Neurotoxic Aβ42 oligomers are believed to be the main cause of Alzheimer’s disease. Previously, we found that the C-terminal fragments (CTFs), Aβ(30–42) and Aβ(31–42) were the most potent inhibitors of Aβ42 oligomerization and toxicity in a series of Aβ(x–42) peptides, (x=28–39). Therefore, we chose these peptides as leads for further development. These CTFs are 12/13-amino-acid long, hydrophobic peptides with limited aqueous solubility. Our first attempt to attach hydrophilic groups to the N-terminus resulted in toxic peptides. Therefore, next we incorporated N-methyl amino acids, which are known to increase the solubility of such peptides by disrupting β-sheet formation. Focusing on Aβ(31–42), we used a two-step N-methyl (N-Me) amino acid substitution strategy to study the structural factors controlling inhibition of Aβ42-induced toxicity. First, each residue was substituted by N-Me-alanine (N-Me-A). In the next step, in positions where substitution produced a significant effect, we restored the original side-chain. This strategy allowed exploring the role of both side-chain structure and N-Me substitution in inhibitory activity. We found that the introduction of N-Me amino acid was an effective way to increase both the aqueous solubility and the inhibitory activity of Aβ(31–42). In particular, N-Me amino acid substitution at positions 9 or 11 increased the inhibitory activity relative to the parent peptide. The data suggest that inhibition of Aβ42 toxicity by short-peptides is highly structure-specific, providing basis for the design of new peptidomimetic inhibitors with improved activity, physicochemical properties, and metabolic stability.

Early diagnosis is the way to improve lung cancer survival rate and is almost impossible today due to the lack of molecular probes that recognize lung cancer cells sensitively and selectively. We have developed a new aptamer approach for the recognition of specific small cell lung cancer (SCLC) cell surface molecular markers. Our approach relies on cell based systematic evolution of ligands by exponential enrichment (cell-SELEX) to evolve aptamers for whole live cells that express a variety of surface markers representing molecular differences among cancer cells. When applied to different lung cancer cells including those from patient samples, these aptamers bind to SCLC cells with high affinity and specificity in different assay formats. When conjugated with magnetic and fluorescent nanoparticles, the aptamer nano-conjugates could effectively extract SCLC cells from mixed cell media for isolation, enrichment, and sensitive detection. These studies demonstrate the potential of the aptamer approach for early lung cancer detection.

Triclosan has been previously shown to inhibit InhA, an essential enoyl acyl carrier protein reductase of mycolic acid biosynthesis, whose inhibition leads to the lysis of Mycobacterium tuberculosis. Using a structure-based drug design approach, a series of 5-substituted derivatives of triclosan was developed. Two groups of triclosan derivatives with alkyl and aryl substituents, respectively, were identified with dramatically enhanced potency against purified InhA. The most efficacious inhibitor displayed an IC50 value of 21 nM, which was 50-fold more potent than triclosan. X-ray crystal structures of InhA in complex with four triclosan derivatives revealed the structural basis for the inhibitory activity. Six selected triclosan derivatives were tested against isoniazid-sensitive and resistant strains of M. tuberculosis. Among those, the best inhibitor had an MIC value of 4.7 µg/mL (13 µM), which represents a tenfold improvement over the bacteriocidal activity of triclosan. A subset of these triclosan analogs was more potent than isoniazid against two isoniazid-resistant M. tuberculosis strains, demonstrating the significant potential for structure-based design in the development of next generation antitubercular drugs.

In the United States, breast cancer affects one in eight women, with mortality that is second only to lung cancer. Although chemotherapy is widely used in breast cancer treatment, its side effects remain a challenge. One way to address this problem is through drug delivery by the internalization of cell type-specific probes. Although nucleic acid aptamers are excellent probes for molecular recognition, only a few reports have demonstrated that aptamers can be internalized into living cells. Therefore, in this work, we report the development of a cancer cell-specific DNA aptamer probe, KMF2-1a. Using the cell-SELEX method, this aptamer was selected against breast cancer cell line MCF-10AT1. Our results showed that KMF2-1a was internalized efficiently and specifically to the endosome of target breast cancer cells. These results indicate that KMF2-1a is a promising agent for cell type-specific intracellular delivery with both diagnostic and therapeutic implications.

