Indiana University-Purdue University Indianapolis (IUPUI) / The human aldehyde dehydrogenase (ALDH) superfamily consists of 19 isoenzymes that are critical for normal physiology as well as the removal of toxic aldehydes. Members of the ALDH1/2 family have vital roles in cell signaling during early development, ethanol metabolism, and the removal of aldehydes derived from oxidative stress. We sought to develop selective compounds toward ALDH2 to help determine its individual contribution to biological function, as many of the ALDH1/2 family possess overlapping substrate preferences. A high-throughput screen of over 100,000 compounds uncovered a class of aromatic lactones which inhibit the ALDH1/2 enzyme family. The lactones were then characterized using a combination of enzyme kinetics, X-ray crystallography, and cell culture experiments. We found that many of the lactones are over ten times more potent toward ALDH2 than daidzin, a previously described ALDH2 inhibitor. Our ability to produce many more ALDH isoenzymes allowed us to determine that daidzin is not as selective as previously believed, inhibiting ALDH2, ALDH1B1, and ALDH1A2 with equal potency. This inhibition pattern was seen with several of the aromatic lactones as well. Structural studies show that many of the lactones bind between key aromatic residues in the ALDH1/2 enzyme substrate-binding sites. One lactone in particular mimics the position of an aldehyde substrate and alters the position of the catalytic cysteine to interfere with the productive binding of NAD+ for enzyme catalysis. Further characterization of related compounds led to the realization that the mechanism of inhibition, potency, and selectivity differs amongst the lactones based off the substituents on the aromatic scaffold and its precise binding location. Two of these compounds were found to be selective for one of the ALDH1/2 family members, BUC22, selective for ALDH1A1, and BUC27, selective for ALDH2. BUC22 demonstrates ten-fold selectivity for ALDH1A1 over ALDH1A2 and does not inhibit the remaining ALDH1/2 enzymes. Additionally, treatment with BUC22 led to decreased growth of triple-negative breast cancer cells in culture. BUC27 inhibits ALDH2 with the same potency as daidzin. Both BUC22 and BUC27 could be further developed to use as chemical tools to better understand the functional roles of ALDH1A1 and ALDH2 in biological systems.

A crucial step in the biosynthesis of membrane proteins is their incorporation into the hydrophobic environment of the lipid bilayer. In eukaryotic cells this event occurs largely in concert with translation on ribosomes bound to the membrane of the endoplasmic reticulum (ER) at a site termed the ER translocon. This dynamic proteinaceous complex forms an aqueous conduit across the ER membrane and is laterally gated to allow transmembrane (TM) segments to partition into the lipid phase. In the case of polytopic membrane proteins, the coordinated release of multiple TM segments by the ER translocon is a poorly defined process and appears to be highly substratespecific. In this study, the ion channel subunit P2X2 was used as a novel model to examine themolecular details of membrane protein integration at the ER translocon. A primarily in vitro approach was taken using stable biosynthetic intermediates to simulate each stage of the membrane translocation and integration of P2X2. Chemical and photoreactive site-specific cross-linking analyses were then conducted to determine the molecular environment of the P2X2 TM segments throughout biosynthesis. Remarkably, both TM1 and TM2 of P2X2 were found to remain directly adjacent to the ER translocon throughout P2X2 biosynthesis and were only dislocated into the lipid phase by artificial termination of translation and disruption of the ribosome-translocon interaction. Retention of P2X2 TM1 at the ER translocon is maintained despite the synthesis of over 300 amino acid residues separating it from the ribosome peptidyl transferase centre. Premature dislocation of TM1 from the ER translocon site resulted in a pronounced aggregation of TM1 fragments both in vitro and in vivo. This is in stark contrast to previous passive-partitioning models of membrane integration and suggests that the ER translocon regulates the integration of polytopic membrane proteins in order to accommodate the specific requirementsof the substrate protein itself. The detailed characterisation of P2X2 biosynthesis was then exploited in order to examine the effect of a novel small inhibitor of ER translocon function. Eeyarestatin 1 (ESI) was found to cause a substantial inhibition of protein secretion in vivo and dramatically reduced the ER translocation of three distinct classes of substrate, including P2X2, in vitro. Using both a cross-linking analysis and a protease-protection assay for a specialised translocation reaction, ESI was shown to prevent the transfer of the nascent polypeptide chain from the membrane delivery machinery to the ER translocon complex. Further evidence that ESI targets the Sec61 complex is presented and a model for ESI-mediated inhibition of ER translocation is suggested. Taken together these data establish ESI as a novel small molecule inhibitor that selectively inhibits protein translocation both in vitroand in vivo.

We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett. 2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-lM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.

Methods for preparing an array of potential small molecule inhibitors of Bone Morphogenetic Protein Receptor 1b (BMPR1b) are described. Target molecules were prepared from two general classes: (1) N9-aryl-N6-ureidoadenines, and (2) dicarbamyl iodoacetamides. Recent data from the Peterson lab indicated that both classes might bind to BMPR1b and thus inhibit this key receptor. Docking studies performed using Sureflex Dock suggested the N9-aryl-N6-ureidoadenines would bind to the active site of BMPR1b. In addition antiproliferative activities of dicarbamyl iodoacetamides previously synthesized in the Peterson lab pointed to this moiety as an attractive target for structure activity relationship (SAR) development. Compounds were prepared in good to excellent yields and 40 derivatives were screened for antiproliferative activity. Of the N9-aryl-N6-ureidoadenine derivatives, N9-phenyl-N6-N-phenylureaadenine was most potent and exhibited selective activity against HeLa cells (IC50 = 11± 1 uM). Dicarbamyl iodoacetamide derivatives had similar activities compared to the previously reported compound (JRS-150).

