Jadhav, Gopal P.
(2008)
Search for Pseudomonas aeruginosa immune-modulatory but non-inducing agents.
PhD thesis, University of Nottingham.
Abstract
Dysfunctional immunity which is the most common antecedent of immunological diseases is central to the common disorders like rheumatoid arthritis, psoriasis, thyroid disease, type-1 diabetes mellitus, multiple sclerosis and other autoimmune conditions that are leading causes of chronic morbidities and disabilities. The currently employed therapies suffer from serious side effects. For example, steroid therapy, being nonselective is seldom used. Myleotoxicity limits azathioprine while cyclosporine-A (CsA) and FK506 do show myleo-, nephro and neuro-toxicities. Antibody therapies cause immune mediated toxicity. Therefore, the need of developing less toxic and specific immune modulatory agents is a top priority.
Recently, Pseudomonas aeruginosa quorum sensing signal molecules (QSSMs), N-(3-oxododecanoyl)-L-homoserine lactone (3O, C12-HSL) and 2-n-heptyl-3-hydroxy-4-(1H)-quinolone (PQS) have been found to modulate eukaryotic immune processes via different targets. The structure activity relationship (SAR) study of 3O, C12-HSL by Chhabra et.al, showed that L-homoserine lactone ring, 3-oxo or hydroxyl group and 12 to14-carbons long acyl chain, apparently conferring optimum lipophilicity and flexibility, are important for immune modulation. However, the new analogues including 3O, C12-HSL negated clinical usefulness as these also participated in bacterial quorum-sensing (QS) activity thus promoting bacterial growth. Also analogues with more than 12-carbons alkyl chain could not be explored by in vitro immune assays due to their limited solubility in the solvents employed.
The present study addresses these issues by making further judicial structural changes in the 3O, C12-HSL in order to optimize its immune modulatory activity while losing its intrinsic QS activity. Accordingly, a series of new ring variants, 3-acetoxy, aza, thia, oxa and fluorine substituted analogues were developed. The later were largely confined in the 1, 3-dicarbonyl segment of the 3O, C12-HSL structure in order to modulate the 3-enolic content of the molecule. Also tetramic acid analogue, a non-enzymatic degradative product of 3O, C12-HSL was synthesized. After purity and identity characterization the new analogues were evaluated for their immune modulation (with murine cells proliferation assay), cytotoxicity (with Trypan dye exclusion assay) and auto-inducing or QS activity (with a specifically designed lux bacterial bio-reporter).
The splenocyte proliferation assay demonstrated that immune suppression in 4-aza analogues increased with increasing alkyl chain (up to C15) yet without any solubility problems in the standard solvents used. Similar trend was observed when N4 was alkyl substituted (n-propyl, being most potent). At least one of the C2 hydrogens was found to be essential for activity since, difluoro substitution lost activity. Retention of immune suppression by N3-OH and increased potency by N3-NH2 (EC50=0.99 microM) demonstrated that C3=O can be replaced by an appropriate H-bond donor/acceptor groups. Complete loss of activity with 3-thia substitution (3-thia C12-HSL) and then regain of potency by 3-sulfoxide/sulfone derivatives further demonstrated the importance of 3-oxo (suitable H-bond acceptor/donor) in immune suppression. Insertion of oxygen at C4 retained activity but additional oxygen(s) in the side chain yielded analogues that were devoid of immune suppression. Analogue with thiazole ring instead of HSL ring retained activity. In case of hetero ring altered analogues of 3O, 4-aza C12-HSL, almost all retained immune suppressive activities except their oxa analogues.
In conclusion, all the above strategies have delivered potent immune modulatory agents yet gratifyingly devoid of QS activity. Some of the most potent and promising non-inducing immune modulatory agents are 3O, 4-aza-C15-HSL (EC50 0.65 microM), 3-NH2-3-aza-C12-HSL (EC50 0.99 microM) and N, N'-dimethyl-2-(3-oxo-4-azadodecanoyl) aminobenzamide (EC50 0.7 microM) and are worthy of further study to be developed as therapeutic agents.
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