Imidazo[2,1-b]thiazole based indoleamine-2,3-dioxygenase 1 (IDO1) inhibitor: Structure based design, synthesis, bio-evaluation and docking studies.

Indoleamine-2,3-dioxygenase 1 (IDO1) is an immunomodulatory enzyme known to catalyse the initial and rate limiting step of kynurenine pathway of l-tryptophan metabolism. IDO1 enzyme over expression plays a crucial role in progression of cancer, malaria, multiple sclerosis and other life-threatening diseases. Several efforts over the last two decades have been invested by the researchers for the discovery of different IDO1 inhibitors and the plasticity of the IDO1 enzyme ligand binding pocket provide ample opportunities to develop new heterocyclic scaffolds targeting this enzyme. In the present work, based on the X-ray crystal structure of human IDO1 coordinated with few ligands, we designed and synthesized new fused heterocyclic compounds and evaluated their potential human IDO1 inhibitory activity (compound 30 and 41 showed IC50 values of 23 and 13 µM, respectively). The identified HITs were observed to be non-toxic to HEK293 cells at 100 µM concentration. The observed activity of the synthesized compounds was correlated with the specific interactions of their structures at the enzyme pocket using docking studies. A detailed analysis of docking results of the synthesized analogues as well as selected known IDO1 inhibitors revealed that most of the inhibitors have some reasonable docking scores in at least two crystal structures and have similar orientation as that of co-crystal ligands.

Genetic interaction between F-box motif encoding YDR131C and retrograde signaling-related RTG1 regulates the stress response and apoptosis in Saccharomyces cerevisiae.

The retrograde signaling pathway is well conserved from yeast to humans, which regulates cell adaptation during stress conditions and prevents cell death. One of its components, RTG1 encoded Rtg1p in association with Rtg3p communicates between mitochondria, nucleus, and peroxisome during stress for adaptation, by regulation of transcription. The F-box motif protein encoded by YDR131C  constitutes a part of SCF Ydr131c -E3 ligase complex, with unknown function; however, it is known that retrograde signaling is modulated by the E3 ligase complex. This study reports epistasis interaction between YDR131C and RTG1, which regulates cell growth, response to genotoxic stress, decreased apoptosis, resistance to petite mutation, and cell wall integrity. The cells of ydr131cΔrtg1Δ genetic background exhibits growth rate improvement however, sensitivity to hydroxyurea, itraconazole antifungal agent and synthetic indoloquinazoline-based alkaloid (8-fluorotryptanthrin, RK64), which disrupts the cell wall integrity in Saccharomyces cerevisiae. The epistatic interaction between YDR131C and RTG1 indicates a link between protein degradation and retrograde signaling pathways.

Socialization During the COVID-19 Pandemic: The Role of Social and Scientific Networks During Social Distancing.

In the COVID-19 era, while we are encouraged to be physically far away from each other, social and scientific networking is needed more than ever. The dire consequences of social distancing can be diminished by social networking. Social media, a quintessential component of social networking, facilitates the dissemination of reliable information and fighting against misinformation by health authorities. Distance learning, telemedicine, and telehealth are among the most prominent applications of networking during this pandemic. Additionally, the COVID-19 pandemic highlights the importance of collaborative scientific efforts. In this chapter, we summarize the advantages of harnessing both social and scientific networking in minimizing the harms of this pandemic. We also discuss the extra collaborative measures we can take in our fight against COVID-19, particularly in the scientific field.

Facile synthesis of C6-substituted benz[4,5]imidazo[1,2-a]quinoxaline derivatives and their anticancer evaluation.

Cancer remains a leading cause of death worldwide, resulting in continuous efforts to discover and develop highly efficacious anticancer drugs. High-throughput screening of heterocyclic compound libraries is one of the promising approaches that provided several new lead molecules with a novel mechanism of action. On the basis of the promising anticancer potential of imidazoquinoxaline as well as the structurally similar imidazoquinoline-derived scaffold, we prepared a set of C6-substituted benzimidazo[1,2-a]quinoxaline derivatives via two novel synthetic routes using commercially available starting materials, with good to excellent yields and evaluated their anticancer activity against the NCI-60 cancer cell lines. The one-dose (10 µM) anticancer screening of the synthesized compounds in the NCI-60 cell line panel revealed that the substituents have a significant role in the activity. In particular, the indole (7f), imidazole (7g), and benzimidazole (7h) derivatives showed significant activity against the triple-negative breast cancer cell line, MDA-MB-468. The lead compounds also exhibited notable IC50 values against another breast cancer cell line, MCF-7. Furthermore, it was observed that these compounds were relatively nontoxic to normal cell lines: HEK293 (human embryonic kidney cell line) and MCF12A (nontumorigenic human breast epithelial cell line). The IC50 values against healthy cells were at least 5- to 11-fold higher, offering a new class of heterocycles that can be further developed as promising therapeutics for cancer treatment.

