Structure-based drug design approach to target toll-like receptor signaling pathways for disease treatment

Toll-like receptor (TLR) signaling pathways are the first line of defence against many microbial organisms. The question of how TLRs recognize endogenous ligands remains controversial. Several studies have shown that TLRs are implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Therefore, in structure-based drug design, TLRs are now viewed as potential therapeutic targets in the treatment of autoimmune diseases. This review shows how proteins, specifically TLRs, are used as therapeutic targets to design inhibitors (drugs) using the structure-based drug design approach for disease treatment.Keywords: Structure-based drug design, Toll-like receptors, Autoimmune diseases, Endogenous ligands, X-ray crystallography, Homology modelin

Crystallization and X-ray diffraction analysis of the N-terminal domain of the Toll-like receptor signalling adaptor protein TRIF/TICAM-1

As part of the mammalian innate immune response, Toll-like receptors 3 and 4 can signal via the adaptor protein TRIF/TICAM-1 to elicit the production of type-I interferons and cytokines. Recent studies have suggested an auto-inhibitory role for the N-terminal domain (NTD) of TRIF. This domain has no significant sequence similarity to proteins of known structure. In this paper, the crystallization and X-ray diffraction analysis of TRIF-NTD and its selenomethionine-labelled mutant TRIF-NTDA66M/L113M are reported. Thin plate-like crystals of native TRIF-NTD obtained using polyethylene glycol 3350 as precipitant diffracted X-rays to 1.9 Å resolution. To facilitate phase determination, two additional methionines were incorporated into the protein at positions chosen based on the occurrence of methionines in TRIF homologues in different species. Crystals of the selenomethionine-labelled protein were obtained under conditions similar to the wild-type protein; these crystals diffracted X-rays to 2.5 Å resolution. The TRIF-NTD and TRIF-NTDA66M/L113M crystals have the symmetry of space groups P2 12121 and P1, and most likely contain two and four molecules in the asymmetric unit, respectively. These results provide a sound foundation for the future structure determination of this novel domain

Knowledge about anesthesia and the role of anesthesiologists among Jeddah citizens

Background: The anesthesiologist has a vital role in the operating theatres. Awareness of the role of the anesthesiologist and the types of anesthesia is essential for every person. This study was made to estimate how much information the general population have about the anesthesiologist and the different types of anesthesia.Methods: This research was a cross sectional non-interventional study. The research team conducted a questionnaire in which each participant in the study was interviewed by the research team. The sample size was 159 participants.Results: From the participants,99 (62.2%) recognized the anesthesiologist as a specialized doctor who administers the anesthetics,62 (38.9%) know that the anesthesiologist has a role in resuscitating the patient with the team if crises occurred. However, 85 (53.4%) believe that the surgeon has the responsibility of postoperative pain management. Physicians were the source of knowledge for most participant’s information.Conclusions: A reasonable percentage of people appreciated the role of the anesthesiologist in administrating the anesthesia, however there is a lack of information about the role of the anesthesiologist intra and postoperatively. The need for more education for people about anesthesia is essential as the amount of information about anesthesia in general is rather low

The TLR signalling adaptor TRIF/TICAM-1 has an N-terminal helical domain with structural similarity to IFIT proteins

TRIF/TICAM-1 (TIR domain-containing adaptor inducing interferon-beta/TIR domain-containing adaptor molecule 1) is the adaptor protein in the Toll-like receptor (TLR) 3 and 4 signalling pathway that leads to the production of type 1 interferons and cytokines. The signalling involves TIR (Toll/interleukin-1 receptor) domain-dependent TRIF oligomerization. A protease-resistant N-terminal region is believed to be involved in self-regulation of TRIF by interacting with its TIR domain. Here, the structural and functional characterization of the N-terminal domain of TRIF (TRIF-NTD) comprising residues 1-153 is reported. The 2.22 angstrom resolution crystal structure was solved by single-wavelength anomalous diffraction (SAD) using selenomethionine-labelled crystals of TRIF-NTD containing two additional introduced Met residues (TRIF-NTDA66M/L113M). The structure consists of eight antiparallel helices that can be divided into two subdomains, and the overall fold shares similarity to the interferon-induced protein with tetratricopeptide repeats (IFIT) family of proteins, which are involved in both the recognition of viral RNA and modulation of innate immune signalling. Analysis of TRIF-NTD surface features and the mapping of sequence conservation onto the structure suggest several possible binding sites involved in either TRIF auto-regulation or interaction with other signalling molecules or ligands. TRIF-NTD suppresses TRIF-mediated activation of the interferon-beta promoter, as well as NF-kappa B-dependent reporter-gene activity. These findings thus identify opportunities for the selective targeting of TLR3- and TLR4-mediated inflammation

