The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We developed a novel MRI probe which targets HER2, a biomarker for various cancers and a target for anti-cancer therapies. This multimodal HER2-targeted MRI probe integrates a rationally designed protein contrast agent with a high affinity HER2 affibody and near IR dye. Our probe can differentially monitor tumors with different HER2 levels in both cells and xenograft mice. In addition to its 10-fold higher dose efficiency compared to clinically-approved agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention as demonstrated by even distribution of the imaging probe across the entire tumor mass. Additionally, a second series of protein contrast agents that included affibody against EFGR developed with the capability to specifically target EGFR. These contrast agents have been utilized to monitor drug treatments and quantitatively analyze biomarker expression level. Furthermore, we anticipate these agents will provide powerful tools for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.

The first section of this work describes the synthesis of a library of novel rhein analogues that are potential anticancer agents. The design of these compounds takes advantage of the ability for rhein to intercalate into DNA and as the incorporation of an alkylating agent, which serves to covalently modify DNA. In three cell lines, these compounds showed potent cytotoxicity with IC50 in the low to mid-μM range. The second project was focused on the development of an efficient synthesis of 6H-Isoindolo[2,1-α]indol-6-one (24), a core structure for a number of biologically active compounds. The approach is metal-free and uses a Beckmann rearrangement followed by an intramolecular cyclization.

Magnetic Resonance Imaging instruments rely on a contrast agent to provide high-resolution images of tissues in vivo. However, current clinical contrast agents are hindered by low relaxivity and fast correlation time, necessitating high injection dosages. These concerns, among others, have driven the development of a class of protein-based contrast agents (ProCAs), by design of lanthanide binding sites into a scaffold protein. ProCA1 has a higher reported relaxivity and dosage efficiency than current contrast agents. In this study, expression and Glutathione-S-Transferase purification procedures were optimized, and a refolding method for rapid production of ProCA1 has been developed to enable studies of conformation, metal binding, relaxivity, and in vivo applications. Several ProCA1 variants with 4-5 charged ligand residues were shown to have strong gadolinium binding affinity (Kd of 10-12 M) and metal selectivity. Several options to improve ProCA1 have been explored, including addition of a polyethylene chain or a bombesin tag.

An intriguing mystery about tryptophan 2, 3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of L-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2, 3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (δ = 0.055 mm/s and ΔEQ = 1.755 mm/s) and a protein-based free radical (g = 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. A Trp-Trp dimer and a monooxygenated L-Trp were both observed as the enzyme reactivation by-products by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism.

The 5-HT7 receptor is the most recent addition to the 5-HT receptor family and has been linked to a variety of physiological and pathophysiological processes. Well established antide-pressant pharmaceuticals have recently been found to activate the 5-HT7 receptor, supporting the role of the 5-HT7 receptor in the antidepressant mechanism. The synthesis of potent selec-tive 5-HT7 receptor antagonists could afford a greater understanding of the 5-HT7 receptor function as well as lead to potential drug candidates. The synthesis of unfused biheteroaryl derivatives as 5-HT7 receptor ligands has been de-scribed within. These compounds have been tested for biological activity on the 5-HT6 and 5-HT7 receptors. 4-(3’-Furyl)-2-(N-substituted-piperazino)pyrimidines were found to be potent 5-HT7 receptor ligands. 4-(2’-Furyl)-2-(N-substituted-piperazino)pyrimidines have shown high se-lectivity for the 5-HT7 receptor over the 5-HT6 receptor.

Boronic acids play an important role in the design and synthesis of chemosensors for carbohydrates due to their ability to reversibly bind with diol-containing compounds. Along this line, the availability of boronic acids that change fluorescence upon sugar binding is critical to a successful sensor design effort. Here, two boronic acids that show strong fluorescent intensity changes upon sugar binding are reported: isoquinoline-7-boronic acid (7-IQBA) and phenoxathiin-4-boronic acid (4-POBA).

In part A, the effect of varying short-chain alkyl substitution on the spectroscopic properties of cyanine dyes was examined. Molar absorptivities and quantum yields were determined for groups of pentamethine and heptamethine dyes for which the substitution of the indole nitrogen was varied. For both sets of dyes, increasing alkyl chain length did not significantly change quantum yield or molar absorptivity. These results may be useful in designing new cyanine dyes. In part B, the effect of structure on the suitability of 2,4,6-trisubstituted pyridines as color pH indicators was studied by determining spectral effects of protonation, molar absorptivities, pKa values, and the structural origin of the spectral behavior. Good color indicating properties result from aniline substitution at the 4 position of pyridine and electron donating substitution at the 2 and 6 positions of pyridine, which provide a strong red shift in the spectra and greater red shifted peak absorptivity, respectively.

