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The immune system uses members of the toll-like receptor (TLR) family to recognize a variety of pathogen- and host-derived molecules in order to initiate immune responses. Although TLR-mediated, pro-inflammatory immune responses are essential for host defense, prolonged and exaggerated activation can result in inflammation pathology that manifests in a variety of diseases. Therefore, small molecule inhibitors of the TLR signaling pathway might have promise as anti-inflammatory drugs. We have previously identified a class of triaryl pyrazole compounds that inhibit TLR signaling by modulation of the protein-protein interactions essential to the pathway. We have now systematically examined the structural features essential for inhibition of this pathway, revealing characteristics of compounds that inhibited all TLRs tested (pan-TLR signaling inhibitors) as well as compounds that selectively inhibited certain TLRs. These findings reveal interesting classes of compounds that could be optimized for particular inflammatory diseases governed by different TLRs.

A major risk for patients having estrogen receptor α (ERα)-positive breast cancer is the recurrence of drug-resistant metastases after initial successful treatment with endocrine therapies. Recent studies have implicated a number of activating mutations in the ligand-binding domain of ERα that stabilize the agonist conformation as a prominent mechanism for this acquired resistance. There are several critical gaps in our knowledge regarding the specific pharmacophore requirements of an antagonist that could effectively inhibit all or most of the different mutant ERs. To address this, we screened various chemotypes for blocking mutant ER-mediated transcriptional signaling and identified RU58668 as a model compound that contains structural elements that support potent ligand-induced inhibition of mutant ERs. We designed and synthesized a focused library of novel antagonists and probed how small and large perturbations in different ligand structural regions influenced inhibitory activity on individual mutant ERs in breast cancer cells. Effective inhibition derives from both non-polar and moderately polar motifs in a multifunctional side chain of the antagonists, with the nature of the ligand core making important contributions by increasing the potency of ligands possessing similar types of side chains. Some of our new antagonists potently blocked the transcriptional activity of the three most common mutant ERs (L536R, Y537S, D538G) and inhibited mutant ER-mediated cell proliferation. Supported by our molecular modeling, these studies provide new insights into the role of specific components, involving both the ligand core and multifunctional side chain, in suppressing wild-type and mutant ER-mediated transcription and breast cancer cell proliferation.

Toll-like receptors (TLRs) recognize various pathogen- and host tissue-derived molecules and initiate inflammatory immune responses. Exaggerated or prolonged TLR activation, however, can promote etiologically diverse diseases, such as bacterial sepsis, metabolic and autoimmune diseases, or stroke. Despite the apparent medical need, no small molecule drugs against TLR-pathways are clinically available. This may be because of the complex signaling mechanisms of TLRs, which are governed by a series of protein interactions initiated by Toll-interleukine-1 receptor (TIR)-domains found in TLRs and the cytoplasmic adapter proteins TIRAP and MyD88. Activation/oligomerization of TLRs and consecutive oligomerization of MyD88 and/or TIRAP leads to the recruitment of IRAK family members and the ubiquitin ligase TRAF6. Here, we developed a phenotypic drug screening system based on inducible dimerization of TIRAP, MyD88 and TRAF6, that ranked hits according to the position of action in the hierarchy of the TLR signaling cascade. From a bioactive compound library, we identified methyl-piperidino-pyrazole (MPP) as a TLR-specific inhibitor. Structure-activity relationship analysis, quantitative proteomics, protein interaction assays, and cellular thermal shift assays strongly suggest that MPP targets the TIR domain of MyD88. Chemical evolution of the original MPP-scaffold generated compounds with selectivity for distinct TLRs that interfered with specific TIR interactions. Administration of a MPP-analog to mice protected against LPS-mediated TNFα release. These results validate this phenotypic screening approach and implicate the MPP scaffold as starting point for anti-inflammatory drug development.

The estrogen receptors (ERs) bind with high affinity to many structurally diverse ligands by significantly distorting the contours of their ligand-binding pockets. This raises a question: To what degree is ER able to distinguish between structurally related regioisomers and enantiomers? We have explored the structural compliance and specificity of ERα with a set of ligands having a 7-oxa-bicyclo[2.2.1]hept-5-ene sulfonate core and basic side chains typical of selective ER modulators (SERMs). These ligands have two regioisomers, each of which is a racemate of enantiomers. Using orthogonal protecting groups and chiral HPLC, we isolated all 4 isomers and assigned their absolute stereochemistry by X-ray analysis. The 1S,2R,4S isomer has a 80-170-fold higher affinity for ERα than the others, and it profiles as a partial agonist/antagonist in cellular reporter gene assays and in suppressing proliferation of MCF-7 breast cancer cells with subnanomolar potency, far exceeding that of the other isomers. It is the only isomer found bound to ERα by X-ray analysis after crystallization with four-isomer mixtures of closely related analogs. Thus, despite the general compliance of this receptor for binding a large variety of ligand structures, ER demonstrates marked structural specificity and stereospecificity by selecting a single component from a mixture of structurally related isomers to drive ER-regulated cellular activity. Our findings lay the necessary groundwork for seeking unique ER-mediated pharmacological profiles by rational structural perturbations of two different types of side chains in this unprecedented class of ER ligands, which may prove useful in developing more effective endocrine therapies for breast cancer.

Novel stilbene-chalcone (S-C) hybrids were synthesized via a sequential Claisen-Schmidt-Knoevenagel-Heck approach and evaluated for antiplasmodial activity in in vitro red cell culture using SYBR Green I assay. The most potent hybrid (11) showed IC(50) of 2.2, 1.4, and 6.4 μM against 3D7 (chloroquine sensitive), Indo, and Dd2 (chloroquine resistant) strains of Plasmodium falciparum, respectively. Interestingly, the respective individual stilbene (IC(50)100 μM), chalcone (IC(50) = 11.5 μM), or an equimolar mixture of stilbene and chalcone (IC(50) = 32.5 μM) were less potent than 11. Studies done using specific stage enriched cultures and parasite in continuous culture indicate that 11 and 18 spare the schizont but block the progression of the parasite life cycle at the ring or the trophozoite stages. Further, 11 and 18 caused chromatin condensation, DNA fragmentation, and loss of mitochondrial membrane potential in Plasmodium falciparum, thereby suggesting their ability to cause apoptosis in malaria parasite.

In the present study, microwave-assisted extraction was compared with conventional approaches for the efficient extraction of juglone and other phenolics from Juglans regia bark. The effect of different solvents was also studied and ethyl acetate was found to be a better solvent in terms of juglone yield and stability. Further, a simple and fast RP-HPLC method was developed and validated for the determination of juglone and other bioactive phenolics like gallic acid, caffeic acid, quercetin, myricetin, and quercitrin in these extracts. In addition, the extracts were tested for antimicrobial activity against 16 microorganisms where all the extracts showed broad spectrum activity.

A new one-pot strategy has been developed, wherein abundantly available methoxylated phenylpropenes are directly transformed into corresponding dienones (1,5-diarylpenta-2,4-dien-1-ones) and enones (chalcones and cinnamic esters) via allylic oxidation-condensation or allylic oxidation-esterification sequences. Preliminary antimalarial activity studies of the above synthesized diaryldienones and enones against Plasmodium falciparum (Pf3D7) have shown them to be promising lead candidates for developing newer and economical antimalarial agents. In particular, two enones (12b and 13b) were found to possess comparatively better activity (IC(50) = 4.0 and 3.4 μM, respectively) than licochalcone (IC(50) = 4.1 μM), a well known natural antimalarial compound.