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LF is a bioactive protein, derived from colostrum and milk that has been found to possess various immunomodulatory, iron chelating, and antimicrobial properties, especially in its apo-form. Recent studies have demonstrated the functionality of LF in attaching to the S proteins of SARS-CoV-2, thereby preventing it from interacting with the ACE-2 receptor. However, the molecular mechanism mediating the process is poorly understood. In this study, molecular docking and MD simulations coupled with free energy calculations were applied to elucidate the key interaction of apo-LF and its N-lobe and C-lobe derivative forms with the RBD of coronavirus S proteins. This has also been extended into evaluating the L452R mutant, which is associated with the delta variant of SARS-CoV-2. The results demonstrate the efficacy of the apo-LF C-lobe in binding to the RBD of both variants, primarily through electrostatic attractions between the acidic residues of the former and the basic residues of each RBD. Furthermore, due to the additional arginine in the L452R variant, the interaction between the C-lobe and the latter is stronger, resulting in a more favourable binding and tightly bound structure. The simulations highlight that the C-lobe, followed by full-length apo-LF can form a multimeric complex with the RBD of SARS-CoV-2, indicating their potential use as novel therapeutics, particularly the cleaved C-lobe of apo-LF to disrupt the S proteins from binding to the host ACE-2 receptor. Communicated by Ramaswamy H. Sarma.

Lactoferrin is a protein, primarily found in milk that has attracted the interest of the food industries due to its health properties. Nevertheless, the instability of lactoferrin has limited its commercial application. Recent studies have focused on encapsulation to enhance the stability of lactoferrin. However, the molecular insights underlying the changes of structural properties of lactoferrin and the interaction with protectants remain poorly understood. Computational approaches have proven useful in understanding the structural properties of molecules and the key binding with other constituents. In this review, comprehensive information on the structure and function of lactoferrin and the binding with various molecules for food purposes are reviewed, with a special emphasis on the use of molecular dynamics simulations. The results demonstrate the application of modeling and simulations to determine key residues of lactoferrin responsible for its stability and interactions with other biomolecular components under various conditions, which are also associated with its functional benefits. These have also been extended into the potential creation of enhanced lactoferrin for commercial purposes. This review provides valuable strategies in designing novel nutraceuticals for food science practitioners and those who have interests in acquiring familiarity with the application of computational modeling for food and health purposes.

Supplementary Methods from Receptor-Mediated DNA-Targeted Photoimmunotherapy

Supplementary Figures 1-2, Tables 1-2 from Receptor-Mediated DNA-Targeted Photoimmunotherapy

DNA-minor groove binding bibenzimidazole ligands, such as ortho-iodoHoechst bind in the minor groove of DNA in AT-rich regions. Upon photodehalogenation of the DNA ligand a carbon-centred radical is formed.

Lactoferrin is a protein, primarily found in milk that has attracted the interest of the food industries due to its health properties. Nevertheless, the instability of lactoferrin has limited its commercial application. Recent studies have focused on encapsulation to enhance the stability of lactoferrin. However, the molecular insights underlying the changes of structural properties of lactoferrin and the interaction with protectants remain poorly understood. Computational approaches have proven useful in understanding the structural properties of molecules and the key binding with other constituents. In this review, comprehensive information on the structure and function of lactoferrin and the binding with various molecules for food purposes are reviewed, with a special emphasis on the use of molecular dynamics simulations. The results demonstrate the application of modeling and simulations to determine key residues of lactoferrin responsible for its stability and interactions with other biomolecular components under various conditions, which are also associated with its functional benefits. These have also been extended into the potential creation of enhanced lactoferrin for commercial purposes. This review provides valuable strategies in designing novel nutraceuticals for food science practitioners and those who have interests in acquiring familiarity with the application of computational modeling for food and health purposes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.529 variant (Omicron), represents a significant deviation in genetic makeup and function compared to previous variants. Following the BA.1 sublineage, the BA.2 and BA.3 Omicron subvariants became dominant, and currently the BA.4 and BA.5, which are quite distinct variants, have emerged. Using molecular dynamics simulations, we investigated the binding characteristics of the Delta and Omicron (BA.1) variants in comparison to wild-type (WT) at the interface of the spike protein receptor binding domain (RBD) and human angiotensin converting enzyme-2 (ACE2) ectodomain. The primary aim was to compare our molecular modelling systems with previously published observations, to determine the robustness of our approach for rapid prediction of emerging future variants. Delta and Omicron were found to bind to ACE2 with similar affinities (-39.4 and -43.3 kcal/mol, respectively) and stronger than WT (-33.5 kcal/mol). In line with previously published observations, the energy contributions of the non-mutated residues at the interface were largely retained between WT and the variants, with F456, F486, and Y489 having the strongest energy contributions to ACE2 binding. Further, residues N440K, Q498R, and N501Y were predicted to be energetically favourable in Omicron. In contrast to Omicron, which had the E484A and K417N mutations, intermolecular bonds were detected for the residue pairs E484:K31 and K417:D30 in WT and Delta, in accordance with previously published findings. Overall, our simplified molecular modelling approach represents a step towards predictive model systems for rapidly analysing arising variants of concern.

Graphical abstract, The SARS-CoV-2 papain-like (PLpro) protease is essential for viral replication. We investigated potential antiviral effects of hypericin relative to the well-known noncovalent PLpro inhibitor GRL-0617. Molecular dynamics and PELE Monte Carlo simulations highlight favourable binding of hypericin and GRL-0617 to the naphthalene binding pocket of PLpro. Although not potent as GRL-0617 (45.8 vs 1.6µM for protease activity, respectively), in vitro fluorogenic enzymatic assays with hypericin show concentration-dependent inhibition of both PLpro protease and deubiquitinating activities. Given its use in supplementations and the FDA conditional approval of a synthetic version, further evaluation of hypericin as a potential SARS-CoV-2 antiviral is warranted.

Major depressive disorder (MDD) is considered a serious public health issue that adversely impacts an individual's quality of life and contributes significantly to the global burden of disease. The clinical heterogeneity that exists among patients limits the ability of MDD to be accurately diagnosed and currently, a symptom-based approach is utilized in many cases. Due to the complex nature of this disorder, and lack of precise knowledge regarding the pathophysiology, effective management is challenging. The aetiology and pathophysiology of MDD remain largely unknown given the complex genetic and environmental interactions that are involved. Nonetheless, the aetiology and pathophysiology of MDD have been the subject of extensive research, and there is a vast body of literature that exists. Here we overview the key hypotheses that have been proposed for the neurobiology of MDD and highlight the need for a unified model, as many of these pathways are integrated. Key pathways discussed include neurotransmission, neuroinflammation, clock gene machinery pathways, oxidative stress, role of neurotrophins, stress response pathways, the endocannabinoid and endovanilloid systems, and the endogenous opioid system. We also describe the current management of MDD, and emerging novel therapies, with particular focus on patients with treatment-resistant depression (TRD).