The emergence of virulent, drug-resistant bacterial strains coupled with a minimal output of new pharmaceutical agents to combat them makes this a critical time for antibacterial research. Aminoglycosides are a well-studied, highly potent class of naturally occurring antibiotics with scaffolds amenable to modification, and therefore, they provide an excellent starting point for the development of semisynthetic, next-generation compounds. To explore the potential of this approach, we synthesized a small library of aminoglycoside derivatives selectively and minimally modified at one or two positions with a guanidine group replacing the corresponding amine or hydroxy functionality. Most guanidino-aminoglycosides showed increased affinity for the ribosomal decoding rRNA site, the cognate biological target of the natural products, when compared with their parent antibiotics, as measured by an in vitro fluorescence resonance energy transfer (FRET) A-site binding assay. Additionally, certain analogues showed improved minimum inhibitory concentration (MIC) values against resistant bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA). An amikacin derivative holds particular promise with activity greater than or equal to the parent antibiotic in the majority of bacterial strains tested.

The microfilament cytoskeleton protein actin plays important role in cell biology and can affect cytokinesis, morphogenesis, and cell migration. These functions usually fail and become abnormal in cancer cells. The marine-derived macrolide latrunculins A and B, from the Red Sea sponge Negombata magnifica, are known to reversibly bind actin monomers, forming 1:1 complex with G-actin, disrupting its polymerization. To identify novel therapeutic agents for effective treatment of metastatic breast cancer, several semisynthetic derivatives of latrunculin A (1) with diverse steric, electrostatic, hydrogen bond donor and acceptor properties were rationally prepared. Analogues were designed to modulate the binding affinity toward G-actin. Examples of these reactions are esterification, acetylation, and N-alkylation. Semisynthetic latrunculins were then tested for their ability to inhibit pyrene-conjugated actin polymerization, and subsequently for their antiproliferative, and anti-invasive properties against MCF7 and MDA-MB-231 cells using MTT and invasion assays, respectively.

Cyclohexylcarbamic acid aryl esters are a class of Fatty Acid Amide Hydrolase (FAAH) inhibitors, which includes the reference compound URB597. The reactivity of their carbamate fragment is involved in pharmacological activity and may affect pharmacokinetic and toxicological properties. We conducted in vitro stability experiments in chemical and biological environments to investigate the structure-stability relationships in this class of compounds. The results show that electrophilicity of the carbamate influences its chemical stability, as suggested by the relation between the rate constant of alkaline hydrolysis (log kpH9) and the energy of lowest unoccupied molecular orbital (LUMO). Introduction of small, electron donor substituents at conjugated positions of the O-aryl moiety increased overall hydrolytic stability of the carbamate group without affecting FAAH inhibitory potency, whereas peripheral nonconjugated hydrophilic groups, which favor FAAH recognition, helped reducing oxidative metabolism in the liver.

Estrogen receptor-alpha (ER) antagonists have been widely used for breast cancer therapy. Despite initial responsiveness, eventually hormone-sensitive ER-positive cancer cells develop resistance to ER antagonists. It has been shown that, in most of these resistant tumor cells, the ER is expressed and continues to regulate tumor growth. Recent studies propose that tamoxifen initially acts as an antagonist but later functions as an ER agonist, promoting tumor growth. This suggests that targeted ER degradation may provide an effective therapeutic approach for breast cancers, even those which are resistant to conventional therapies. With this in mind, we previously demonstrated that PROTACs effectively induce degradation of the ER as a proof of concept experiment. Herein, we further refined the PROTAC approach to target the ER for degradation. The ER-targeting PROTACs are composed of an estradiol on one end and Hypoxia Inducing Factor 1α (HIF-1α)-derived synthetic pentapeptide on the other. The pentapeptide is recognized by an E3 ubiquitin ligase called the von Hippel Lindau tumor suppressor protein (pVHL), thereby recruiting the ER to this E3 ligase for ubiquitination and degradation. Specifically, the pentapeptide is attached at three different locations on estradiol to generate three different types of PROTACs. When the pentapeptide is linked through the C-7α position of estradiol, the resulting PROTAC showed the most effective ER degradation and best affinity for the estrogen receptor. This result provides an opportunity to develop a novel type of ER antagonist that may overcome the resistance of breast tumor to conventional drugs, such as tamoxifen and fulvestrant (Faslodex™).

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) via a lysophospholipase D activity that leads to the generation of the growth factor-like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy-resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Here we report the synthesis and pharmacological characterization of inhibitors of ATX based on the 4-tetradecanoylaminobenzyl phosphonic acid scaffold that was previously found to lack sufficient stability in cellular systems. The new 4-substituted benzyl phosphonic acid and 6-substituted naphthalen-2-yl-methyl phosphonic acid analogs blocked ATX with Ki values in the low-micromolar-nanomolar range against FS-3, LPC, and nucleotide substrates through a mixed-mode mechanism of inhibition. None of the compounds tested inhibited the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor subtypes. Analogs 22 and 30b, the two most potent ATX inhibitors, dose-dependently inhibited the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro. The average terminal half-life for compound 22 was 10h ± 5.4h and it caused a long-lasting reduction plasma LPA levels. Compounds 22 and 30b significantly reduced lung metastasis of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment paradigm. The described 4-substituted benzyl phosphonic acids and 6-substituted naphthalen-2-yl-methyl phosphonic acids represent new lead compounds that effectively inhibit the ATX-LPA-LPA receptor axis both in vitro and in vivo.

