Therapeutic potential of combating cancer by restoring wild-type p53 through mRNA nanodelivery.

Among the tumor suppressor genes, TP53 is the most frequently mutated in human cancers, and most mutations are missense mutations causing production of mutant p53 (mutp53) proteins. TP53 mutations not only results in loss of function (LOH) as a transcription factor and a tumor suppressor, but also gain wild-type p53 (WTp53)-independent oncogenic functions that enhance cancer metastasis and progression (Yamamoto and Iwakuma, 2018; Zhang et al., 2022). TP53 has extensively been studied as a therapeutic target as well as for drug development and therapies, however with limited success. Achieving targeted therapies for restoration of WTp53 function and depletion or repair of mutant p53 (mutp53) will have far reaching implication in cancer treatment and therapies. This review briefly discusses the role of p53 mutation in cancer and the therapeutic potential of restoring WTp53 through the advances in mRNA nanomedicine.

Elucidating the Elementary Reaction Pathways and Kinetics of Hydroxyl Radical-Induced Acetone Degradation in Aqueous Phase Advanced Oxidation Processes.

Advanced oxidation processes (AOPs) that produce highly reactive hydroxyl radicals are promising methods to destroy aqueous organic contaminants. Hydroxyl radicals react rapidly and nonselectively with organic contaminants and degrade them into intermediates and transformation byproducts. Past studies have indicated that peroxyl radical reactions are responsible for the formation of many intermediate radicals and transformation byproducts. However, complex peroxyl radical reactions that produce identical transformation products make it difficult to experimentally study the elementary reaction pathways and kinetics. In this study, we used ab initio quantum mechanical calculations to identify the thermodynamically preferable elementary reaction pathways of hydroxyl radical-induced acetone degradation by calculating the free energies of the reaction and predicting the corresponding reaction rate constants by calculating the free energies of activation. In addition, we solved the ordinary differential equations for each species participating in the elementary reactions to predict the concentration profiles for acetone and its transformation byproducts in an aqueous phase UV/hydrogen peroxide AOP. Our ab initio quantum mechanical calculations found an insignificant contribution of Russell reaction mechanisms of peroxyl radicals, but significant involvement of HO2• in the peroxyl radical reactions. The predicted concentration profiles were compared with experiments in the literature, validating our elementary reaction-based kinetic model.

The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis.

Ribosomal protein uS5 is an essential component of the small ribosomal subunit that is involved in subunit assembly, maintenance of translational fidelity, and the ribosome's response to the antibiotic spectinomycin. While many of the characterized uS5 mutations that affect decoding map to its interface with uS4, more recent work has shown that residues distant from the uS4-uS5 interface can also affect the decoding process. We targeted one such interface-remote area, the loop 2 region (residues 20 to 31), for mutagenesis in Escherichia. coli and generated 21 unique mutants. A majority of the loop 2 alterations confer resistance to spectinomycin and affect the fidelity of translation. However, only a minority show altered rRNA processing or ribosome biogenesis defects.

Mechanistic Insight into the Reactivity of Chlorine-Derived Radicals in the Aqueous-Phase UV-Chlorine Advanced Oxidation Process: Quantum Mechanical Calculations.

The combined ultraviolet (UV) and free chlorine (UV-chlorine) advanced oxidation process that produces highly reactive hydroxyl radicals (HO•) and chlorine radicals (Cl•) is an attractive alternative to UV alone or chlorination for disinfection because of the destruction of a wide variety of organic compounds. However, concerns about the potential formation of chlorinated transformation products require an understanding of the radical-induced elementary reaction mechanisms and their reaction-rate constants. While many studies have revealed the reactivity of oxygenated radicals, the reaction mechanisms of chlorine-derived radicals have not been elucidated due to the data scarcity and discrepancies among experimental observations. We found a linear free-energy relationship quantum mechanically calculated free energies of reaction and the literature-reported experimentally measured reaction rate constants, kexp, for 22 chlorine-derived inorganic radical reactions in the UV-chlorine process. This relationship highlights the discrepancy among literature-reported rate constants and aids in the determination of the rate constant using quantum mechanical calculations. We also found linear correlations between the theoretically calculated free energies of activation and kexp for 31 reactions of Cl• with organic compounds. The correlation suggests that H-abstraction and Cl-adduct formation are the major reaction mechanisms. This is the first comprehensive study on chlorine-derived radical reactions, and it provides mechanistic insight into the reaction mechanisms for the development of an elementary reaction-based kinetic model.