Oxidative stress is one of the pathophysiological mechanisms implicated in drug-resistant epilepsy. Recurrent seizures and prolonged treatment with anti-seizure medicines (ASMs) can produce reactive oxygen species (ROS) resulting in neuronal cell damage, cell toxicity, and cell death. This damage may contribute to the loss of efficacy of anti-seizure medicines. Add-on therapy with antioxidants, neuroimmunophilins, and polyphenols may thus be beneficial in drug-resistant epilepsy. In vitro and in vivo studies have shown a significant improvement in drug efficacy and seizure suppression using co-treatment of anti-seizure medication with naturally available antioxidants including alpha-lipoic acid (α-lipoic acid) from walnut; however, the underlying mechanisms of action remain to be fully understood.We undertook molecular docking and molecular dynamics simulations to determine whether alpha-lipoic acid and related analogues interacted with the human manganese superoxide dismutase (MnSOD) protein, a member of the oxidative metabolic pathway. The 3D structure of the compounds and the protein were retrieved from protein and chemical databases, binding sites were identified and ligand-protein interactions were performed.Alpha-lipoic acid and various analogues docked within a human MnSOD binding region. Docking results were validated by molecular dynamic simulation. The CMX-2043 analogue showed strong binding with MnSOD compared to alpha-lipoic acid and other analogues.Our findings provide new insights into additional mechanisms of action, which may in part, account for the antioxidant properties associated with alpha-lipoic acid and related analogues. The results support further in vitro and in vivo evaluation of these compounds to better understand their potential as add-on therapy for ASM treatment in epilepsy.

L-sulforaphane (LSF) is a naturally derived isothiocyanate derived from cruciferous vegetables that has long been known for its health benefiting effects. These include its anti-carcinogenic, antioxidant and anti-inflammatory effects. LSF also possesses antimicrobial properties, although the evidence for this is limited. Respiratory pathogens such as Streptococcus pneumoniae, Haemophilus influenzae, S. pyogenes and respiratory syncytial virus (RSV) are leading causes of illness and death among children under five-years-old globally, especially in resource-poor countries where access to vaccines are limited or in the case of S. pyogenes and RSV, have not been licensed for use in humans. Therefore, alternate strategies to prevent and/or treat these common infectious diseases are urgently needed. This study investigated the antimicrobial effects of LSF against common respiratory pathogens, S. pneumoniae (serotypes 1 and 6B), H. influenzae type B (HiB) and non-typeable H. influenzae (NTHi), S. pyogenes and RSV in relevant human cell-based models. LSF significantly inhibited the growth of H. influenzae, but not S. pneumoniae or S. pyogenes. LSF did not improve opsonophagocytic capacity or killing by human phagocytic cell lines (HL-60s and THP-1 macrophages) for S. pneumoniae yet showed some improved killing for H. influenzae species in THP-1 macrophages. However, LSF significantly reduced RSV infection in human lung epithelial cells, associated with increased expression of cyclin D1 (CCND1) gene as well as the antioxidant genes nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HMOX-1). Overall, LSF represents an exciting avenue for further antimicrobial research, particularly as a novel therapy against H. influenzae species and RSV.

COVID-19 is an ongoing pandemic caused by the SARS-CoV-2 virus with important political, socio-economic, and public health consequences. Inhibiting replication represents an important antiviral approach, and in this context two viral proteases, the SARS-CoV-2 main and papain-like proteases (PLpro), which cleave pp1a and pp1ab polypeptides, are critical. Along with protease activity, the PLpro possesses deubiquitinating activity, which is important in immune regulation. Naphthalene-based inhibitors, such as the well-investigated GRL-0617 compound, have been shown to possess dual effects, inhibiting both protease and deubiquitinating activity of the PLpro. Rather than binding to the canonical catalytic triad, these type of non-covalent inhibitors target an adjacent pocket, the naphthalene-inhibitor binding site. Using a high-throughput screen, we have previously identified the dietary hypericin, rutin, and cyanidin-3-O-glucoside compounds as potential protease inhibitors targeting the naphthalene-inhibitor binding site. Here, our aim was to investigate the binding characteristics of these compounds to the PLpro, and to evaluate deubiquitinating activity, by analyzing seven different PLpro crystal structures. Molecular docking highlighted the relatively high affinity of GRL-0617 and dietary compounds. In contrast binding of the small molecules was abolished in the presence of ubiquitin in the palm subdomain of the PLpro. Further, docking the small molecules in the naphthalene-inhibitor binding site, followed by protein-protein docking revealed displacement of ubiquitin in a conformation inconsistent with functional activity. Finally, the deubiquitinating activity was validated in vitro using an enzymatic activity assay. The findings indicated that the dietary compounds inhibited deubiquitinase activity in the micromolar range with an order of activity of GRL-0167, hypericin >> rutin, cyanidin-3-O-glucoside > epigallocatechin gallate, epicatechin gallate, and cefotaxime. Our findings are in accordance with mechanisms and potential antiviral effects of the naphthalene-based, GRL-0617 inhibitor, which is currently progressing in preclinical trials. Further, our findings indicate that in particular hypericin, rutin, and cyanidin-3-O-glucoside, represent suitable candidates for subsequent evaluation as PLpro inhibitors.

Graphical abstract, The SARS-CoV-2 papain-like protease (PLpro) is a suitable target for drug development, and its deubiquitinating and deISGylating activities have also been reported. In this study, molecular docking was used to investigate the binding properties of a selection of dietary compounds and naphthalene-based inhibitors to the previously characterised binding site of GRL-0617. The structures of the SARS-CoV-2 and SARS-CoV PLpro in complex with interferon-stimulated gene 15 (ISG15) and lysine 48 (K48)-linked diubiquitin were utilised. To predict whether compounds could potentially interfere with the binding of these cellular modifiers, docking was conducted in the absence and presence of ISG15 and K48-linked diubiquitin.