Design, synthesis and bio-evaluation of C-1 alkylated tetrahydro-ß-carboline derivatives as novel antifungal lead compounds.

The field of antifungal agent has become static and development of resistance by the pathogen as well as limited clinical efficacy of marketed drugs demand the constant development of new antifungals. The presence of hydrocarbon chain of specific length linked with various different heterocycles was found to be an important structural feature in various antifungal lead compounds. Based on the prominent antimicrobial activity of ß-carboline derivatives, a set of C1 alkylated tetrahydro-ß-carboline derivatives were proposed to be active against fungi. To validate and confirm the role of suitable alkyl chains linked to a ß-carboline scaffold, few related analogues having C1 aryl substituents were also synthesized in one step via classic Pictet-Spengler reaction. The synthesized library was evaluated for its antifungal activity against C. albicans, C. krusei, C. parapsilosis, C. kefyr, C. glabrata, C. tropicalis and C. neoformans. One of the library members (compound 12c), with n-alkyl chain of eight carbons exhibited potent antifungal activity against C. glabrata and C. kefyr. The lead compound, being selectively toxic also demonstrated prominent synergy enhancing the potency of antifungal drugs up to 10-fold. The time kill kinetic studies confirmed the efficacy of compound 12c, where the results obtained were comparable to that of Amp B. FE-SEM analysis revealed the increased asymmetry, disintegration and roughness of cell surface which could be because of the possible interaction of compound 12c at membrane level or interference in cell wall structure. Apoptosis/necrosis detection assay confirmed the significant apoptotic activity in C. glabrata cells after 12c treatment which was responsible for the rapid killing of C. glabrata cells.

Efficacy of TLR7 agonistic imidazoquinoline as immunochemotherapeutic agent against P. Berghei ANKA infected rodent host.

Malaria is a serious disease and is one of the most alarming public health issues. Plasmodium is being resistant to various antimalarials including Chloroquine (CQ) which was the first-line therapy for malaria treatment. WHO recommended several combination therapies but declining efficacy was reported to many of these therapies. Despite a great amount of research, efficient malaria vaccine still seems to be a distant dream. Immunochemotherapy could be an alternate strategy to deal with malaria. Based on the differential activity of various cytokines in the pathogenesis of and protection against malaria, the efficacy of highly active TLR7 agonistic imidazoquinoline (BBIQ) in combination with a suboptimal dose of CQ against P. berghei ANKA (PbA) in vivo was investigated. In mice treated with CQ alone, parasite appeared on Day 17 and all mice of this group died by Day 21. Whereas, mice treated with BBIQ along with CQ exhibited no appearance of parasite till Day 23. Frequencies of T cells (CD3+, CD4+and CD8+) and T regulatory cells (CD4+, CD25 +and FoxP3+) were found to be lower in brain of BBIQ + CQ treated mice as compared to BBIQ alone and CQ alone treated mice on Day 10. Inhibition of infiltration of inflammatory T cells and activation of T helper and T cytotoxic cells against the parasite was observed in the mice treated with this combination therapy. Serum levels of IFN-γ and IL-12 were found to be higher on same day in mice treated with BBIQ + CQ which revealed the generation of strong Th1 immune response in mice against the infection. Overall, TLR7 agonist acted as an efficient partner when combined with potent antimalarial drug.

Structure-activity relationship in NOD2 agonistic muramyl dipeptides.

Nucleotide-binding oligomerization domain 2 (NOD2) is a receptor of the innate immune system that is capable of perceiving bacterial and viral infections. Muramyl dipeptide (MDP, N-acetyl muramyl L-alanyl-d-isoglutamine), identified as the minimal immunologically active component of bacterial cell wall peptidoglycan (PGN) is recognized by NOD2. In terms of biological activities, MDP demonstrated vaccine adjuvant activity and stimulated non-specific protection against bacterial, viral, and parasitic infections and cancer. However, MDP has certain drawbacks including pyrogenicity, rapid elimination, and lack of oral bioavailability. Several detailed structure-activity relationship (SAR) studies around MDP scaffolds are being carried out to identify better NOD2 ligands. The present review elaborates a comprehensive SAR summarizing structural aspects of MDP derivatives in relation to NOD2 agonistic activity.

