate the release of drug payload. The extent of drug release is quantified by the fluorescence intensity of the coumarin formed. Further, the photoactivatable prodrug accumulates in the mitochondria and shows light-triggered temporally controlled cell death. In the future, our platform can be tuned for any biological application of interest, offering immense value in biomedicine.
Lipid‐based palmitoylation is a post‐translation modification (PTM) which acts as a biological rheostat in life cycle progression of a deadly human malaria parasite, Plasmodium falciparum. P. falciparum palmitoylation is catalyzed by 12 putative palmitoyl acyl‐transferase enzymes containing the conserved DHHC‐CRD (DHHC motif within a cysteine‐rich domain) which can serve as a druggable target. However, the paucity of high‐throughput assays has impeded the design of drugs targeting palmitoylation. We have developed a novel strategy which involves engineering of Escherichia coli, a PTM‐null system, to enforce ectopic expression of palmitoyl acyl‐transferase in order to study Plasmodium‐specific palmitoylation and screening of inhibitors. In this study, we have developed three synthetic E. coli strains expressing Plasmodium‐specific DHHC proteins (PfDHHC7/8/9). These cells were used for validating acyl‐transferase activity via acyl‐biotin exchange (ABE) and clickable chemistry methods. E. coli proteome was found to be palmitoylated in PfDHHC‐expressing clones, suggesting that plasmodium DHHC can catalyze palmitoylation of E. coli proteins. Upon treatment with generic inhibitor 2‐bromopalmitate (2‐BMP), a predominant reduction in palmitic acid incorporation is detected. Overall, these findings suggest that synthetic E. coli strains expressing PfDHHCs can enforce global palmitoylation in the E. coli proteome. Interestingly, this finding was corroborated by our in silico palmitoylome profiling, which revealed that out of the total E. coli proteome, 108 proteins were predicted to be palmitoylated as represented by the presence of three cysteine consensus motifs (cluster type I, II, III). In summary, our study reports a proof of concept for screening of chemotherapeutics targeting the palmitoylation machinery using a high‐throughput screening platform.
The sphingolipid pool is key regulator of vital cellular functions in Plasmodium falciparum a causative agent for deadly malaria. Erythrocytes, the host for asexual stage of Plasmodium, are major reservoir for Sphingosine-1-phosphate (S1P). Erythrocyte possesses Sphingosine kinase (SphK) that catalyzed its biosynthesis from sphingosine (Sph). Since, Plasmodium lacks SphK homologous protein it can be envisaged that it co-opts sphingolipids from both intraerythrocytic as well as extracellular pools for its growth and development. Herein, by sphingosine-NBD probing, we report that infected erythrocytes imports Sph from extracellular pool, which is converted to S1P and thereby taken by P. falciparum. Next, by targeting of the SphK through specific inhibitor N,N-Dimethylsphingosine DMS, we show a reduction in erythrocyte endogenous S1P pool and SphK-phosphorylation that led to inhibition in growth and development of ring stage P. falciparum. Owing to the role of S1P in erythrocyte glycolysis we analyzed uptake of NBD-Glucose and production of lactate in DMS treated and untreated plasmodium. DMS treatment led to decreased glycolysis in Plasmodium. Interestingly the host free Plasmodium did not show any effect on glycolysis with DMS treatment indicating its host-mediated effect. Further to understand the in-vivo anti-plasmodial effects of exogenous and endogenous erythrocyte S1P level, Sphingosine-1-phosphate lyase (S1PL) inhibitor (THI), S1P and SphK-1 inhibitor (DMS), were used in Plasmodium berghei ANKA (PbA) mice model. DMS treatment led to reduction of endogenous S1P conferred significant decrease in parasite load, whereas the plasma level S1P modulated by (THI) and exogenous S1P have no effect on growth of Plasmodium. This suggested erythrocyte endogenous S1P pool is important for Plasmodium growth whereas the plasma level S1P has no effect. Altogether, this study provides insight on cellular processes regulated by S1P in P. falciparum and highlights the novel mechanistically distinct molecular target i.e. SphK-1.
Sphingosine-1-phosphate (S1P), a bioactive lipid mediator is involved in an array of biological processes and linked to pathological manifestations. Erythrocyte is known as the major reservoir for S1P as they lack S1P-degrading enzymes (S1P lyase and S1P phosphohydrolase) and harbor sphingosine kinase-1 (SphK-1) essential for sphingosine conversion to S1P. Reduced S1P concentration in serum was correlated with disease severity in patients with Plasmodium falciparum and Plasmodium vivax infections. Herein, we aimed to identify the underlying mechanism and contribution of host erythrocytes toward depleted S1P levels in Plasmodium-infected patients vs. healthy individuals. The level and activity of SphK-1 were measured in vitro in both uninfected and cultured P. falciparum-infected erythrocytes. Infected erythrocytes demonstrated a significant decrease in SphK-1 level in a time-dependent manner. We found that 10-42 h post invasion (hpi), SphK1 level was predominantly reduced to ∼50% in rings, trophozoites, and schizonts compared to uninfected erythrocytes. We next analyzed the phosphorylation status of SphK-1, a modification responsible for its activity and S1P production, in both uninfected control and Plasmodium-infected erythrocytes. Almost ∼50% decrease in phosphorylation of SphK-1 was observed that could be corroborated with significant reduction in the production and release of S1P in infected erythrocytes. Serum S1P levels were studied in parallel in P. falciparum (N = 15), P. vivax (N = 36)-infected patients, and healthy controls (N = 6). The findings revealed that S1P concentration was significantly depleted in uncomplicated malaria cases and was found to be lowest in complicated malaria and thrombocytopenia in both P. falciparum and P. vivax-infected groups ( * * p < 0.01). The lower serum S1P level could be correlated with the reduced platelet count defining the role of S1P level in platelet formation. In conclusion, erythrocyte SphK-1 and S1P levels were studied in Plasmodium-infected individuals and erythrocytes that helped in characterizing the complications associated with malaria and thrombocytopenia, providing insights into the contribution of host erythrocyte biology in malaria pathogenesis. Finally, this study proposes the use of S1P and its analog as a novel adjunct therapy for malaria complications.
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