Circadian rhythms are responsible for regulating a number of physiological processes. The central oscillator is located within the suprachiasmatic nucleus (SCN) of the hypothalamus and the SCN synchronises the circadian clocks that are found in our peripheral organs through neural and humoral signalling. At the molecular level, biological clocks consist of transcription-translation feedback loops (TTFLs) and these pathways are influenced by transcription factors, post-translational modifications, signalling pathways and epigenetic modifiers. When disruptions occur in the circadian machinery, the activities of the proteins implicated in this network and the expression of core clock or clock-controlled genes (CCGs) can be altered. Circadian misalignment can also arise when there is desychronisation between our internal clocks and environmental stimuli. There is evidence in the literature demonstrating that disturbances in the circadian rhythm contribute to the pathophysiology of several diseases and disorders. This includes the metabolic syndrome and recently, it has been suggested that the 'circadian syndrome' may be a more appropriate term to use to not only describe the cardio-metabolic risk factors but also the associated comorbidities. Here we overview the molecular architecture of circadian clocks in mammals and provide insight into the effects of shift work, exposure to artificial light, food intake and stress on the circadian rhythm. The relationship between circadian rhythms, metabolic disorders and depression is reviewed and this is a topic that requires further investigation. We also describe how particular proteins involved in the TTFLs can be potentially modulated by small molecules, including pharmacological interventions and dietary compounds.

As highlighted in the OliveNet™ library, Olea europaea consists of a diverse collection of chemical compounds. We have classified over 600 compounds into 13 main classes and 47 subclasses. Various compounds, including oleuropein and hydroxytyrosol, have been investigated for their potential beneficial effects in multiple human pathologies. However, the vast majority of compounds remain largely unexplored and approximately 50% are currently non-commercially available.Here, we utilise conventional software to characterise the absorption, distribution, metabolism, excretion, and toxicity profile of OliveNet™ compounds. Molecular docking was performed for assessment of P-glycoprotein (P-gp) inhibition and interactions with the human ether-à-go-go-related gene (hERG) channel. Potential hERG ion channel inhibition was calibrated using in vitro patch clamp assays and steered molecular dynamics (SMD) simulations were used to examine membrane permeability of a subset of compounds.Our findings indicated that 313 out of 675 olive compounds were predicted to be absorbed by the gastrointestinal tract. Hydroxytyrosol required the least amount of force to pass through the lipid bilayer compared to elenolic acid diglucoside in SMD simulations. Based on the ADMET and molecular docking data, the hERG inhibitory activities of verbascoside, oleuropein, and hydroxytyrosol were investigated using patch clamp assays and they were identified as non-inhibitors.While the favourable properties of well-known compounds were confirmed, we identified oleuropein aglycone decarboxymethyl dialdehyde acetal form, decarboxymethyl elenolic acid dialdehyde, acetal of decarboxymethyl elenolic acid dialdehyde, methyl malate-β-hydroxytyrosol ester, hydroxytyrosil elenolate, D-(+)-erythro-1-(4-hydroxy-3-methoxy)-214-phenyl-1,2,3-propantriol, (+)-1-acetoxypinoresinol-4″-O-methyl ether, and 3-[1-(hydroxymethyl)-(E)-1-propenyl] glutaric acid as potential candidates for synthesis and further evaluation.

Graphical abstract, Highlights • The SARS-CoV-2 main protease (Mpro) has an important role in the viral life cycle. • Inhibition of the active site or dimerization site of Mpro can mitigate activity. • Mapping reveals a reactive pocket in the dimerization pocket at the apex of Mpro. • Blind docking shows that ligands may preferentially bind at the apex of Mpro. • Stable ligand interactions are formed at the active and apex sites of Mpro., The SARS-CoV-2 virus is causing COVID-19 resulting in an ongoing pandemic with serious health, social, and economic implications. Much research is focused in repurposing or identifying new small molecules which may interact with viral or host-cell molecular targets. An important SARS-CoV-2 target is the main protease (Mpro), and the peptidomimetic α-ketoamides represent prototypical experimental inhibitors. The protease is characterised by the dimerization of two monomers each which contains the catalytic dyad defined by Cys145 and His41 residues (active site). Dimerization yields the functional homodimer. Here, our aim was to investigate small molecules, including lopinavir and ritonavir, α-ketoamide 13b, and ebselen, for their ability to interact with the Mpro. The sirtuin 1 agonist SRT1720 was also used in our analyses. Blind docking to each monomer individually indicated preferential binding of the ligands in the active site. Site-mapping of the dimeric protease indicated a highly reactive pocket in the dimerization region at the domain III apex. Blind docking consistently indicated a strong preference of ligand binding in domain III, away from the active site. Molecular dynamics simulations indicated that ligands docked both to the active site and in the dimerization region at the apex, formed relatively stable interactions. Overall, our findings do not obviate the superior potency with respect to inhibition of protease activity of covalently-linked inhibitors such as α-ketoamide 13b in the Mpro active site. Nevertheless, along with those from others, our findings highlight the importance of further characterisation of the Mpro active site and any potential allosteric sites.

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used to reduce pain, and function by targeting cyclooxygenase (COX) enzymes to inhibit the production of prostaglandins that facilitate inflammation. Since oleocanthal derived from Olea europaea is known to inhibit COX, we sought to characterise novel olive compounds with COX inhibitory activity using in silico techniques. Following on from part 1 of this study which identified 1-oleyltyrosol (1OL) and ligstroside derivative 2 (LG2) with COX inhibitory potential, the mechanisms of COX interactions by these selected compounds were further examined using molecular dynamics (MD) simulations. Classical MD simulations were carried out on COX-1 and COX-2 complexed with 1OL and LG2 to determine the stability and protein backbone fluctuation. Protein dynamics were examined using essential dynamics methods and network analysis, which identified that the N-terminal epidermal growth factor-like domain and membrane bound domains of COX-1 and -2 exhibited altered motions when ligands were bound. Distinct dynamical modules were identified, and that COX-2 inter-residue communications were more sensitive to ligand binding compared to COX-1. The use of various network metrics presents a novel approach in the characterisation of network behaviour of different ligands. It is proposed that inter-residue network metrics provide additional measures of the potential bioactivity of ligands, which may form a useful adjunct to conventional direct predictions of binding affinity, in determining the efficacy of potential small-molecule inhibitors. Overall, this two-part study characterises anti-inflammatory effects of low dosage dietary COX inhibitors, and provides a possible avenue for the development of therapeutics in inflammatory diseases.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to reduce pain. These target cyclooxygenase (COX) enzymes which produce inflammatory mediators. Adverse effects associated with the use of traditional NSAIDs have led to a rise in the development of alternative therapies. Derived from Olea Europaea, olive oil is a main component of the Mediterranean diet, containing phenolic compounds that contribute to its antioxidant and anti-inflammatory properties. It has previously been found that oleocanthal, a phenolic compound derived from the olive, had similar effects to ibuprofen, a commonly used NSAID. There is an abundance of olive phenolic compounds that have yet to be investigated for their anti-inflammatory properties. In this study, it was sought to identify potential olive-derived compounds with the ability to inhibit COX enzymes, and study the mechanisms using in silico approaches. Molecular docking was employed to determine the COX inhibitory potential of an olive phenolic compound library. From docking, it was determined that 1-oleyltyrosol (1OL) and ligstroside derivative 2 (LG2) demonstrated the greatest binding affinity to both COX-1 and COX-2. Interactions with these compounds were further examined using molecular dynamics simulations. The residue contributions to binding free energy were computed using Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) methods, revealing that residues Leu93, Val116, Leu352, and Ala527 in COX-1 and COX-2 were key determinants of potential inhibition. Along with part 2 of this study, this work aims to identify and characterise novel phenolic compounds which may possess COX inhibitory properties.