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the TIR domain from the Brucella melitensis TIR-domain-containing protein TcpB

In mammals, Toll-like receptors (TLRs) recognize conserved microbial molecular signatures and induce an early innate immune response in the host. TLR signalling is mediated by interactions between the cytosolic TIR (Toll/interleukin-1 receptor) domains of the receptor and the adaptor proteins. Increasingly, it is apparent that pathogens target this interaction via pathogen-expressed TIR-domain-containing proteins to modulate immune responses. A TIR-domain-containing protein TcpB has been reported in the pathogenic bacterium Brucella melitensis. Studies have shown that TcpB interferes with the TLR2 and TLR4 signalling pathways to inhibit TLR-mediated inflammatory responses. Such interference may involve TIR-TIR-domain interactions between bacterial and mammalian proteins, but there is a lack of information about these interactions at the molecular level. In this study, the cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the protein construct corresponding to the TIR domain of TcpB (residues 120-250) are reported. The crystals diffracted to 2.6 angstrom resolution, have the symmetry of the monoclinic space group P2(1) and are most likely to contain four molecules in the asymmetric unit. The structure should help in understanding the molecular basis of how TcpB affects the innate immunity of the host

Expression and production optimization of the cationic antimicrobial peptide : indolicidin by the recombinant E. coli C41 (DE3) clones

The cytoplasmic granules of bovine neutrophils naturally possess indolicidin - a promising cationic antimicrobial peptide as it displays inherent inhibitory activities against a broad type of microbial pathogens. In this study, a shake flask level production and expression optimizations of the indolicidin by the recombinant Escherichia coli C41 (DE3) clones (transformed with pET21a(+) plasmid carrying indolicidin gene) were carried out under standard conditions, as to determine the conditions required for maximal production. It was determined that a concentration of 1 mM of IPTG was effective, the 2×YT with salts and LB media at pH 7.5 with 3-6 h of incubation were required for maximal indolicidin expression

In silico structural homology modeling and characterization of multiple N-terminal domains of selected bacterial Tcps

Several bacterial pathogens produce Toll/interleukin-1 receptor (TIR) domain-containing protein homologs that are important for subverting the Toll-like receptor (TLR) signaling cascades in hosts. Consequently, promoting the persistence and survival of the bacterial pathogens. However, the exact molecular mechanisms elucidating the functional characteristics of these bacterial proteins are not clear. Physicochemical and homology modeling characterization studies have been conducted to predict the conditions suitable for the stability and purification of these proteins and to predict their structural properties. The outcomes of these studies have provided important preliminary data for the drug discovery pipeline projects. Here, using in silico physicochemical and homology modeling tools, we have reported the primary, secondary and tertiary structural characteristics of multiple N-terminal domains of selected bacterial TIR domain-containing proteins (Tcps). The results show variations between the primary amino acid sequences, secondary structural components and three-dimensional models of the proteins, suggesting the role of different molecular mechanisms in the functioning of these proteins in subverting the host immune system. This study could form the basis of future experimental studies advancing our understanding of the molecular basis of the inhibition of the host immune response by the bacterial Tcps.</jats:p

Brucellosis: An Investigation of the Knowledge, Attitudes and Behaviors Among a Selected Population in Majmaah Saudi Arab

Brucellosis is a zoonotic bacterial disease carried by animals such as sheep, cows, and camels. It is transmitted to humans through the consumption of affected animals or their by-products. Dairy products form a major part of the Arabic diet and are very widely consumed in Saudi Arabia, where brucellosis is endemic. This study has analyzed the current knowledge, attitudes, and behaviors toward brucellosis of adults aged 18 years and older among Majmaah University students and staff. A cross-sectional study with 181 participants was conducted using an online survey during March–April 2021. The results showed that the majority of people knew about brucellosis and its transmission, but only a fraction of them took the required precautionary measures. This study highlights that knowledge alone is insufficient to promote healthy behaviors. It is thus important to raise awareness in a way that can convert knowledge into action.</jats:p