The marine-derived α-galactosylceramide is an exogenous ligand for natural killer T cells and leads to the secretion of both T help 1 (Th1) and Th2 cytokines. The relationship between the sugar moiety structure and invariant natural killer T (iNKT) cell stimulation ability has not been fully understood. With the series α-galactosylceramide analogues varied on C3′ and C4′ position, subjected to a murine system, we discovered that the 3′ hydroxyl is very crucial in maintaining the molecule’s immunogenicity. Any modification on this position will lead to the losing of activity. We also found that the C4′ position is not so sensitive and can tolerate some small modifications on it. Moreover, the C4′ substituted analogues induced biased Th2 cytokines release was observed.

Urotensin-II (U-II) has been shown to be the most potent mammalian vasoconstrictor known.[1, 2] Thus a U-II antagonist might be of therapeutic value in a number of cardiovascular disorders.[3] However, interspecies variability of several nonpeptidic ligands complicates the interpretation of in vivo studies of such antagonists in pre-clinical animal models of disease. Thus compound ACT058362 is a selective antagonist for human U-II receptor (hUT2R) with a reported Kd ~ 4 nM in a molecular binding assay, but it is reported to bind weakly to rat UT2R (rUT2R), with Kd ~ 1,500 nM.[4] In contrast, the arylsulphonamide SB706375 is a selective antagonist against both hUT2R (Kd: ~ 9 nM) and rUT2R (Kd: ~ 21 nM).[3] To understand the species selectivity of the UT2R, we investigated the binding site of ACT058362 and SB706375 complex with both hUT2R and rUT2R to explain the dramatic (~ 400-fold) lower affinity of ACT058362 for rUT2R and the similar (~10 nM) affinity of SB706375 for both UT2R. These studies.used MembStruk and MSCDock to predict the UT2R structure and the binding site for ACT058362 and SB706375. Based on binding energy, we found two binding modes each with D1303.32 as the crucial anchoring point. We predict that ACT058362 (an aryl-amine-aryl or ANA ligand) binds in the TM 3456 region while we predict that SB706375 (an aryl-aryl-amine or AAN ligand) binds in the TM 1237 region. These predicted sites explain the known differences in binding the ANA ligand to rat and human while explaining the similar binding of the AAN compound to rat and human. Moreover the predictions explain currently available SAR data. To further validate the predicted binding site of these ligands to hUT2R and rUT2R, we propose several mutations that would help define the structural origins of differential responses of UT2R among species potentially indicating novel UT2R antagonists with cross-species high affinity.

Small molecules that increase the cellular level of melanin can be used to study melanogenesis, and have therapeutic potential for melanin-related diseases such as albinism. We describe the identification of a potent activator of melanogenesis from a targeted combinatorial library. Treating melanocytes with our most active molecule results in a 1.8-fold increase in melanin, and an increase in tyrosinase-catalyzed oxidation of L-tyrosine, a key step in melanin biosynthesis.

PPARγ is involved in expression of genes that control glucose and lipid metabolism. PPARγ is the molecular target of the thiazolidinedione (TZD) class of antidiabetic drugs. However, despite their clinical use these drugs are related to numerous adverse effects, which are related to their full activation of PPARγ transcriptional responses. PPARγ partial agonists are the focus of development efforts towards second-generation PPARγ modulators with favourable pharmacology, potent insulin sensitization without the severe full agonists’ adverse effects. In order to identify novel PPARγ partial agonist lead compounds, we developed a virtual screening protocol based on 3D-ligand shape similarity and docking. 235 compounds were prioritized for experimental screening from a 340,000 MLSMR chemical library. Seven novel potent partial agonists were confirmed in cell-based transactivation and competitive binding assays. Our results illustrate a well-designed virtual screening campaign successfully identifying novel lead compounds as potential entry points for the development of antidiabetic drugs.