Graphical abstract, Highlights • The SARS-CoV-2 main protease (Mpro) is an important target against COVID-19. • Peptidomimetic α-ketoamides represent prototypical inhibitors of the SARS-CoV-2 Mpro. • We confirmed the high affinity of the α-ketoamide 13b inhibitor to SARS-CoV-2 Mpro • MD simulations highlight α-ketoamide 13b stability in Mpro compared to amoxicillin • The α-ketoamide compound class could be pursued as potential antivirals against COVID-19, The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes an illness known as COVID-19, which has been declared a global pandemic with over 2 million confirmed cases and 137,000 deaths in 185 countries and regions at the time of writing (16 April 2020), over a quarter of these cases being in the United States. In the absence of a vaccine, or an approved effective therapeutic, there is an intense interest in repositioning available drugs or designing small molecule antivirals. In this context, in silico modelling has proven to be an invaluable tool. An important target is the SARS-CoV-2 main protease (Mpro), involved in processing translated viral proteins. Peptidomimetic α-ketoamides represent prototypical inhibitors of Mpro. A recent attempt at designing a compound with enhanced pharmacokinetic properties has resulted in the synthesis and evaluation of the α-ketoamide 13b analogue. Here, we performed molecular docking and molecular dynamics simulations to further characterize the interaction of α-ketoamide 13b with the active site of the SARS-CoV-2 Mpro. We included the widely used antibiotic, amoxicillin, for comparison. Our findings indicate that α-ketoamide 13b binds more tightly (predicted GlideScore = -8.7 and -9.2 kcal/mol for protomers A and B, respectively), to the protease active site compared to amoxicillin (-5.0 and -4.8 kcal/mol). Further, molecular dynamics simulations highlight the stability of the interaction of the α-ketoamide 13b ligand with the SARS-CoV-2 Mpro (ΔG = -25.2 and -22.3 kcal/mol for protomers A and B). In contrast, amoxicillin interacts unfavourably with the protease (ΔG = +32.8 kcal/mol for protomer A), with unbinding events observed in several independent simulations. Overall, our findings are consistent with those previously observed, and highlight the need to further explore the α-ketoamides as potential antivirals for this ongoing COVID-19 pandemic.

Streptococcus pneumoniae infection can lead to pneumococcal disease, a major cause of mortality in children under the age of five years. In low- and middle-income country settings where pneumococcal disease burden is high, vaccine use is low and widespread antibiotic use has led to increased rates of multi-drug resistant pneumococci. l -sulforaphane (LSF), derived from broccoli and other cruciferous vegetables, has established anti-inflammatory, antioxidant, and anti-microbial properties. Hence, we sought to investigate the potential role of LSF against pneumococcal infection. Using a combination of in vitro and computational methods, the results showed that LSF and relevant metabolites had a potential to reduce pneumococcal adherence through modulation of host receptors, regulation of inflammation, or through direct modification of bacterial factors. Treatment with LSF and metabolites reduced pneumococcal adherence to respiratory epithelial cells. Synchrotron-Fourier transform infrared microspectroscopy (S-FTIR) revealed biochemical changes in protein and lipid profiles of lung epithelial cells following treatment with LSF or metabolites. Molecular docking studies of 116 pneumococcal and 89 host factors revealed a potent effect for the metabolite LSF-glutathione (GSH). A comprehensive list of factors involved in interactions between S. pneumoniae and host cells was compiled to construct a bacterium and host interaction network. Network analysis revealed plasminogen, fibronectin, and RrgA as key factors involved in pneumococcal-host interactions. Therefore, we propose that these constitute critical targets for direct inhibition by LSF and/or metabolites, which may disrupt pneumococcal-host adherence. Overall, our findings further enhance understanding of the potential role of LSF to modulate pneumococcal-host dynamics.

The ovalbumin-induced (OVA) chronic allergic airways murine model is a well-established model for investigating pre-clinical therapies for chronic allergic airways diseases, such as asthma. Here, we examined the effects of several experimental compounds with potential anti-asthmatic effects including resveratrol (RV), relaxin (RLN), l-sulforaphane (LSF), valproic acid (VPA), and trichostatin A (TSA) using both a prevention and reversal model of chronic allergic airways disease. We undertook a novel analytical approach using focal plane array (FPA) and synchrotron Fourier-transform infrared (S-FTIR) microspectroscopic techniques to provide new insights into the mechanisms of action of these experimental compounds. Apart from the typical biological effects, S-FTIR microspectroscopy was able to detect changes in nucleic acids and protein acetylation. Further, we validated the reduction in collagen deposition induced by each experimental compound evaluated. Although this has previously been observed with conventional histological methods, the S-FTIR technique has the advantage of allowing identification of the type of collagen present. More generally, our findings highlight the potential utility of S-FTIR and FPA-FTIR imaging techniques in enabling a better mechanistic understanding of novel asthma therapeutics.

