Christopher C. Lai

PROTEIN TYROSINE KINASE INHIBITORS DECREASE LIPOPOLYSACCHARIDE-INDUCED PROINFLAMMATORY CYTOKINE PRODUCTION IN MIXED GLIA, MICROGLIA-ENRICHED OR ASTROCYTE-ENRICHED CULTURES

Proinflammatory cytokines, tumor necrosis factor-alpha (TNF alpha), interleukin-1 (IL-1), and interleukin-6 (IL-6), produced by glial cells have been implicated in the neuropathogenesis of various diseases. However, the signal transduction pathway(s) for the production of these cytokines in glial cells are not well understood. This study examined the effects of two potent protein tyrosine kinase inhibitors, genistein and tyrphostin A25, on lipopolysaccharide (LPS)-induced production of TNF alpha, IL-1 alpha, and IL-6 in mouse primary mixed glia, microglia- or astrocyte-enriched cultures. LPS dose-dependently increased the production of TNF alpha, IL-1 alpha, and IL-6 from the mixed glia cultures. Genistein or tyrphostin A25 significantly inhibited the LPS-induced production of these cytokines. The LPS-induced TNF alpha, IL-1 alpha, and IL-6 production in microglia- or astrocyte-enriched cultures were also inhibited by tyrphostin A25. These results demonstrate that protein tyrosine kinases are involved in the signaling events of the LPS-induced production of TNF alpha, IL-1 alpha, or IL-6 in microglia or astrocytes, which may provide insights into therapeutic interventions in the pathway for cytokine production in the brain.

A Novel Side-Bridged Hybrid Phosphonate/Acetate Pendant Cyclam: Synthesis, Characterization, and 64Cu Small Animal PET Imaging

Copper-64 (t(1/2)=12.7h; beta(+): 0.653 MeV, 17.4%; beta(-): 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. A novel side-bridged cyclam featuring both pendant acetate and phosphonate groups was synthesized using a Kabachnik-Fields approach followed by hydrobromic acid deprotection. The Cu(II) complex of the novel ligand was synthesized, radiolabeling with (64)Cu was demonstrated, and in vitro (serum) stability was performed. In addition, in vivo distribution and clearance of the (64)Cu-labeled complex was visualized by positron emission tomography (PET) imaging. This novel chelate may be useful in (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as targeted radiotherapeutic applications.

Synthesis and biological evaluation of novel dihydro-aryl/alkylsulfanyl-cyclohexylmethyl-oxopyrimidines (S-DACOs) as high active anti-HIV agents

A novel dihydro-aryl/alkylsulfanyl-cyclohexylmethyl-oxopyrimidines (S-DACOs) combinatory library was synthesized and evaluated with C8166 cells infected by the HIV-1(IIIB) in vitro, using Nevirapine (NVP) and Zidovudine (AZT) as positive control. The anti-HIV screening results revealed that C-6-cyclohexylmethyl substituted pyrimidinones possessed higher selective index than its 6-arylmethyl counterparts. Compounds 1g, 1c, 1e and 1b showed potent anti-HIV activities with EC(50) values of 0.012, 0.025, 0.088 and 0.162nM, respectively.

Mono‐anionic phosphopeptides produced by unexpected histidine alkylation exhibit high plk1 polo‐box domain‐binding affinities and enhanced antiproliferative effects in hela cells

Binding of polo‐like kinase 1 (Plk1) polo‐box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)‐containing sequences is critical for the proper function of Plk1. Although high‐affinity synthetic pThr‐containing peptides provide starting points for developing PBD‐directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di‐anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on‐resin N(τ)‐alkylation of histidine residues already bearing a N(π)‐ alkyl group. This resulted in cationic imidazolium‐containing pThr peptides, several of which exhibit single‐digit nanomolar PBD‐binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio‐reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD‐binding peptides and peptide mimetics.

DB-02, a C-6-Cyclohexylmethyl Substituted Pyrimidinone HIV-1 Reverse Transcriptase Inhibitor with Nanomolar Activity, Displays an Improved Sensitivity against K103N or Y181C Than S-DABOs

6-(cyclohexylmethyl)-5-ethyl-2-((2-oxo-2-phenylethyl)thio)pyrimidin-4(3H)-one (DB-02) is a member of the newly reported synthetic anti-HIV-1 compounds dihydro-aryl/alkylsulfanyl-cyclohexylmethyl-oxopyrimidines, S-DACOs. In vitro anti-HIV-1 activity and resistance profile studies have suggested that DB-02 has very low cytotoxicity (CC50>1mM) to cell lines and peripheral blood mononuclear cells (PBMCs). It displays potent anti-HIV-1 activity against laboratory adapted strains and primary isolated strains including different subtypes and tropism strains (EC50s range from 2.40 to 41.8 nM). Studies on site-directed mutagenesis, genotypic resistance profiles revealed that V106A was the major resistance contributor for the compound. Molecular docking analysis showed that DB-02 located in the hydrophobic pocket with interactions of Lys101, Val106, Leu234, His235. DB-02 also showed non-antagonistic effects to four approved antiretroviral drugs. All studies indicated that DB-02 would be a potential NNRTI with low cytotoxicity and improved activity.

