Lipid‐Embedded Molecular Dynamics Simulation Model for Exploring the Reverse Prostaglandin D2 Agonism of CT‐133 towards CRTH2 in the Treatment of Type‐2 Inflammation Dependent Diseases

Chemoattractant receptor‐homologous molecule expressed on Th2 cells (CRTH2) has been involved in several inflammation dependent diseases by mediating the chemotaxis of pro‐inflammatory cells in response to allergy and other responses through PGD2 ligation. This CRTH2‐PGD2 signaling pathway has become a target for treating allergic and type 2 inflammation dependent diseases, with many inhibitors developed to target the PGD2 binding pocket. One of such inhibitors is the ramatroban analog, CT‐133, which exhibited therapeutic potency cigarette smoke‐induced acute lung injury in patients. Nonetheless, the molecular mechanism and structural dynamics that accounts for its therapeutic prowess remain unclear. Employing computational tools, this study revealed that although the carboxylate moiety in CT‐133 and the native agonist PGD2 aided in their stability within the CRTH2 binding pocket, the tetrahydrocarbazole group of CT‐133 engaged in strong interactions with binding pocket residues which could have formed as the basis of the antagonistic advantage of CT‐133. Tetrahydrocarbazole group interactions also enhanced the relative stability CT‐133 within the binding pocket which consequently favored CT‐133 binding affinity. CT‐133 binding also induced an inactive or ‘desensitized’ state in the helix 8 of CRTH2 which could conversely favor the recruitment of arrestin. These revelations would aid in the speedy development of small molecule inhibitors of CRTH2 in the treatment of type 2 inflammation dependent diseases.     

Analysis of purine receptor expression and functionality in alveolar epithelial cells

Despite its fundamental role in providing an extensive surface for gas exchange, the alveolar epithelium (AE) serves as an immunological barrier through, e.g., the release of proinflammatory cytokines and secretion of surfactant to prevent alveolar collapse. Thus, AE is important for sustaining lung homeostasis. Extracellular ATP secreted by alveolar epithelial cells (AECs) is involved in physiological and pathological conditions and acts mainly through the activation of purine receptors (P2Rs). When studying P2R-mediated processes, primary isolated type II AECs (piAECs) still represent the gold standard in in vitro research, although their preparation is time-consuming and requires the sacrifice of many animals. Hence, cultivated immortalized and tumor-derived AEC lines may constitute a valuable alternative. In this work, we examined P2R expression and functionality in piAECs, in immortalized and tumor-derived AEC lines with the purpose of gaining a better understanding of purinergic signaling in different cell systems and assisting researchers in the choice of a suitable cell line with a certain P2R in demand. We combined mRNA and protein analysis to evaluate the expression of P2R. For pharmacological testing, we conducted calcium ([Ca²⁺]) measurements and siRNA receptor knockdown. Interestingly, the mRNA and protein levels of P2Y₂, P2Y_6, and P2X₄ were detected on all cell lines. Concerning functionality, P2XR could be narrowed to L2 and piAECs while P2YR were active in all cell lines.

     

Investigating the Mechanistic Inhibitory Discrepancies of Novel Halogen and Alkyl Di-Substituted Oxadiazole-Based Dibenzo-Azepine-Dione Derivatives on Poly (ADP-Ribose) Polymerase-1

Numerous studies have established the involvement of Poly (ADP-ribose) Polymerase-1 (PARP-1) in cancer presenting it as an important therapeutic target over recent years. Although homology among the PARP protein family makes selective targeting difficult, two compounds [d11 (0.939 μM) and d21 (0.047 μM)] with disparate inhibitory potencies against PARP-1 were recently identified. In this study, free energy calculations and molecular simulations were used to decipher underlying mechanisms of differential PARP-1 inhibition exhibited by the two compounds. The thermodynamics calculation revealed that compound d21 had a relatively higher ΔG_bind than d11. High involvement of van der Waal and electrostatic effects potentiated the affinity of d21 at PARP-1 active site. More so, incorporated methyl moiety in d11 accounted for steric hindrance which, in turn, prevented complementary interactions of key site residues such as TYR889, MET890, TYR896, TYR907. Conformational studies also revealed that d21 is more stabilized for interactions in the active site compared to d11. We believe that findings from this study would provide an important avenue for the development of selective PARP-1 inhibitors.     