Structure-Activity Relationships toward the Identification of a High-Potency Selective Human Toll-like Receptor-7 Agonist.

Toll-like receptor (TLR)-7 agonists are immunostimulatory vaccine adjuvants. A systematic structure-activity relationship (SAR) study of TLR7-active 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine led to the identification of a potent hTLR7-specific p-hydroxymethyl IMDQ 23 with an EC50 value of 0.22 µM. The SAR investigation also resulted in the identification of TLR7 selective carboxamide 12 with EC50 values of 0.32 µM for hTLR7 and 18.25 µM for hTLR8. In the vaccination study, TLR7-specific compound 23 alone or combined with alum (aluminum hydroxide wet gel) showed adjuvant activity for a spike protein immunogen in mice, with enhanced anti-spike antibody production. Interestingly, the adjuvant system comprising carboxamide 12 and alum showed prominent adjuvant activity with high levels of IgG1, IgG2b, and IgG2c in immunized mice, confirming a balanced Th1/Th2 response. In the absence of any apparent toxicity, the TLR7 selective agonists in combination with alum may make a suitable vaccine adjuvant.

Synthesis of Triazolo[4',5':4,5]furo[2,3-c]pyridine via Post Modification of an Unusual Groebke-Blackburn-Bienaymé Multicomponent Reaction.

The Groebke-Blackburn-Bienaymé (GBB) reaction is a well-established three-component reaction for synthesizing imidazofused scaffolds from heterocyclic amidines, aldehydes, and isonitriles. However, the replacement of pyridoxal as an aldehyde component in this reaction results in the formation of the furo[2,3-c]pyridine skeleton as an "unusual GBB product". Despite the interesting nature of this unusual reaction, not much work was further reported. The present research investigates the optimization strategy for the synthesis of novel tricyclic triazolo[4',5':4,5]furo[2,3-c]pyridines via diazotization of 2,3-diamino-furo[2,3-c]pyridines specifically synthesized utilizing the chemistry of tert-alkyl isocyanide.

Design and Synthesis of Polyphenolic Imidazo[4,5-c]quinoline Derivatives to Modulate Toll Like Receptor-7 Agonistic Activity and Adjuvanticity.

TLR-7/8 agonists are a well-known class of vaccine adjuvants, with a leading example now included in Covaxin, a licensed human COVID-19 vaccine. This thereby provides the opportunity to develop newer, more potent adjuvants based on structure-function studies of these classes of compounds. Imidazoquinoline-based TLR7/8 agonists are the most potent, but when used as a vaccine adjuvant side effects can arise due to diffusion from the injection site into a systemic circulation. In this work, we sought to address this issue through structural modifications in the agonists to enhance their adsorption capacity to the classic adjuvant alum. We selected a potent TLR7-selective agonist, BBIQ (EC50 = 0.85 µM), and synthesized polyphenolic derivatives to assess their TLR7 agonistic activity and adjuvant potential alone or in combination with alum. Most of the phenolic derivatives were more active than BBIQ and, except for 12b, all were TLR7 specific. Although the synthesized compounds were less active than resiquimod, the immunization data on combination with alum, specifically the IgG1, IgG2b and IgG2c responses, were superior in comparison to BBIQ as well as the reference standard resiquimod. Compound 12b was 5-fold more potent (EC50 = 0.15 µM in TLR7) than BBIQ and induced double the IgG response to SARS-CoV-2 and hepatitis antigens. Similarly, compound 12c (EC50 = 0.31 µM in TLR7) was about 3-fold more potent than BBIQ and doubled the IgG levels. Even though compound 12d exhibited low TLR7 activity (EC50 = 5.13 µM in TLR7), it demonstrated superior adjuvant results, which may be attributed to its enhanced alum adsorption capability as compared with BBIQ and resiquimod. Alum-adsorbed polyphenolic TLR7 agonists thereby represent promising combination adjuvants resulting in a balanced Th1/Th2 immune response.

Toll-like receptor-8 agonistic activities in C2, C4, and C8 modified thiazolo[4,5-c]quinolines.