Modulation of decoding fidelity by ribosomal proteins S4 and S5.

Ribosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype. Structural analyses of ribosomal complexes indicate that the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins. In agreement with this observation, several of the mutations that promote miscoding alter residues located at the S4-S5 interface. Here, the Escherichia coli rpsD and rpsE genes encoding the S4 and S5 proteins were targeted for mutagenesis and screened for accuracy-altering mutations. While a majority of the 38 mutant proteins recovered decrease the accuracy of translation, error-restrictive mutations were also recovered; only a minority of the mutant proteins affected rRNA processing, ribosome assembly, or interactions with antibiotics. Several of the mutations affect residues at the S4-S5 interface. These include five nonsense mutations that generate C-terminal truncations of S4. These truncations are predicted to destabilize the S4-S5 interface and, consistent with the domain closure model, all have ribosomal-ambiguity phenotypes. A substantial number of the mutations alter distant locations and conceivably affect tRNA selection through indirect effects on the S4-S5 interface or by altering interactions with adjacent ribosomal proteins and 16S rRNA.

Accelerating AI Innovation in Healthcare Through Mentorship.

The adoption of Artificial Intelligence (AI) in the Canadian healthcare system falls behind that of other countries. Socio-technological considerations such as organizational readiness and a limited understanding of the technology are a few barriers impeding its adoption. To address this need, this study implemented a five-month AI mentorship program with the primary objective of developing participants' AI toolset. The analysis of our program's effectiveness resulted in recommendations for a successful mentorship and AI development and implementation program. 12 innovators and 11 experts from diverse backgrounds were formally matched and two symposiums were integrated into the program design. 8 interviewed participants revealed positive perceptions of the program underscoring its contribution to their professional development. Recommendations for future programs include: (1) obtaining organizational commitment for each participant; (2) incorporating structural supports throughout the program; and (3) adopting a team-based mentorship approach. The findings of this study offer a foundation rooted in evidence for the formulation of policies necessary to promote the integration of AI in Canada.

Diversity in the C3b [corrected] contact residues and tertiary structures of the staphylococcal complement inhibitor (SCIN) protein family.

To survive in immune-competent hosts, the pathogen Staphylococcus aureus expresses and secretes a sophisticated array of proteins that inhibit the complement system. Among these are the staphylococcal complement inhibitors (SCIN), which are composed of three active proteins (SCIN-A, -B, and -C) and one purportedly inactive member (SCIN-D or ORF-D). Because previous work has focused almost exclusively on SCIN-A, we sought to provide initial structure/function information on additional SCIN proteins. To this end we determined crystal structures of an active, N-terminal truncation mutant of SCIN-B (denoted SCIN-B18-85) both free and bound to the C3c fragment of complement component C3 at 1.5 and 3.4 Å resolution, respectively. Comparison of the C3c/SCIN-B18-85 structure with that of C3c/SCIN-A revealed that both proteins target the same functional hotspot on the C3b/C3c surface yet harbor diversity in both the type of residues and interactions formed at their C3b/C3c interfaces. Most importantly, these structures allowed identification of Arg44 and Tyr51 as residues key for SCIN-B binding to C3b and subsequent inhibition of the AP C3 convertase. In addition, we also solved several crystal structures of SCIN-D to 1.3 Å limiting resolution. This revealed an unexpected structural deviation in the N-terminal α helix relative to SCIN-A and SCIN-B. Comparative analysis of both electrostatic potentials and surface complementarity suggest a physical explanation for the inability of SCIN-D to bind C3b/C3c. Together, these studies provide a more thorough understanding of immune evasion by S. aureus and enhance potential use of SCIN proteins as templates for design of complement targeted therapeutics.

Synthesis and biological activity of novel MIF antagonists.

Two series of novel furan and indole compounds were synthesized and probed for inhibition of macrophage migration inhibitory factor (MIF) activity. Several compounds from both series inhibited the enzymatic activity of MIF at levels equal to or significantly better than ISO-1 (an early MIF inhibitor). The majority of the compounds that robustly inhibited the spontaneous secretion/release/recognition of MIF from freshly isolated human peripheral blood mononuclear cells were from the furan series (compounds 5, 9, 13, 15, and 16). In contrast, compounds that markedly inhibited the MIF-induced production of pro-inflammatory cytokines were predominantly from the indole series (compounds 26, 29, and 32).