Lactoferrin (LF) is a multi-functional protein that is primarily found in milk. The apo form of LF (apo-LF) is devoid of iron, and as a result, may serve as an iron scavenger with attendant anti-pathogenic properties, thereby, endowing apo-LF with promising health benefits. Despite these advantages, the structural stability of apo-LF may be perturbed by various processing conditions. One of the strategies employed to stabilise the activity of apo-LF is to encapsulate it as part of an oligomeric complex, composed of apo-LF, α-lactalbumin (α-La) and β-lactoglobulin (β-Lg). Accordingly, the aim of this study is to investigate the structural changes of apo-LF and its interactions with α-La and β-Lg under the temperature conditions of freeze-drying (−30, 0, and 20 °C). Using in silico approaches, molecular dynamics simulations showed apo-LF had limited structural perturbation when processed at these temperatures. Both α-La and β-Lg stabilise apo-LF by enhancing and maintaining the structural integrity and cohesion of the tertiary structure of apo-LF. These stabilities are the result of electrostatic interactions, binding apo-LF and the protective proteins into a multimeric complex. The secondary structure and the anti-bacterial derivatives in apo-LF were also sustained under these simulated conditions. Furthermore, inter-residue contact maps in apo-LF predict an increase in intramolecular contacts at simulated freeze-drying conditions, resulting in agglomeration and, therefore potential proteins dispersibility during subsequent rehydration. Overall, this work demonstrates the advantages of computational approaches to understand the macromolecular conformations, interactions, and functional implications of a multimeric complex composed of apo-LF, α-La, and β-Lg at low temperatures.

Increasingly, lactic acid bacteria have been encapsulated into food formulations because of their health advantages, including modulation of the gut microflora. However, the molecular mechanism on how these bacteria interact with the encapsulating agent, which is typically composed of milk proteins is not fully comprehended. Accordingly, molecular dynamics simulations were employed to investigate the interaction between the cell wall component of these bacteria, specifically the peptidoglycan layer and a protein complex, comprising apo-lactoferrin, β-lactoglobulin, and α-lactalbumin at spray drying temperatures (68 and 110 °C). The results demonstrate that the peptidoglycan model interacts with the posterior N-lobe of apo-lactoferrin and the three-turn α-helix of β-lactoglobulin via van der Waals, electrostatic, and non-polar solvation interactions. Free energy calculations also show higher binding affinity at 110 °C than 68 °C, indicating that elevated temperatures are essential to facilitate hydrophobic interactions between the peptidoglycan component and the N-lobe of apo-lactoferrin. Furthermore, the secondary structure and the antibacterial regions in apo-lactoferrin are maintained following thermal simulations. The simulations not only highlight the formation of a stable complex, involving the peptidoglycan component, apo-lactoferrin, β-lactoglobulin, and α-lactalbumin, but also the preservation of the functionalities of apo-lactoferrin, which may provide valuable insights into the development of novel nutraceuticals.

Graphical abstract, The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) is a promising target for antiviral drugs. In this study, a chemical library (n = 300) was screened against the nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain. Blind docking was performed using a selection of 30 compounds and nine ligands were chosen based on their docking scores, safety profile, and availability. Using cluster analysis on a 10 microsecond molecular dynamics simulation trajectory (from D.E. Shaw Research), the compounds were docked to the different conformations. On the basis of our modelling studies, oleuropein was identified as a potential lead compound.

This study employs computational techniques to investigate whether the inclusion of β-lactoglobulin and α-lactalbumin can enhance the molecular stability of apo-lactoferrin in conditions of gastric pH. Atomistic molecular dynamics simulations reveal that acidic pH results in weakened interactions between lactoferrin and the other proteins compared to those under neutral pH. Nevertheless, protein-protein dissociation free energy calculations using the umbrella sampling calculations confirm that some contacts are maintained between apo-lactoferrin and the other whey proteins, specifically the β-lactoglobulin units. This is manifested as a broad energy minimum at the intermediate inter-molecular distance of the inter-protein free energy profiles; thereby, demonstrating the formation of a nano-scale flocculant, followed by a potential gel, composed of protein particles. Practically, this property could be exploited to entrap further bioactive components within the protein matrix for novel engineered food materials. Furthermore, it was found that the anti-bacterial peptide regions of apo-lactoferrin are unaffected by the acidic pH, indicating the potential capability of apo-lactoferrin to act against harmful bacteria. Overall, this work unveils an understanding at a molecular level regarding the structural changes of the aforementioned proteins that maybe beneficial for future advancement of nutraceuticals.

The SARS-CoV-2 virus is causing COVID-19, an ongoing pandemic, with extraordinary global health, social, and political implications. Currently, extensive research and development efforts are aimed at producing a safe and effective vaccine. In the interim, small molecules are being widely investigated for antiviral effects. With respect to viral replication, the papain-like (PLpro) and main proteases (Mpro), are critical for processing viral replicase polypeptides. Further, the PLpro possesses deubiquitinating activity affecting key signalling pathways, including inhibition of interferon and innate immune antagonism. Therefore, inhibition of PLpro activity with small molecules is an important research direction. Our aim was to focus on identification of potential inhibitors of the protease activity of SARS-CoV-2 PLpro. We investigated 300 small compounds derived predominantly from our OliveNet™ library (222 phenolics) and supplemented with synthetic and dietary compounds with reported antiviral activities. An initial docking screen, using the potent and selective noncovalent PLpro inhibitor, GRL-0617 as a control, enabled a selection of 30 compounds for further analyses. From further in silico analyses, including docking to scenes derived from a publicly available molecular dynamics simulation trajectory (100 μs PDB 6WX4; DESRES-ANTON-11441075), we identified lead compounds for further in vitro evaluation using an enzymatic inhibition assay measuring SARS-CoV-2 PLpro protease activity. Our findings indicate that hypericin possessed inhibition activity, and both rutin and cyanidin-3-O-glucoside resulted in a concentration-dependent inhibition of the PLpro, with activity in the micromolar range. Overall, hypericin, rutin, and cyanidin-3-O-glucoside can be considered lead compounds requiring further characterisation for potential antiviral effects in appropriate model systems., Graphical abstract Image 1

The SARS-CoV-2 papain-like protease (PLpro) is a suitable target for drug development, and its deubiquitinating and deISGylating activities have also been reported. In this study, molecular docking was used to investigate the binding properties of a selection of dietary compounds and naphthalene-based inhibitors to the previously characterised binding site of GRL-0617. The structures of the SARS-CoV-2 and SARS-CoV PLpro in complex with interferon-stimulated gene 15 (ISG15) and lysine 48 (K48)-linked diubiquitin were utilised. To predict whether compounds could potentially interfere with the binding of these cellular modifiers, docking was conducted in the absence and presence of ISG15 and K48-linked diubiquitin.