Toll-like receptor (TLR)-8 agonists typified by the 2-alkylthiazolo[4,5-c]quinolin-4-amine (CL075) chemotype are uniquely potent in activating adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds could be promising candidate vaccine adjuvants, especially for neonatal vaccines. Alkylthiazoloquinolines with methyl, ethyl, propyl and butyl groups at C2 displayed comparable TLR8-agonistic potencies; activity diminished precipitously in the C2-pentyl compound, and higher homologues were inactive. The C2-butyl compound was unique in possessing substantial TLR7-agonistic activity. Analogues with branched alkyl groups at C2 displayed poor tolerance of terminal steric bulk. Virtually all modifications at C8 led to abrogation of agonistic activity. Alkylation on the C4-amine was not tolerated, whereas N-acyl analogues with short acyl groups (other than acetyl) retained TLR8 agonistic activity, but were substantially less water-soluble. Immunization in rabbits with a model subunit antigen adjuvanted with the lead C2-butyl thiazoloquinoline showed enhancements of antigen-specific antibody titers.

Green synthesis of lignin-based nanoparticles as a bio-carrier for targeted delivery in cancer therapy.

Polymeric magnetic nanoparticles have shown higher efficacy in cancer diagnosis and treatment than conventional chemotherapies. Lignin is an abundantly available natural polymer that can be selectively modified using a rapidly expanding toolkit of biocatalytic and chemical reactions to yield 'intelligent' theranostic-nanoprobes. We aim to valorize lignin to develop a natural polymeric-magnetic-nano-system for the targeted delivery of methotrexate. In the current study, we synthesized nanoparticles of lignin and iron oxide with methotrexate using a new approach of anti-solvent precipitation with ultrasonication. The ensuing nanoparticles are magnetic, smooth, polyhedral with characteristic dimension of 110-130 nm. The drug loading and encapsulation efficiencies were calculated to be 66.06 % and 64.88 %, respectively. The nanoparticles exhibit a concentration-dependent release of methotrexate for the initial 24 h, followed by sustained release. Moreover, formulation is non-hemolytic and scavenges radicals owing to the antioxidant property of lignin. Additionally, methotrexate delivered using the nanoparticles exhibited higher cytotoxicity in cellular-viability assays employing breast cancer and macrophage cell lines compared to the pure form of the drug. Synergistic action of lignin, iron oxide, and methotrexate contribute to enhanced caspase-3 activity and reduced glutathione levels in the breast cancer cells, as well as elevated internalization of the drug on account of increased receptor-mediated endocytosis.

Development of nanostructured lipid carriers as a promising tool for methotrexate delivery: physicochemical and in vitro evaluation.

The aim of the present study is to fabricate the stable nanostructured lipid carriers (NLCs) using biocompatible excipients for the encapsulation of Methotrexate (MTX), a chemotherapeutic agent for breast cancer treatment. MTX has restricted clinical applications owing to its low solubility, non-specific targeting and adverse side effects. Glyceryl Monostearate (GMS) and Miglyol 812 (MI1) were chosen as solid and liquid lipids, respectively, for the fabrication of NLCs, and the influence of variation of solid and liquid composition was investigated. The prepared NLCs exhibited long-term stability and spherical shape morphology as characterized by electron microscopy. The internal structure of fabricated NLCs was arranged into cubic crystalline as confirmed by small-angle X-ray scattering (SAXS) analysis. MTX's encapsulation efficiency of ∼85 ± 0.9%. and sustained in vitro release of MTX ∼ 52% ± 3.0 in 24 h was achieved. Classical molecular dynamics (MD) simulations were performed to study the structural stability of the MTX encapsulated NLCs. Hemolysis carried out on the NLCs showcased the biosafety of the formulation under the tolerance limit (<10%). Further, the MTT assay demonstrates that MTX-loaded NLCs exhibited toxicity against HeLa and MCF-7 cell lines as compared to blank NLCs. The finding demonstrates NLCs as promising vehicles for MTX delivery to address cancer.Communicated by Ramaswamy H. Sarma.

Recent advances in pharmaceutical and biotechnological applications of lignin-based materials.