A preferential p110alpha/gamma PI3K inhibitor attenuates experimental inflammation by suppressing the production of proinflammatory mediators in a NF-kappaB-dependent manner.

A promising therapeutic approach to diminish pathological inflammation is to inhibit the increased production and/or biological activity of proinflammatory cytokines (e.g., TNF-alpha, IL-6). The production of proinflammatory cytokines is controlled at the gene level by the activity of transcription factors, such as NF-kappaB. Phosphatidylinositol 3-kinase (PI3K), a lipid kinase, is known to induce the activation of NF-kappaB. Given this, we hypothesized that inhibitors of PI3K activation would demonstrate anti-inflammatory potential. Accordingly, we studied the effects of a preferential p110alpha/gamma PI3K inhibitor (compound 8C; PIK-75) in inflammation-based assays. Mechanism-based assays utilizing human cells revealed that PIK-75-mediated inhibition of PI3K activation is associated with dramatic suppression of downstream signaling events, including AKT phosphorylation, IKK activation, and NF-kappaB transcription. Cell-based assays revealed that PIK-75 potently and dose dependently inhibits in vitro and in vivo production of TNF-alpha and IL-6, diminishes the induced expression of human endothelial cell adhesion molecules (E-selectin, ICAM-1, and VCAM-1), and blocks human monocyte-endothelial cell adhesion. Most importantly, PIK-75, when administered orally in a therapeutic regimen, significantly suppresses the macroscopic and histological abnormalities associated with dextran sulfate sodium-induced murine colitis. The efficacy of PIK-75 in attenuating experimental inflammation is mediated, at least in part, due to the downregulation of pertinent inflammatory mediators in the colon. Collectively, these results provide first evidence that PIK-75 possesses anti-inflammatory potential. Given that PIK-75 is known to exhibit anti-cancer activity, the findings from this study thus reinforce the cross-therapeutic functionality of potential drugs.

Novel derivatives of ISO-1 as potent inhibitors of MIF biological function.

A series of novel 1,2,4-oxadiazole, phthalimide, amide and other derivatives of ISO-1 were synthesized and probed for inhibition of macrophage migration inhibitory factor (MIF) activity. Several compounds inhibited MIF enzymatic activity at levels better than ISO-1. Of note, compounds 7, 22, 23, 24, 25 and 27 inhibited the spontaneous secretion/release/recognition of MIF from freshly isolated human peripheral blood mononuclear cells and, more importantly, inhibited the MIF-induced production of interleukin-6 (IL-6) and/or interleukin-1beta (IL-1beta) significantly better than ISO-1.

The C-terminus of ribosomal protein uS4 contributes to small ribosomal subunit biogenesis and the fidelity of translation.

Ribosomal protein uS4 is an essential ribosomal component involved in multiple functions, including mRNA decoding. Structural analyses indicate that during decoding, the interface between the C-terminus of uS4 and protein uS5 is disrupted and in agreement with this, C-terminal uS4 truncation mutants are readily isolated on the basis of their increased miscoding phenotypes. The same mutants can also display defects in small subunit assembly and 16S rRNA processing and some are temperature sensitive for growth. Starting with one such temperature sensitive Escherichia coli uS4 mutant, we have isolated temperature insensitive derivatives carrying additional, intragenic mutations that restore the C-terminus and ameliorate the ribosomal defects. At least one of these suppressors has no detectable ribosome biogenesis phenotype, yet still miscodes, suggesting that the C-terminal requirements for ribosome assembly are less rigid than for mRNA decoding. In contrast to the uS4 C-terminal mutants that increase miscoding, two Salmonella enterica uS4 mutants with altered C-termini have been reported as being error-restrictive. Here, reconstruction experiments demonstrate that contrary to the previous reports, these mutants have a distinct error-prone, increased misreading phenotype, consistent with the behavior of the equivalent E. coli mutants and their likely structural effects on uS4-uS5 interactions.

Alterations in ribosomal protein L19 that decrease the fidelity of translation.