Graphical abstract, The COVID-19 pandemic caused by SARS-CoV-2 has resulted in an international health emergency. The SARS-CoV-2 nsp16 is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase, and with its cofactor nsp10, is responsible for RNA cap formation. This study aimed to identify small molecules binding to the SAM-binding site of the nsp10-nsp16 heterodimer for potential inhibition of methyltransferase activity. By screening a library of 300 compounds, 30 compounds were selected based on binding scores, side-effects, and availability. Following more advanced docking, six potential lead compounds were further investigated using molecular dynamics simulations. This revealed the dietary compound oleuropein as a potential methyltransferase inhibitor.

Although olive oil polyphenols are minor components of olive oil, they contribute substantially to the numerous health benefits of olive oil including against cancer, diabetes, osteoporosis, cardiovascular, and neurodegenerative disease. Studies describing these effects including some mechanisms of action are summarized, as well as details about clinical trials related to promising polyphenols. The powerful antioxidants, hydroxytyrosol and oleuropein, are the focus of this chapter and have been the focus of various health-related studies on olive oil polyphenols that have reported antiinflammatory effects, and other health benefits including enhanced wound healing, and increased digestive and respiratory health. Many of the studies selected for this synopsis involve bioactivities of orally administered olive oil polyphenols to provide further support for the identification of olive oil as a functional food. We propose that more clinical trials are needed to further emphasize the importance of incorporating olive oil or polyphenol supplements into a healthy diet.

According to the World Health Organisation, 70% of all deaths globally can be attributed to chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, respiratory conditions, cardiovascular diseases, diabetes and cancer. Chronic inflammation has a significant impact on the quality of life of affected individuals with an increased risk of developing other chronic inflammatory diseases. Given the limitations of current pharmaceuticals, there is an intense research interest in identifying novel dietary interventions that can regulate and alleviate inflammation. A diet rich in cruciferous vegetables has been extensively studied for its immediate and long-term health benefits, particularly in the context of cardiovascular disease and cancer. Cruciferous vegetables contain the precursor glucoraphanin, which is hydrolysed upon consumption to form l-sulforaphane (LSF), the primary active compound that mediates potential cardio-protective and anti-carcinogenic effects. LSF has been shown to have beneficial effects in vitro and in animal studies through its classical antioxidant and anti-inflammatory properties, and more recently its chromatin modifying effects. This review discusses the clinical evidence to date in relation to the use of LSF in the context of chronic inflammatory diseases as well as provide key mechanistic insights for these effects.

The dietary isothiocyanate L-sulforaphane (LSF), derived from cruciferous vegetables, is reported to have several beneficial biological properties, including anti-inflammatory and immunomodulatory effects. However, there is limited data on how LSF modulates these effects in human immune cells. The present study was designed to investigate the immunomodulatory effects of LSF (10 µM and 50 µM) on peripheral blood mononuclear cell (PBMC) populations and cytokine secretion in healthy adult volunteers (n = 14), in the presence or absence of bacterial (lipopolysaccharide) and viral (imiquimod) toll-like receptor (TLRs) stimulations. Here, we found that LSF reduced pro-inflammatory cytokines interleukin (IL)-6, IL-1b, and chemokines monocyte chemoattractant protein (MCP)-1 irrespective of TLR stimulations. This result was associated with LSF significantly reducing the proportion of natural killer (NK) cells and monocytes while increasing the proportions of dendritic cells (DCs), T cells and B cells. We found a novel effect of LSF in relation to reducing cluster of differentiation (CD) 14+ monocytes while simultaneously increasing monocyte-derived DCs (moDCs: lineage-Human Leukocyte Antigen-DR isotype (HLA-DR)+CD11blow-high CD11chigh). LSF was also shown to induce a 3.9-fold increase in the antioxidant response element (ARE) activity in a human monocyte cell line (THP-1). Our results provide important insights into the immunomodulatory effects of LSF, showing in human PBMCs an ability to drive differentiation of monocytes towards an immature monocyte-derived dendritic cell phenotype with potentially important biological functions. These findings provide insights into the potential role of LSF as a novel immunomodulatory drug candidate and supports the need for further preclinical and phase I clinical studies.

Graphical abstract, Highlights • The SARS-CoV-2 Mpro is an important viral target for the ongoing COVID-19 pandemic. • Initial screen by docking 300 small molecules to the Mpro active site. • Preferential binding of 3 leads to the Mpro active site revealed by blind docking. • Stability of lead compounds with the Mpro active site confirmed by MD simulations. • Hypericin and cyanidin-3-O-glucoside inhibit Mpro in vitro in micromolar range., Caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pandemic is ongoing, with no proven safe and effective vaccine to date. Further, effective therapeutic agents for COVID-19 are limited, and as a result, the identification of potential small molecule antiviral drugs is of particular importance. A critical antiviral target is the SARS-CoV-2 main protease (Mpro), and our aim was to identify lead compounds with potential inhibitory effects. We performed an initial molecular docking screen of 300 small molecules, which included phenolic compounds and fatty acids from our OliveNet™ library (224), and an additional group of curated pharmacological and dietary compounds. The prototypical α-ketoamide 13b inhibitor was used as a control to guide selection of the top 30 compounds with respect to binding affinity to the Mpro active site. Further studies and analyses including blind docking were performed to identify hypericin, cyanidin-3-O-glucoside and SRT2104 as potential leads. Molecular dynamics simulations demonstrated that hypericin (ΔG = -18.6 and -19.3 kcal/mol), cyanidin-3-O-glucoside (ΔG = -50.8 and -42.1 kcal/mol), and SRT2104 (ΔG = -8.7 and -20.6 kcal/mol), formed stable interactions with the Mpro active site. An enzyme-linked immunosorbent assay indicated that, albeit, not as potent as the covalent positive control (GC376), our leads inhibited the Mpro with activity in the micromolar range, and an order of effectiveness of hypericin and cyanidin-3-O-glucoside > SRT2104 > SRT1720. Overall, our findings, and those highlighted by others indicate that hypericin and cyanidin-3-O-glucoside are suitable candidates for progress to in vitro and in vivo antiviral studies.

