TNF-α Inhibitors from Natural Compounds: An Overview, CADD Approaches, and their Exploration for Anti-inflammatory Agents.

Inflammation is a natural process that occurs in the organism in response to harmful external agents. Despite being considered beneficial, exaggerated cases can cause severe problems for the body. The main inflammatory manifestations are pain, increased temperature, edema, decreased mobility, and quality of life for affected individuals. Diseases such as arthritis, cancer, allergies, infections, arteriosclerosis, neurodegenerative diseases, and metabolic problems are mainly characterized by an exaggerated inflammatory response. Inflammation is related to two categories of substances: pro- and anti-inflammatory mediators. Among the pro-inflammatory mediators is Tumor Necrosis Factor-α (TNF-α). It is associated with immune diseases, cancer, and psychiatric disorders which increase its excretion. Thus, it becomes a target widely used in discovering new antiinflammatory drugs. In this context, secondary metabolites biosynthesized by plants have been used for thousands of years and continue to be one of the primary sources of new drug scaffolds against inflammatory diseases. To decrease costs related to the drug discovery process, Computer-Aided Drug Design (CADD) techniques are broadly explored to increase the chances of success. In this review, the main natural compounds derived from alkaloids, flavonoids, terpene, and polyphenols as promising TNF-α inhibitors will be discussed. Finally, we applied a molecular modeling protocol involving all compounds described here, suggesting that their interactions with Tyr59, Tyr119, Tyr151, Leu57, and Gly121 residues are essential for the activity. Such findings can be useful for research groups worldwide to design new anti-inflammatory TNF-α inhibitors.

TNF-α: The shape of small molecules to come?

In 2020, the anti-tumor necrosis factor (TNF) monoclonal antibody Humira® generated US$165.8 billion in cumulative sales and snatched the crown for the industry's most successful drug from Lipitor (atorvastatin). TNF-α is a major component in beneficial and disease-related inflammation and TNF-α-inhibitor biologics have gained widespread use in autoimmune diseases, such as rheumatoid arthritis (RA). Many more diseases could benefit from TNF-α inhibitors, such as Alzheimer's disease (AD) or major depression. However, the nature of TNF-α-inhibitor biologics prohibits central nervous system (CNS) applications. Moreover, high drug production costs and pricing, together with antidrug immune reactions and insufficient patient coverage, argue for the development of small-molecule drugs. Recently, drug-like orally available small molecules were described with high activity in animal disease models with activities comparable to those of antibodies.

In Silico Identification and Evaluation of Natural Products as Potential Tumor Necrosis Factor Function Inhibitors Using Advanced Enalos Asclepios KNIME Nodes.

Tumor necrosis factor (TNF) is a regulator of several chronic inflammatory diseases, such as rheumatoid arthritis. Although anti-TNF biologics have been used in clinic, they render several drawbacks, such as patients' progressive immunodeficiency and loss of response, high cost, and intravenous administration. In order to find new potential anti-TNF small molecule inhibitors, we employed an in silico approach, aiming to find natural products, analogs of Ampelopsin H, a compound that blocks the formation of TNF active trimer. Two out of nine commercially available compounds tested, Nepalensinol B and Miyabenol A, efficiently reduced TNF-induced cytotoxicity in L929 cells and production of chemokines in mice joints' synovial fibroblasts, while Nepalensinol B also abolished TNF-TNFR1 binding in non-toxic concentrations. The binding mode of the compounds was further investigated by molecular dynamics and free energy calculation studies, using and advancing the Enalos Asclepios pipeline. Conclusively, we propose that Nepalensinol B, characterized by the lowest free energy of binding and by a higher number of hydrogen bonds with TNF, qualifies as a potential lead compound for TNF inhibitors' drug development. Finally, the upgraded Enalos Asclepios pipeline can be used for improved identification of new therapeutics against TNF-mediated chronic inflammatory diseases, providing state-of-the-art insight on their binding mode.

Expression of a Tagless Single-Chain Variable Fragment (scFv) of Anti-TNF-α by a Salt Inducible System and its Purification and Characterization.