Lignin, a versatile and abundant biomass-derived polymer, possesses a wide array of properties that makes it a promising material for biotechnological applications. Lignin holds immense potential in the biotechnology and pharmaceutical field due to its biocompatibility, high carbon content, low toxicity, ability to be converted into composites, thermal stability, antioxidant, UV-protectant, and antibiotic activity. Notably, lignin is an environmental friendly alternative to synthetic plastic and fossil-based materials because of its inherent biodegradability, safety, and sustainability potential. The most important findings related to the use of lignin and lignin-based materials are reported in this review, providing an overview of the methods and techniques used for their manufacturing and modification. Additionally, it emphasizes on recent research and the current state of applications of lignin-based materials in the biomedical and pharmaceutical fields and also highlights the challenges and opportunities that need to be overcome to fully realize the potential of lignin biopolymer. An in-depth discussion of recent developments in lignin-based material applications, including drug delivery, tissue engineering, wound dressing, pharmaceutical excipients, biosensors, medical devices, and several other biotechnological applications, is provided in this review article.

Imidazo[1,2-a]pyrimidine as a New Antileishmanial Pharmacophore against Leishmania amazonensis Promastigotes and Amastigotes.

Leishmania poses a substantial threat to the human population all over the globe because of its visceral and cutaneous spread engendered by all 20 species. Unfortunately, the available drugs against leishmania are already hobbled with toxicity, prolonged treatment, and increasing instances of acquirement of resistance. Under these grave circumstances, the development of new drugs has become imperative to keep these harmful microbes at bay. To this end, a Groebke-Blackburn-Bienaymé multicomponent reaction-based library of different imidazo-fused heterocycles has been synthesized and screened against Leishmania amazonensis promastigotes and amastigotes. Among the library compounds, the imidazo-pyrimidine 24 has been found to be the most effective (inhibitory concentration of 50% (IC50) < 10 µM), with selective antileishmanial activity on amastigote forms, a stage of the parasite related to human disease. The compound 24 has exhibited an IC50 value of 6.63 µM, being ∼two times more active than miltefosine, a reference drug. Furthermore, this compound is >10 times more destructive to the intracellular parasites than host cells. The observed in vitro antileishmanial activity along with suitable in silico physicochemical and absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of compound 24 reinforce the imidazo-pyrimidine scaffold as a new antileishmanial pharmacophore and encourage further murine experimental leishmaniasis studies.

Fangchinoline inhibits SARS-CoV-2 and MERS-CoV entry.

The COVID-19 pandemic caused by SARS-CoV-2, lead to mild to severe respiratory illness and resulted in 6.9 million deaths worldwide. Although vaccines are effective in preventing COVID-19, they may not be sufficient to protect immunocompromised individuals from this respiratory illness. Moreover, novel emerging variants of SARS-CoV-2 pose a risk of new COVID-19 waves. Therefore, identification of effective antivirals is critical in controlling SARS and other coronaviruses, such as MERS-CoV. We show that Fangchinoline (Fcn), a bisbenzylisoquinoline alkaloid, inhibits replication of SARS-CoV, SARS-CoV-2, and MERS-CoV in a range of in vitro assays, by blocking entry. Therapeutic use of Fcn inhibited viral loads in the lungs, and suppressed associated airway inflammation in hACE2. Tg mice and Syrian hamster infected with SARS-CoV-2. Combination of Fcn with remdesivir (RDV) or an anti-leprosy drug, Clofazimine, exhibited synergistic antiviral activity. Compared to Fcn, its synthetic derivative, MK-04-003, more effectively inhibited SARS-CoV-2 and its variants B.1.617.2 and BA.5 in mice. Taken together these data demonstrate that Fcn is a pan beta coronavirus inhibitor, which possibly can be used to combat novel emerging coronavirus diseases.

Antibacterial activities of Groebke-Blackburn-Bienaymé-derived imidazo[1,2-a]pyridin-3-amines.

We sought to explore the imidazo[1,2-a]pyridin-3-amines for TLR7 (or 8)-modulatory activities. This chemotype, readily accessed via the Groebke-Blackburn-Bienaymé multi-component reaction, resulted in compounds that were TLR7/8-inactive, but exhibited bacteriostatic activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). To investigate the mechanism of antibacterial activity of this new chemotype, a resistant strain of S. aureus was generated by serially passaging the organism in escalating doses of the most active analogue. A comparison of minimum inhibitory concentrations (MICs) of known bacteriostatic agents in wild-type and resistant strains indicates a novel mechanism of action. Structure-activity relationship studies have led to the identification of positions on the scaffold for additional structural modifications that should allow for the introduction of probes designed to examine cognate binding partners and molecular targets, while not significantly compromising antibacterial potency.

Toll-like receptor (TLR) agonists as a driving force behind next-generation vaccine adjuvants and cancer therapeutics.