Alzheimer’s disease (AD) is a debilitating neurodegenerative disease that affects over 47 million people worldwide, and is the most common form of dementia. There is a vast body of literature demonstrating that the disease is caused by an accumulation of toxic extracellular amyloid-β (Aβ) peptides and intracellular neurofibrillary tangles that consist of hyperphosphorylated tau. Adherence to the Mediterranean diet has been shown to reduce the incidence of AD and the phenolic compounds in extra virgin olive oil, including oleocanthal, have gained a significant amount of attention. A large number of these ligands have been described in the pre-existing literature and 222 of these compounds have been characterised in the OliveNet™ database. In this study, molecular docking was used to screen the 222 phenolic compounds from the OliveNet™ database and assess their ability to bind to various forms of the Aβ and tau proteins. The phenolic ligands were found to be binding strongly to the hairpin-turn of the Aβ1−40 and Aβ1−42 monomers, and binding sites were also identified in the tau fibril protein structures. Luteolin-4′-O-rutinoside, oleuricine A, isorhoifolin, luteolin-7-O-rutinoside, cyanidin-3-O-rutinoside and luteolin-7,4-O-diglucoside were predicted to be novel lead compounds. Molecular dynamics (MD) simulations performed using well-known olive ligands bound to Aβ1−42 oligomers highlighted that future work may examine potential anti-aggregating properties of novel compounds in the OliveNet™ database. This may lead to the development and evaluation of new compounds that may have efficacy against Alzheimer’s disease.

There has been increasing interest in lactoferrin as a bioactive compound from milk, as it functions as an antimicrobial and also in delivering iron. Despite the potential as a food ingredient, the low bioavailability of lactoferrin has limited opportunities for commercialisation. Therefore, protective encapsulation of lactoferrin has been explored as a means of enhancing its bioavailability. In this study, a novel in silico approach was adopted to explore the possibility of employing whey protein components to encapsulate lactoferrin. The most unstable form of the protein, apo-lactoferrin has been studied, particularly interactions with the whey proteins β-lactoglobulin and α-lactalbumin. Temperature conditions that might be encountered during spray drying (95 and 180 °C) have been modelled in order to provide an in-depth understanding of the interactions at the molecular level. The findings indicate that β-lactoglobulin and α-lactalbumin are able to preserve the two antibacterial regions within the apo-lactoferrin structure (lactoferricin and lactoferampin). Intra-molecular residue contact analyses within apo-lactoferrin illustrate the tendency for agglomeration under these temperature conditions, indicating that it is most likely to be readily dispersible during subsequent rehydration. Furthermore, free energy calculations found electrostatic attractions to be the primary mode of interaction between apo-lactoferrin, β-lactoglobulin, and α-lactalbumin. This data is confirmed by a network analysis that demonstrates how interactions amongst apo-lactoferrin, β-lactoglobulin, and α-lactalbumin are predominantly a result of the acidic and basic amino acid residues of the proteins. Overall, this study provides molecular insights in understanding the interactions between lactoferrin with β-lactoglobulin and α-lactalbumin for potential encapsulation strategies via spray drying.

The antidepressant medications that are currently prescribed to patients suffering from major depressive disorder (MDD) have limitations and as a result, there is an urgent need to increase the options that are available. A number of studies have found that natural polyphenols have neuroprotective properties and there is evidence to suggest that they modulate neurotransmitter systems. There are more than 200 phenolic compounds that have been identified in Olea europaea, many of which have not yet been investigated for their potential biological effects. In this study, in silico methods were used to screen the phenolic library from the OliveNet™ database and identify novel lead compounds for proteins implicated in the pathophysiology of MDD. The molecular docking results revealed that the monoamine oxidase enzyme isoforms (MAO-A/MAO-B) had binding specificities for certain phenolic subclasses. The lead ligands that were identified from these subclasses were positioned near the flavin adenine dinucleotide (FAD) cofactor, interacting in a similar manner as known inhibitors. In addition to the MAO enzymes, several phenolic compounds were docked to neurotransmitter transporters and postsynaptic receptors, as well as proteins involved in neuroinflammation, oxidative stress and the endocannabinoid system. Based on the binding affinity, position, orientation and interactions of the lead phenolic compounds identified in this study, it is predicted that they may have antidepressant properties. The results should be validated further using molecular dynamics (MD) simulations, as well as in vivo and in vitro techniques.

Accumulated epidemiological, clinical and experimental evidence has indicated the beneficial health effects of the Mediterranean diet, which is typified by the consumption of virgin olive oil (VOO) as a main source of dietary fat. At the cellular level, compounds derived from various olive (Olea europaea), matrices, have demonstrated potent antioxidant and anti-inflammatory effects, which are thought to account, at least in part, for their biological effects. Research efforts are expanding into the characterization of compounds derived from Olea europaea, however, the considerable diversity and complexity of the vast array of chemical compounds have made their precise identification and quantification challenging. As such, only a relatively small subset of olive-derived compounds has been explored for their biological activity and potential health effects to date. Although there is adequate information describing the identification or isolation of olive-derived compounds, these are not easily searchable, especially when attempting to acquire chemical or biological properties. Therefore, we have created the OliveNet™ database containing a comprehensive catalogue of compounds identified from matrices of the olive, including the fruit, leaf and VOO, as well as in the wastewater and pomace accrued during oil production. From a total of 752 compounds, chemical analysis was sufficient for 676 individual compounds, which have been included in the database. The database is curated and comprehensively referenced containing information for the 676 compounds, which are divided into 13 main classes and 47 subclasses. Importantly, with respect to current research trends, the database includes 222 olive phenolics, which are divided into 13 subclasses. To our knowledge, OliveNet™ is currently the only curated open access database with a comprehensive collection of compounds associated with Olea europaea. Database URL: https://www.mccordresearch.com.au

Significance: Sulforaphane, produced by the hydrolytic conversion of glucoraphanin after ingestion of cruciferous vegetables, particularly broccoli and broccoli sprouts, has been extensively studied due to its apparent health-promoting properties in disease and limited toxicity in normal tissue. Recent Studies: Recent identification of a sub-population of tumor cells with stem cell-like self-renewal capacity that may be responsible for relapse, metastasis, and resistance, as a potential target of the dietary compound, may be an important aspect of sulforaphane chemoprevention. Evidence also suggests that sulforaphane may target the epigenetic alterations observed in specific cancers, reversing aberrant changes in gene transcription through mechanisms of histone deacetylase inhibition, global demethylation, and microRNA modulation. Critical Issues: In this review, we discuss the biochemical and biological properties of sulforaphane with a particular emphasis on the anticancer properties of the dietary compound. Sulforaphane possesses the capacity to intervene in multistage carcinogenesis through the modulation and/or regulation of important cellular mechanisms. The inhibition of phase I enzymes that are responsible for the activation of pro-carcinogens, and the induction of phase II enzymes that are critical in mutagen elimination are well-characterized chemopreventive properties. Furthermore, sulforaphane mediates a number of anticancer pathways, including the activation of apoptosis, induction of cell cycle arrest, and inhibition of NFκB. Future Directions: Further characterization of the chemopreventive properties of sulforaphane and its capacity to be selectively toxic to malignant cells are warranted to potentially establish the clinical utility of the dietary compound as an anti-cancer compound alone, and in combination with clinically relevant therapeutic and management strategies. Antioxid. Redox Signal. 22, 1382–1424.