Calmodulin (CaM) is a eukaryotic Ca2+ signaling protein which can interact with more than 300 enzymes in the cell including membrane proteins Ryanodine receptor1 (RyR1) and gap junction protein connexin 43 (Cx43). By binding to Ca2+, CaM undergoes a conformational change which exposed the hydrophobic patch that access to the target protein. wt-CaM and three mutant CaM (isolate C and N domain of CaM, deletion of five residues from the central linker of CaM) are designed for studying the specific contributions to calcium binding affinity and calcium induced conformational change. wt-CaM exhibits metal binding affinity to calcium analog Tb3+ with a Kd of 3.97 nM using FRET assay and metal-buffer system and activates target protein phosphodiesterase assay. The Kd values of domain specific calcium binding affinity of CaM probed by intrinsic Phe or Tyr are 12.2 and 2.77 uM, respectively. In addition, Ca2+ also induces helicity for both w.t. CaM and C-terminal domain variant. Further, conditions such as medium and Glucose amount for isotopic labeling of CaM by 15N, 13C and D2O have been optimized with relatively high yield of hetero-isotopic labeled CaM. This prepared us to probe the detailed interaction of CaM and its target protein and calcium induced conformational change by high resolution NMR. Furthermore, RyR1 mini domain, which contains two CaM binding regions of RyR1 was designed to study the binding mode of the two regions with CaM. Obtaining bacterial expressed and purified RyR1 mini domain was achieved by engineered with a His-Tag which overcomes the insoluble issue that occurred in the initial study of expression and purification with a GST tag. Moreover, to probe the interaction of CaM to the cytosolic loop of Cx43 that contains two putative CaM binding sites as well as the role of transmembrane region of Cx43, we have successfully expressed and purified fragments Cx4388-154 and Cx4399-154 as a His-tag protein encompassing regions 88-154 and 99-154 of Cx43 with predicted CaM binding sites with and without additional transmembrane region from Cx43. Both fragments were obtained with high yield after expressed as inclusion body and His-tag purification. Fragment Cx4388-154 was shown to bind dansylated CaM with a Kd of 0.107 μM using florescence spectroscopy.

Protein translocation is essential for bacterial survival and the most important translocation mechanism in bacteria is the secretion (Sec) pathway. Thus targeting Sec pathway is a promising strategy for developing novel antibacterial therapeutics. We report the design, syntheses, mechanistic studies and structure-activity relationship studies using HQSAR and 3-D QSAR Topomer CoMFA analyses of 4-oxo-5-cyano thiouracil derivatives. In summary, introduction of polar group such as –N3 and linker groups such as –CH2-O- enhanced the potency as well as logP and logS several fold. We also report the discovery, optimization and structure-activity relationship study of 1,2,4-triazole containing pyrimidines as novel, highly potent antimicrobial agents. A number of inhibitors have been found to inhibit microbial growth at high nanomolar concentrations.

Cancer is responsible for one fourth of the total deaths and is the second leading cause of death, behind heart disease, in the United States. However, there are as many approaches to curing cancer as there are types of cancer. One important issue in solid tumors is hypoxia, a lack of oxygen, which promotes angiogenesis and anaerobic metabolism, which can increase cancer progression and metastasis. The HIF transcription factor is responsible for the mediation of many processes involved during hypoxia and is linked to poor patient prognosis, increased cancer progression, and invasiveness of tumors. With this in mind, the HIF pathway has become an attractive target for small molecule inhibition. Herein, we describe the design and synthesis of small molecules that inhibit the HIF pathway. These compounds are based off an initial “hit” compound, KCN-1, from screening of a 10,000 compound library. KCN1 is both highly effective and has a low toxicity profile. Over 200 compounds have been synthesized by the Wang lab, with the best compound IVSR64b having an IC50 of 0.28 μM. Of special interest is that these compounds do not appear to have any inherent toxicity toward healthy tissues, but only affect cancer cells. Moreover, x-ray crystal structures for both KCN-1 and IVSR64b were obtained and used as the basis for computational modeling, which is still in progress.