BACKGROUND: Anti-TNF-α scFv is gaining acceptance as an effective drug for various diseases, such as rheumatoid arthritis and Crohn's disease that involve elevated levels of TNF-α. The single-chain variable fragment (scFv) consists of variable regions of heavy and light chains of monoclonal antibodies (mAb). Due to its smaller size, it curbs the mAb's auto-antibody effects and their limitation of penetration into the tissues during the neutralization of TNF-α. OBJECTIVE: In this work, a cDNA coding for anti-TNF-α scFv was successfully cloned into a pRSET-B vector and efficiently expressed in an E. coli strain GJ1158, a salt inducible system that uses sodium chloride instead of IPTG as an inducer. METHODS: The protein was expressed in the form of inclusion bodies (IB), solubilized using urea, and refolded by pulse dilution. Further, the amino acid sequence coverage of scFv was confirmed by ESI-Q-TOF MS/MS and MALDI-TOF. Further studies on scaling up the production of scFv and its application of scFv are being carried out. RESULTS: The soluble fraction of anti-TNF-α scFv was then purified in a single chromatographic step using CM-Sephadex chromatography, a weak cation exchanger with a yield of 10.3 mg/L. The molecular weight of the scFv was found to be ~ 28 kDa by SDS PAGE, and its presence was confirmed by western blot analysis and mass spectrometry. CONCLUSION: Anti-TNF-α scFv has been successfully purified in a salt inducible system GJ1158. As per the best of our knowledge, this is the first report of purification of Anti-TNF-α scFv in a salt inducible system from soluble fractions as well as inclusion bodies.

A cross-linked anti-TNF-α aptamer for neutralization of TNF-α-induced cutaneous Shwartzman phenomenon: A simple and novel approach for improving aptamers' affinity and efficiency.

To increase the efficiency of aptamers to their targets, a simple and novel method has been developed based on aptamer oligomerization. To this purpose, previously anti-human TNF-α aptamer named T1-T4 was trimerized through a trimethyl aconitate core for neutralization of in vitro and in vivo of TNF-α. At first, 54 mer T1-T4 aptamers with 5'-NH2 groups were covalently coupled to three ester residues in the trimethyl aconitate. In vitro activity of novel anti-TNF-α aptamer and its dissociation constant (Kd ) was done using the L929 cell cytotoxicity assay. In vivo anti-TNF-α activity of new oligomerized aptamer was assessed in a mouse model of cutaneous Shwartzman. Anchoring of three T1-T4 aptamers to trimethyl aconitate substituent results in formation of the 162 mer fragment, which was well revealed by gel electrophoresis. In vitro study indicated that the trimerization of T1-T4 aptamer significantly improved its anti-TNF-α activity compared to non-modified aptamers (P < 0.0001) from 40% to 60%. The determination of Kd showed that trimerization could effectively enhance Kd of aptamer from 67 nM to 36 nM. In vivo study showed that trimer aptamer markedly reduced mean scar size from 15.2 ± 1.2 mm to 1.6 ± 0.1 mm (P < 0.0001), which prevent the formation of skin lesions. In vitro and in vivo studies indicate that trimerization of anti-TNF-α aptamer with a novel approach could improve the anti-TNF-α activity and therapeutic efficacy. According to our findings, a new anti-TNF-α aptamer described here could be considered an appropriate therapeutic agent in treating several inflammatory diseases.

Modulation of inflammation by anti-TNF α mAb-dendrimer nanoparticles loaded in tyramine-modified gellan gum hydrogels in a cartilage-on-a-chip model.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease characterized by joint inflammation. Since the inflammatory condition plays an important role in the disease process, it is important to develop and test new therapeutic approaches that specifically target and treat joint inflammation. In this study, a human 3D inflammatory cartilage-on-a-chip model was established to test the therapeutic efficacy of anti-TNFα mAb-CS/PAMAM dendrimer NPs loaded-Tyramine-Gellan Gum in the treatment of inflammation. The results showed that the proposed therapeutic approach applied to the human monocyte cell line (THP-1) and human chondrogenic primary cells (hCH) cell-based inflammation system revealed an anti-inflammatory capacity that increased over 14 days. It was also possible to observe that Coll type II was highly expressed by inflamed hCH upon the culture with anti-TNF α mAb-CS/PAMAM dendrimer NPs, indicating that the hCH cells were able maintain their biological function. The developed preclinical model allowed us to provide more robust data on the potential therapeutic effect of anti-TNF α mAb-CS/PAMAM dendrimer NPs loaded-Ty-GG hydrogel in a physiologically relevant model.