Neighbor-directed histidine N (τ)-alkylation: A route to imidazolium-containing phosphopeptide macrocycles.

Our recently discovered, selective, on‐resin route to N(τ)‐alkylated imidazolium‐containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring‐forming junctions. Interestingly, these cationic moieties subsequently serve to charge‐mask the phosphoamino acid group that directed their formation. Neighbor‐directed histidine N(τ)‐alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.

North- and south-bicyclo[3.1.0]hexene nucleosides: the effect of ring planarity on anti-HIV activity.

The syntheses of new conformationally locked North‐ and South‐bicyclo[3.1.0]hexene nucleosides is reported. The North analogues were synthesized by a convergent approach from the known (1S,2R,5R)‐5‐[(tert‐butyldiphenylsilyloxy)methyl]bicyclo[3.1.0]hex‐3‐en‐2‐ol by Mitsunobu coupling with the nucleobases. The South analogues were synthesized from their bicyclo[3.1.0]hexane nucleoside precursors by the selective protection of the primary hydroxy group, conversion of the secondary alcohol into a good leaving group, and base‐catalyzed elimination to generate the olefin. The transformation of a bicyclo[3.1.0]hexane nucleoside into a bicyclo[3.1.0]hexene nucleoside flattens the five‐membered ring of the bicyclic system and rescues anti‐HIV activity for North‐D4T, North‐D4A, and South‐D4C. The relationship between planarity and the anti/syn disposition of the nucleobase that is favored by a particular pseudosugar platform are proposed as key parameters in controlling biological activity.

Exploring the influence of indololactone structure on selectivity for binding to the C1 domains of PKCα, PKCε, and RasGRP

C1 domain-containing proteins, such as protein kinase C (PKC), have a central role in cellular signal transduction. Their involvement in many diseases, including cancer, cardiovascular disease, and immunological and neurological disorders has been extensively demonstrated and has prompted a search for small molecules to modulate their activity. By employing a diacylglycerol (DAG)-lactone template, we have been able to develop ultra potent analogs of diacylglycerol with nanomolar binding affinities approaching those of complex natural products such as phorbol esters and bryostatins. One current challenge is the development of selective ligands capable of discriminating between different protein family members. Recently, structure-activity relationship studies have shown that the introduction of an indole ring as a DAG-lactone substituent yielded selective Ras guanine nucleotide-releasing protein (RasGRP1) activators when compared to PKCα and PKCε. In the present work, we examine the effects of ligand selectivity relative to the orientation of the indole ring and the nature of the DAG-lactone template itself. Our results show that the indole ring must be attached to the lactone moiety through the sn-2 position in order to achieve RasGRP1 selectivity.

Design and synthesis of Fmoc-Thr[PO(OH)(OPOM)] for the preparation of peptide prodrugs containing phosphothreonine in fully protected form.

The design and efficient synthesis of N‐Fmoc‐phosphothreonine protected by a mono‐(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc‐Thr[PO(OH)(OPOM)]‐OH, 1, is reported. This reagent is suitable for solid‐phase syntheses employing acid‐labile resins and Fmoc‐based protocols. It allows the preparation of phosphothreonine (pThr)‐containing peptides bearing bis‐POM‐phosphoryl protection. The methodology allows the first reported synthesis of pThr‐containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.

Synthesis and biological evaluation of novel anti-hepatitis C virus (HCV) agents: 2-hydroxylphenethyl sulfanyl-oxopyrimidines

A novel series of dihydro-hydroxyl-phene-thylsulfanyl-ω-cyclohexyl/phenyl-oxopyrimidine derivatives have been synthesized and their in vitro anti-hepatitis C virus activities have been evaluated using Huh 7.5.1 cells. Some of the compounds showed moderate anti-hepatitis C virus activities, with EC50 range from 7.53 to 0.13 μM. Among all the compounds, 6-(cyclohexylmethyl)-5-ethyl-2-((2-hydroxy-2-phenylethyl)thio)-pyrimidin-4 (3H)-one (3a) had the most promising potential in inhibiting hepatitis C virus with an EC50 value of 0.13 μM and SI value of 121. It was noticed that some of these compounds are both active on hepatitis C virus and human immunodeficiency virus. In addition to experimental evaluation, structure-activity relationships and the molecular modeling analysis of these new congeners are also discussed.