Halting ionic shuttle to disrupt the synthetic machinery—Structural and molecular insights into the inhibitory roles of Bedaquiline towards Mycobacterium tuberculosis ATP synthase in the treatment of tuberculosis

Therapeutic targeting of the adenosine triphosphate (ATP) machinery of Mycobacterium tuberculosis (Mtb) has recently presented a potent and alternative measure to halt the pathogenesis of tuberculosis. This has been potentiated by the development of bedaquiline (BDQ), a novel small molecule inhibitor that selectively inhibits mycobacterial F₁F_o-ATP synthase by targeting its rotor c-ring, resulting in the disruption of ATP synthesis and consequential cell death. Although the structural resolution of the mycobacterial C₉ ring in co`mplex with BDQ provided the first-hand detail of BDQ interaction at the c-ring region of the ATP synthase, there still remains a need to obtain essential and dynamic insights into the mechanistic activity of this drug molecule towards crucial survival machinery of Mtb. As such, for the first time, we report an atomistic model to describe the structural dynamics that explicate the experimentally reported antagonistic features of BDQ in halting ion shuttling by the mycobacterial c-ring, using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Surface Area methods. Results showed that BDQ exhibited a considerably high ΔG while it specifically maintained high-affinity interactions with Glu65_B and Asp32_B, blocking their crucial roles in proton binding and shuttling, which is required for ATP synthesis. Moreover, the bulky nature of BDQ induced a rigid and compact conformation of the rotor c-ring, which impedes the essential rotatory motion that drives ion exchange and shuttling. In addition, the binding affinity of a BDQ molecule was considerably increased by the complementary binding of another BDQ molecule, which indicates that an increase in BDQ molecule enhances inhibitory potency against Mtb ATP synthase. Taken together, findings provide atomistic perspectives into the inhibitory mechanisms of BDQ coupled with insights that could enhance the structure-based design of novel ATP synthase inhibitors towards the treatment of tuberculosis.     

Dual Drug Targeting of Mutant Bcr‐Abl Induces Inactive Conformation: New Strategy for the Treatment of Chronic Myeloid Leukemia and Overcoming Monotherapy Resistance

Bcr‐Abl is an oncogenic fusion protein which expression enhances tumorigenesis, and has been highly associated with chronic myeloid leukemia (CML). Acquired drug resistance in mutant Bcr‐Abl has enhanced pathogenesis with the use of single therapy agents such as nilotinib. Moreover, allosteric targeting has been identified to consequentially inhibit Bcr‐Abl activity, which led to the recent development of ABL‐001 (asciminib) that selectively binds the myristoyl pocket. Experimental studies have revealed that the combination of nilotinib and ABL‐001 induced a ‘bent’ conformation in the C‐terminal helix of Bcr‐Abl; a benchmark of inhibition, thereby exhibiting a greater potency in the treatment of CML, surmounting the setbacks of drug resistance, disease regression and relapse. Therefore, we report the first account of the dynamics and conformational analysis of oncogenic T334I Bcr‐Abl by dual targeting. Our findings revealed that unlike in the Bcr‐Abl‐Nilotinib complex, dual targeting by both inhibitors induced the bent conformation in the C‐terminal helix that varied with time. This was coupled with significant alteration in Bcr‐Abl stability, flexibility, and compactness and an overall structural re‐orientation inwards towards the hydrophobic core, which reduced the solvent‐exposed residues indicative of protein folding. This study will facilitate allosteric targeting and the design of more potent allosteric inhibitors for resistive target proteins in cancer.     

Stable and Unstable Malaria Hotspots in Longitudinal Cohort Studies in Kenya

Background

Infectious diseases often demonstrate heterogeneity of transmission among host populations. This heterogeneity reduces the efficacy of control strategies, but also implies that focusing control strategies on “hotspots” of transmission could be highly effective.