Ribosomal protein L19 is an essential ribosomal protein and is a component of bridge B8, one of the protein-RNA bridges linking the large and small ribosomal subunits. Bridge B8 also contributes to the accuracy of translation by affecting GTPase activation by ribosome-bound aminoacyl tRNA-EF-Tu•GTP ternary complexes. Previous work has identified a limited number of accuracy-altering alterations in protein L19 of Salmonella enterica and Thermus thermophilus. Here, we have targeted the Escherichia coli rplS gene encoding L19 for mutagenesis and have screened for mutants with altered levels of miscoding. We have recovered 14 distinct L19 mutants, all of which promote increased stop codon readthrough, but do not have major effects on subunit association or cell growth. Examination of the E. coli 70S ribosome structure indicates that the amino acid substitutions cluster in three distinct regions of L19 and thereby potentially affect its interactions with L14 and 16S rRNA.

NF-κB dependent anti-inflammatory activity of chlorojanerin isolated from Saussurea heteromalla.

Medicinal plants have shown great promise as a source of novel drug compounds for the treatment of inflammatory disorders. In our search for new entities with anti-inflammatory potential, the extracts of the whole plant of Saussurea heteromalla (family-Asteraceae), collected from Himalayas, were evaluated in the high throughput screen for TNF-α and IL-6 inhibitors. The extract blocked TNF-α and IL-6 production in LPS stimulated THP-1 cells (human acute monocyte leukemia cell line) completely at 10 and 30 µg/ml. The plant has been found as a new source of chlorojanerin, a guaianolide type of sesquiterpene lactone. Chlorojanerin was shown to be significantly effective in inhibiting TNF-α and IL-6 production in LPS-stimulated THP-1 cells (IC(50)=2.3±0.2 µM and 1.8±0.7 µM respectively). The compound also blocked TNF-α and IL-6 production from LPS-stimulated human monocytes (IC(50)=1.5±0.4 and 0.7±0.2 µM respectively) and synovial cells from a patient with rheumatoid arthritis (IC(50)<0.03 and 0.5 µM respectively). Transcriptional profiling of the LPS stimulated THP-1 cells revealed that chlorojanerin exerted its anti-inflammatory effect by inhibiting the expression of 8 genes involved in activating the transcription factor - NF-κB. Real time analysis of these genes validated the effect of chlorojanerin on the classical downstream targets of NF-κB. Thus, this study clearly delineated 8 genes which were specifically mitigated due to the effect of chlorojanerin on NF-κB induced signaling at the mRNA level. Further, chlorojanerin at 5 µM also inhibited the binding of NF-κB in a GFP reporter assay system by 55.5% thus validating the microarray gene expression data. This work is a step towards the isolation and characterization of lead anti-inflammatory agents from the extract of Saussurea heteromalla, which can be developed into better therapeutic molecules targeted towards some specific inflammatory diseases.

A fluorinated analog of ISO-1 blocks the recognition and biological function of MIF and is orally efficacious in a murine model of colitis.

A promising therapeutic approach to diminish pathological inflammation is to inhibit the synthesis and/or biological activity of macrophage migration inhibitory factor (MIF). Prior studies have shown that intraperitoneal administration of small-molecule inhibitors targeting the catalytic pocket of MIF (e.g., ISO-1) elicits a therapeutic effect in mouse inflammation models. However, it remains to be elucidated whether these tautomerase activity inhibitors block the synthesis and/or biological activity of MIF. In this study, we investigated and compared the activity of representative MIF inhibitors from isoxazole series (fluorinated analog of ISO-1; ISO-F) and substituted quinoline series (compound 7E; 7E). Our results demonstrate that ISO-F is a more potent MIF inhibitor than 7E. Both ISO-F and 7E do not inhibit MIF synthesis but "bind-onto" MIF thereby blocking its recognition. However, in contrast to 7E, ISO-F docks well in the active site of MIF and also has a stronger binding affinity towards MIF. In line with these observations, ISO-F, but not 7E, robustly inhibits the biological function of MIF. Most importantly, ISO-F, when administered orally in a therapeutic regimen, significantly suppresses dextran sulphate sodium (DSS)-induced murine colitis. This study, which provides mechanistic insights into the anti-inflammatory efficacy of ISO-F, is the first documented report of in vivo anti-inflammatory efficacy of a MIF inhibitor upon oral administration. Moreover, the findings from this study reinforce the potential of catalytic site of MIF as a target for eliciting therapeutic effect in inflammatory disorders. Compounds (e.g., ISO-F) that block not only the recognition but also the biological function of MIF are potentially attractive for reducing pathological inflammation.