In this report we describe synthesis and biological evaluation of a series of asymmetric 4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperidin-3-ol based dihydroxy compounds where the hydroxy groups are located both on the piperidine ring and also on the N-phenylethyl side chain exo-cyclically. In vitro uptake inhibition data indicates high affinity of these molecules for the dopamine transporter (DAT) in addition to their moderate to high affinity for the norepinephrine transporter (NET). Interestingly, compounds 9b and 9d exhibited affinities for all three monoamine transporters with highest potency at DAT and NET and moderate potency at the serotonin transporter (SERT) (Ki 2.29, 78.4 and 155 nM for 9b and 1.55, 14.1 and 259 nM for 9d, respectively). Selected compounds, 9a, 9d and 9d’ were tested for their locomotor activity effects in mice, and for their ability to occasion the cocaine discriminative stimulus in rats. These test compounds generally exhibited a much longer duration of action than cocaine for elevating locomotor activity, and dose-dependently completely generalized the cocaine discriminative stimulus.

A novel class of isochroman dopamine analogues, 1, originally reported by Abbott Laboratories, had greater than 100-fold selectivity for D1-like vs. D2-like receptors. We synthesized a parallel series of chroman compounds, 2, and showed that repositioning the oxygen in the heterocyclic ring reduced potency and conferred D2-like receptor selectivity to these compounds. In silico modeling supported the hypothesis that the altered pharmacology for 2 was due to potential intramolecular hydrogen bonding between the oxygen in the chroman ring and the meta-hydroxyl of the catechol moiety. This interaction realigns the catechol hydroxyl groups and disrupts key interactions between these ligands and critical serine residues in TM5 of the D1-like receptors. This hypothesis was tested by the synthesis and pharmacological evaluation of a parallel series of carbocyclic compounds, 3. Our results suggest that when the potential for intramolecular hydrogen bonding is removed, D1-like receptor potency and selectivity is restored.

Affibodies are a class of polypeptide ligands that are potential candidates for cell- or tissue-specific targeting of drug-encapsulated controlled release polymeric nanoparticles (NPs). Here we report the development of drug delivery vehicles comprised of polymeric NPs that are surface modified with Affibody ligands that bind to the extracellular domain of the trans-membrane human epidermal growth factor receptor 2 (HER-2) for targeted delivery to cells which over express the HER-2 antigen. NPs lacking the anti-HER-2 Affibody did not show significant uptake by these cells. Using paclitaxel encapsulated NP-Affibody (1 wt% drug loading), we demonstrated increased cytotoxicity of these bioconjugates in SK-BR-3 and SKOV-3 cell lines. These targeted, drug encapsulated NPAffibody bioconjugates may be efficacious in treating HER-2 expressing carcinoma.

Isoform-selective agonists and antagonists of the lysophosphatidic acid (LPA) G-protein-coupled receptors (GPCRs) have important potential applications in cell biology and therapy. LPA GPCRs regulate cancer cell proliferation, invasion, angiogenesis, and biochemical resistance to chemotherapy- and radiotherapy-induced apoptosis. LPA and its analogues are also feedback inhibitors of the enzyme lysophospholipase D (lysoPLD, also known as autotaxin), a central regulator of invasion and metastasis. For cancer therapy, the ideal therapeutic profile would be a metabolically stabilized pan-LPA receptor antagonist that also inhibits lysoPLD. Herein we describe the synthesis of a series of novel α-substituted methylene phosphonate analogues of LPA. Each of these analogues contains a hydrolysis-resistant phosphonate mimic of the labile monophosphate of natural LPA. The pharmacological properties of these phosphono-LPA analogues were characterized in terms of LPA receptor subtype-specific agonist and antagonist activity using Ca2+ mobilization assays in RH7777 and CHO cells expressing the individual LPA GPCRs. In particular, the methylene phosphonate LPA analogue is a selective LPA2 agonist, whereas the corresponding α-hydroxymethylene phosphonate is a selective LPA3 agonist. Most importantly, the α-bromomethylene and α-chloromethylene phosphonates show pan-LPA receptor subtype antagonist activity. The α-bromomethylene phosphonates are the first reported antagonists for the LPA4 GPCR. Each of the α-substituted methylene phosphonates inhibits lysoPLD, with the unsubstituted methylene phosphonate showing the most potent inhibition. Finally, unlike many LPA analogues, none of these compounds activate the intracellular LPA receptor PPARγ.

Methionine aminopeptidase (MetAP) carries out an essential function of protein N-terminal processing in many bacteria and is a promising target to develop novel antitubercular agents. Natural bengamides potently inhibit proliferation of mammalian cells by targeting MetAP enzymes, and the X-ray structure of human type 2 MetAP in complex with a bengamide derivative revealed the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1a and MtMetAP1c in different metalloforms, inhibition of growth of M. tuberculosis in replicating and non-replicating states, and inhibition of growth of human K562 cells. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. X-ray structures of MtMetAP1c in complex with two of the derivatives provided the valuable structural information for improvement of these inhibitors for potency and selectivity.