Fucosylation and Sialylation of Fc-Fragment of anti-Tumour Necrosis Factor Alpha Antibodies do not Influence Their Immunogenicity in Monocyte-Derived Dendritic Cells.

BACKGROUND AND AIMS: Antibodies targeting tumor necrosis factor-alpha [TNF-alpha] are a mainstay in the treatment of inflammatory bowel disease. However, they fail to demonstrate efficacy in a considerable proportion of patients. On the other hand, glycosylation of antibodies might influence not only their immunogenicity but also their structure and function. We investigated whether specific glycosylation patterns of the Fc-fragment would affect the immunogenicity of anti-TNF-alpha antibody in monocyte-derived dendritic cells. METHODS: The effect of a specific Fc-glycosylation pattern on antibody uptake by monocyte-derived dendritic cells [mo-DCs] and how this process shapes the immunologic profile of mo-DCs was investigated. Three N-glycoforms of the anti-TNF-alpha antibody adalimumab, that differed in the content of fucose or sialic acid, were tested: [1] mock treated Humira, abbreviated 'Fuc-G0', where the N-glycan mainly consist of fucose and N-acetylglucosamine [GlcNAc], without sialic acid; [2] 'Fuc-G2S1/G2S2' with fucose and alpha 2,6 linked sialic acid; and [3] 'G2S1/G2S2' with alpha 2,6 linked sialic acid, without fucose. RESULTS: Our data demonstrated that neither fucosylation nor sialylation of anti-TNF-Abs [Fuc-G0, FucG2S1/G2S2, G2S1/G2S2] influence their uptake by mo-DCs. Additionally, none of the differentially glycosylated antibodies altered CD80, CD86, CD273, CD274 levels on mo-DCs stimulated in with lipopolysaccharide in the presence of antibodies. Next, we evaluated the levels of cytokines in the supernatant of mo-DCs stimulated with lipopolysaccharide in the presence of Fuc-G0, Fuc-G2S1/G2S2 or G2S1/G2S2-glycosylated anti-TNF antibodies. Only IL-2 and IL-17 levels were downregulated, and IL-5 production was upregulated by uptake of Fuc-G0 antibodies, as compared to control without antibodies. CONCLUSIONS: The specific modification in the Fc-glycosylation pattern of anti-TNF-alpha Abs does not affect their immunogenicity under the tested conditions. As this study was limited to mo-DCs, further investigation is required to clarify whether Ab uptake into mo-DCs might change the immunological profile of T- and B-cells, in order to ultimately reduce the formation of anti-drug antibodies and to improve the patient care.

Generation and Evaluation of Bispecific Anti-TNF Antibodies Based on Single-Chain VHH Domains.

Systemic cytokine inhibition may be an effective therapeutic strategy for several autoimmune diseases. However, recent studies suggest that tissue or cell type-specific targeting of certain cytokines, including TNF, may have distinct advantages and show fewer side effects. Here we describe protocols for generating and testing bispecific cytokine inhibitors using variable domain of single-chain antibodies from Camelidae (VHH) with a focus on cell-specific TNF inhibitors.

TAK1: a potent tumour necrosis factor inhibitor for the treatment of inflammatory diseases.

Aberrant tumour necrosis factor (TNF) signalling is a hallmark of many inflammatory diseases including rheumatoid arthritis (RA), irritable bowel disease and lupus. Maladaptive TNF signalling can lead to hyper active downstream nuclear factor (NF)-κß signalling in turn amplifying a cell's inflammatory response and exacerbating disease. Within the TNF intracellular inflammatory signalling cascade, transforming growth factor-ß-activated kinase 1 (TAK1) has been shown to play a critical role in mediating signal transduction and downstream NF-κß activation. Owing to its role in TNF inflammatory signalling, TAK1 has become a potential therapeutic target for the treatment of inflammatory diseases such as RA. This review highlights the current development of targeting the TNF-TAK1 signalling axis as a novel therapeutic strategy for the treatment of inflammatory diseases.