Until recently, the development of new human adjuvants was held back by a poor understanding of their mechanisms of action. The field was revolutionized by the discovery of the toll-like receptors (TLRs), innate immune receptors that directly or indirectly are responsible for detecting pathogen-associated molecular patterns (PAMPs) and respond to them by activating innate and adaptive immune pathways. Hundreds of ligands targeting various TLRs have since been identified and characterized as vaccine adjuvants. This work has important implications not only for the development of vaccines against infectious diseases but also for immuno-therapies against cancer, allergy, Alzheimer's disease, drug addiction and other diseases. Each TLR has its own specific tissue localization and downstream gene signalling pathways, providing researchers the opportunity to precisely tailor adjuvants with specific immune effects. TLR agonists can be combined with other TLR or alternative adjuvants to create combination adjuvants with synergistic or modulatory effects. This review provides an introduction to the various classes of TLR adjuvants and their respective signalling pathways. It provides an overview of recent advancements in the TLR field in the past 2-3 years and discusses criteria for selecting specific TLR adjuvants based on considerations, such as disease mechanisms and correlates of protection, TLR immune biasing capabilities, route of administration, antigen compatibility, new vaccine technology platforms, and age- and species-specific effects.

Combined delivery of TLR2 and TLR7 agonists by Nanostructured lipid carriers induces potent vaccine adjuvant activity in mice.

Toll-like receptor (TLR) agonists are promising adjuvants and the combination of TLR agonists enhance immune responses by providing synergistic immune activity via triggering different signalling pathways. However, systematic cytotoxicity due to the immediate release of such immune potentiators from the site of injection hampers its clinical performance. Nanostructured lipid carriers (NLCs) offer a possibility to incorporate multiple TLR agonists with high encapsulation efficiency and slow drug release. Herein, we synthesized NLCs from didodecyldimethylammonium bromide (D12DAB) and oleic acid and used these to co-encapsulate a Pam2CS derivative (T-2, TLR2 agonist) with an imidazoquinoline derivative (T-7, TLR7 agonist) as a combination vaccine adjuvant. Hydrodynamic diameter and zeta potential of the prepared NLCs were found to be in the range of 200-500 nm and 23-27 mV, respectively. Spherical shape and size of prepared NLCs were also assessed through Field Emission Scanning Electron Microscopy (FE-SEM) and Transmission Electron Microscopy (TEM) analysis. In-vitro release studies of T-7 demonstrated sustained release and the addition of lipopeptide T-2 augmented encapsulation efficiency (from 84 to 92.9%) with a slight trigger in the release percentage. All NLC formulations were screened in TLR2/1, TLR2/6, TLR7 and TLR8 reporter cell lines and loaded NLC formulation showed high TLR2 and TLR7 agonistic activity. Adjuvant potency was evaluated through intramuscular immunization of female C57BL/6 mice with recombinant hepatitis B surface antigen and influenza hemagglutinin protein. T-2 and T-7 loaded NLCs induced good protective efficacy in mice challenged with a lethal dose of influenza virus.

Stereoisomeric Pam2CS based TLR2 agonists: synthesis, structural modelling and activity as vaccine adjuvants.

Lipopeptides including diacylated Pam2CSK4 as well as triacylated Pam3CSK4 act as ligands of toll-like receptor (TLR)-2, a promising target for the development of vaccine adjuvants. The highly investigated Pam2CSK4 and Pam3CSK4, despite their aqueous solubility have not performed well as vaccine adjuvants which may be attributable to potential denaturation of protein antigens by these cationic surfactant-like lipopeptides. In the present investigation, we synthesized (R), (S) and racemic Pam2CS(OMe) analogs and their N-acetyl derivatives without the tetralysine component to systematically investigate the effect of stereochemistry at the thio-glycerol lipopeptide core of these lipopeptide based TLR2 agonists. The resulting compounds were compared using TLR2 reporter cell-based assays and the ability of the synthesized lipopeptides to stimulate cytokine production (IL-6, IL-10 and TNF-α) by freshly collected human PBMCs and CD40 and CD86 expressions by mouse spleen cells was also investigated. Notably, few synthesized lipopeptides were found to be potent TLR2/6 agonists, inducing cytokine production and upregulating CD40 and CD86 expressions. The TLR2/6 agonistic lipopeptides were further assessed for vaccine adjuvant effects in mice. The results confirmed that the R-stereochemistry at the thio-glycerol lipopeptide core was preferred for maximal TLR2/6 activity, as reflected in Th1 immune deviation, higher antibody levels and enhanced vaccine protection against a lethal influenza challenge.