Methods and Findings

In order to identify hotspots of malaria transmission, we analysed longitudinal data on febrile malaria episodes, asymptomatic parasitaemia, and antibody titres over 12 y from 256 homesteads in three study areas in Kilifi District on the Kenyan coast. We examined heterogeneity by homestead, and identified groups of homesteads that formed hotspots using a spatial scan statistic. Two types of statistically significant hotspots were detected; stable hotspots of asymptomatic parasitaemia and unstable hotspots of febrile malaria. The stable hotspots were associated with higher average AMA-1 antibody titres than the unstable clusters (optical density [OD] = 1.24, 95% confidence interval [CI] 1.02–1.47 versus OD = 1.1, 95% CI 0.88–1.33) and lower mean ages of febrile malaria episodes (5.8 y, 95% CI 5.6–6.0 versus 5.91 y, 95% CI 5.7–6.1). A falling gradient of febrile malaria incidence was identified in the penumbrae of both hotspots. Hotspots were associated with AMA-1 titres, but not seroconversion rates. In order to target control measures, homesteads at risk of febrile malaria could be predicted by identifying the 20% of homesteads that experienced an episode of febrile malaria during one month in the dry season. That 20% subsequently experienced 65% of all febrile malaria episodes during the following year. A definition based on remote sensing data was 81% sensitive and 63% specific for the stable hotspots of asymptomatic malaria.

Conclusions

Hotspots of asymptomatic parasitaemia are stable over time, but hotspots of febrile malaria are unstable. This finding may be because immunity offsets the high rate of febrile malaria that might otherwise result in stable hotspots, whereas unstable hotspots necessarily affect a population with less prior exposure to malaria. Please see later in the article for the Editors'' Summary     

Defining clinical malaria: the specificity and incidence of endpoints from active and passive surveillance of children in rural Kenya

Background

Febrile malaria is the most common clinical manifestation of P. falciparum infection, and is often the primary endpoint in clinical trials and epidemiological studies. Subjective and objective fevers are both used to define the endpoint, but have not been carefully compared, and the relative incidence of clinical malaria by active and passive case detection is unknown.

Methods

We analyzed data from cohorts under active and passive surveillance, including 19,462 presentations with fever and 5,551 blood tests for asymptomatic parasitaemia. A logistic regression model was used to calculate Malaria Attributable Fractions (MAFs) for various case definitions. Incidences of febrile malaria by active and passive surveillance were compared in a subset of children matched for age and location.

Results

Active surveillance identified three times the incidence of clinical malaria as passive surveillance in a subset of children matched for age and location. Objective fever (temperature≥37.5°C) gave consistently higher MAFs than case definitions based on subjective fever.

Conclusion

The endpoints from active and passive surveillance have high specificity, but the incidence of endpoints is lower on passive surveillance. Subjective fever had low specificity and should not be used in primary endpoint. Passive surveillance will reduce the power of clinical trials but may cost-effectively deliver acceptable sensitivity in studies of large populations.     

The Relationship between RTS,S Vaccine-Induced Antibodies,CD4+ T Cell Responses and Protection against Plasmodium falciparum Infection

Vaccination with the pre-erythrocytic malaria vaccine RTS,S induces high levels of antibodies and CD4⁺ T cells specific for the circumsporozoite protein (CSP). Using a biologically-motivated mathematical model of sporozoite infection fitted to data from malaria-naive adults vaccinated with RTS,S and subjected to experimental P. falciparum challenge, we characterised the relationship between antibodies, CD4⁺ T cell responses and protection from infection. Both anti-CSP antibody titres and CSP-specific CD4⁺ T cells were identified as immunological surrogates of protection, with RTS,S induced anti-CSP antibodies estimated to prevent 32% (95% confidence interval (CI) 24%–41%) of infections. The addition of RTS,S-induced CSP-specific CD4⁺ T cells was estimated to increase vaccine efficacy against infection to 40% (95% CI, 34%–48%). This protective efficacy is estimated to result from a 96.1% (95% CI, 93.4%–97.8%) reduction in the liver-to-blood parasite inoculum, indicating that in volunteers who developed P. falciparum infection, a small number of parasites (often the progeny of a single surviving sporozoite) are responsible for breakthrough blood-stage infections.