Chronic myeloid leukemia (CML) involves the malignant transformation of hematopoietic stem cells, defined largely by the Philadelphia chromosome and expression of the breakpoint cluster region-Abelson (BCR-ABL) oncoprotein. Pharmacological tyrosine kinase inhibitors (TKIs), including imatinib mesylate, have overcome limitations in conventional treatment for the improved clinical management of CML.Accumulated evidence has led to the identification of a subpopulation of quiescent leukemia progenitor cells with stem-like self renewal properties that may initiate leukemogenesis, which are also shown to be present in residual disease due to their insensitivity to tyrosine kinase inhibition.The characterization of quiescent leukemia progenitor cells as a unique cell population in CML pathogenesis has become critical with the complete elucidation of mechanisms involved in their survival independent of BCR-ABL that is important in the development of novel anticancer strategies. Understanding of these functional pathways in CML progenitor cells will allow for their selective therapeutic targeting. In addition, disease pathogenesis and drug responsiveness is also thought to be modulated by epigenetic regulatory mechanisms such as DNA methylation, histone acetylation, and microRNA expression, with a capacity to control CML-associated gene transcription.A number of compounds in combination with TKIs are under preclinical and clinical investigation to assess their synergistic potential in targeting leukemic progenitor cells and/or the epigenome in CML. Despite the collective promise, further research is required in order to refine understanding, and, ultimately, advance antileukemic therapeutic strategies.

Cancer cells have been shown to have altered metabolism when compared to normal non-malignant cells. The Warburg effect describes a phenomenon in which cancer cells preferentially metabolize glucose by glycolysis, producing lactate as an end product, despite being the presence of oxygen. The phenomenon was first described by Otto Warburg in the 1920s, and has resurfaced as a controversial theory, with both supportive and opposing arguments. The biochemical aspects of the Warburg effect outline a strong explanation for the cause of cancer cell proliferation, by providing the biological requirements for a cell to grow. Studies have shown that pathways such as phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) as well as hypoxia inducible factor-1 (HIF-1) are central regulators of glycolysis, cancer metabolism and cancer cell proliferation. Studies have shown that PI3K signaling pathways have a role in many cellular processes such as metabolism, inflammation, cell survival, motility and cancer progression. Herein, the cellular aspects of the PI3K pathway are described, as well as the influence HIF has on cancer cell metabolism. HIF-1 activation has been related to angiogenesis, erythropoiesis and modulation of key enzymes involved in aerobic glycolysis, thereby modulating key processes required for the Warburg effect. In this review we discuss the molecular aspects of the Warburg effect with a particular emphasis on the role of the HIF-1 and the PI3K pathway.

Asthma is a chronic respiratory disease characterised by airway inflammation, remodeling and hyperresponsiveness. The ability to replicate these asthma traits in the well-established ovalbumin induced chronic model of allergic airways disease is an important tool for asthma research and preclinical drug development. Here, spectra derived from focal plane array and Synchrotron-Fourier transform infrared maps were used to analyse biochemical changes in lung tissue from an ovalbumin-induced murine chronic allergic airways disease model. Analysis of the chemical maps resulted in distinct clusters and significant changes in the lipid and proteins regions of the spectra between the saline control and diseased lung tissue samples. Overall, the utilisation of conventional histological methodologies and Synchrotron infrared microspectroscopy has the ability to expand the characterisation of murine models of asthma.

It has been about nine decades since the proposal of Otto Warburg on the metabolism of cancer cells. Unlike normal cells which undergo glycolysis and oxidative phosphorylation in the presence of oxygen, proliferating and cancer cells exhibit an increased uptake of glucose and increased rate of glycolysis and predominantly undergo lactic acid fermentation. Whether this phenomenon is the consequence of genetic dysregulation in cancer or is the cause of cancer still remains unknown. However, there is certainly a strong link between the genetic factors, epigenetic modulation, cancer immunosurveillance and the Warburg effect, which will be discussed in this review. Dichloroacetate and 3-bromopyruvate are among the substances that have been studied as potential cancer therapies. With our expanding knowledge of cellular metabolism, therapies targeting the Warburg effect appear very promising. This review discusses different aspects of these emerging therapies.

Oxidative stress and inflammation are inextricably linked and play essential roles in the initiation and progression of diabetes complications such as diabetes-associated atherosclerosis and nephropathy. Bolstering antioxidant defenses is an important mechanism to lessen oxidative stress and inflammation. In this study, we have used a novel analog of the NFE2-related factor 2 (Nrf2) agonist bardoxolone methyl, dh404, to investigate its effects on diabetic macrovascular and renal injury in streptozotocin-induced diabetic apolipoprotein E−/− mice. We show that dh404, at lower but not higher doses, significantly lessens diabetes-associated atherosclerosis with reductions in oxidative stress (in plasma, urine, and vascular tissue) and proinflammatory mediators tumor necrosis factor-α, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and monocyte chemotactic protein-1 (MCP-1). We demonstrate that dh404 attenuates functional (urinary albumin-to-creatinine ratio) and structural (mesangial expansion) glomerular injury and improves renal tubular injury. Liver functional and structural studies showed that dh404 is well tolerated. Complementary in vitro studies in normal rat kidney cells showed that dh404 significantly upregulates Nrf2-responsive genes, heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and glutathione-S transferase, with inhibition of transforming growth factor-β–mediated profibrotic fibronectin, collagen I, and proinflammatory interleukin-6. Higher doses of dh404 were associated with increased expression of proinflammatory mediators MCP-1 and nuclear factor-κB. These findings suggest that this class of compound is worthy of further study to lessen diabetes complications but that dosage needs consideration.