Discovered in 1980, HTLV-1 (Human T-cell Leukemia Virus-1), was the first identified human retrovirus and is shown to be associated with a variety of diseases including: adult T-cell leukemia lymphoma (ATLL), tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM), chronic arthropathy, uveitis, infective dermatitis, and polymyositis. The mechanism by which the virus causes disease is still unknown. HTLV- 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan, the Caribbean basin, Central and West Africa, the Southeastern United States, Melanesia, parts of South Africa, the Middle East and India. Approximately 30 million people are infected by HTLV-1 worldwide, although only 3-5% of the infected individuals evolve Adult T-cell Leukemia (ATL) during their life and the prognosis for those infected is still poor. The retroviral proteases (PRs) are essential for viral replication because they process viral Gag and Gag-(Pro)-Pol polyproteins during maturation, much like the PR from Human Immunodeficiency Virus-1 (HIV-1). Various antiviral inhibitors are in clinical use and one of the most significant classes is HIV-1 PR inhibitors, which have used for antiretroviral therapy in the treatment of AIDS. HTLV-1 PR and HIV-1 PR are homodimeric aspartic proteases with 125 and 99 residues, respectively. Even though substrate specificities of these two enzymes are different, HTLV-1 PR shares 28% similarity with HIV-1 PR overall and the substrate binding sites have 45% similarity. In addition to the 125-residue full length HTLV-1 PR, constructs with various C- terminal deletions (giving proteases with lengths of 116, 121, or 122 amino acids) were made in order to elucidate the effect of the residues in the C-terminal region. It was suggested that five amino acids in the C-terminal region are not necessary for the enzymatic activity in Hayakawa et al. 1992. In 2004 Herger et al. had suggested that 10 amino acids at the C-terminal region are not necessary for catalytic activity. A recent paper suggested that C-terminal residues are essential; and that catalytic activity lowers upon truncation, with even the last 5 amino acids necessary for full catalytic activity (1). The mutation L40I has been made to prevent autoproteolysis and the W98V mutation was made to make the active site of HTLV-1 PR similar to HIV-1 PR. We have characterized C-terminal amino acids of HTLV-1 PR as not being essential for full catalytic activity. We have discovered potential new inhibitors by in silico screening of 116-HTLV-1 PR. These small molecules were tested kinetically for various constructs including the 116, 121 and 122-amino acid forms of HTLV-1 PR. Inhibitors with the best inhibition constants were used in HTLV-1 infected cells and one of the inhibitors seems to inhibit gag processing.

Deregulation of platelet-derived growth factor receptor (PDGFR) signaling is a driving event in glioblastoma, promotes tumor progression epithelial to mesenchymal transition (EMT) in multiple cancers, modulates the tumor stroma to facilitate tumorigenesis and reduces tumor uptake of chemotherapeutics. Previous studies identified the 105 kDa isoform of ankyrin3 (Ank105) as a binding partner of the PDGFR signaling machinery and demonstrated that expression of Ank105 promoted PDGFR degradation (Ignatiuk et al., 2006)(Ignatiuk et al., 2006)(Ignatiuk et al., 2006). Receptor tyrosine kinases are targeted for degradation via endocytosis and ubiquitin-dependent trafficking to the lysosome. It was hypothesized that Ank105 promoted the constitutive degradation of the PDGFR and attenuation of PDGFR signaling by facilitating endocytosis of the PDGFR and targeting the PDGFR for lysosomal degradation via an ubiquitin-dependent mechanism. The studies in this thesis characterized the effects of Ank105 expression on PDGFR signaling and protein expression levels, determined the endocytic pathways involved in Ank105-mediated PDGFR degradation and studied the role of ubiquitin binding in Ank105 function. The most robust effect of Ank105 expression on the PDGFR was constitutive degradation as PDGFR protein expression levels in Ank105-expressing cells were significantly reduced compared to NIH 3T3 cells in the absence of PDGF ligand. Low constitutive PDGFR levels resulted in attenuated pro-proliferative AKT and mitogen-activated protein kinase (MAPK) signaling in response to ligand stimulation. To determine the endocytic requirements for Ank105-mediated constitutive PDGFR degradation, a constitutive PDGFR degradation assay was developed and the effects of several small molecule endocytosis inhibitors were evaluated. Additionally, the small molecule endocytosis inhibitors were validated by determining the effects of these inhibitors on low density lipoprotein (LDL) uptake and ligand-induced PDGFR degradation in Ank105-expressing cells. Both LDL uptake and ligand induced PDGFR degradation are known to proceed by a clathrin and dynamin dependent mechanism of endocytosis. In Ank105-expressing cells, both LDL uptake and ligand incuded PDGFR degradation were dependent upon clathrin and dynamin function. Interestingly, constitutive PDGFR degradation in Ank105-expressing cells was not dependent upon CME, but required dynamin activity. Expression of Ank105 may promote clathrin-independent, dynamin-dependent, constitutive endocytosis of the PDGFR. Additionally, acute inhibition of either lysosomal or proteasomal degradation strongly impaired constitutive PDGFR degradation, whereas ligand-induced PDGFR degradation was less sensitive to protein degradation inhibitors, while LDL uptake was unaffected. It was unclear if PDGFR was degraded in the proteasome or if the proteasome was involved in sorting of PDGFR to the lysosome for degradation. Ubiquitination of receptors is required to target them for degradation. Ank105 was assayed for the ability to interact with ubiquitin and ubiquitinated proteins. Interestingly, Ank105 bound ubiquitin in vitro via the spectrin binding domain and co-immunoprecipitated with several ubiquitinated proteins, suggesting a role for Ank105 in the sorting of ubiquitinated proteins for degradation. Furthermore, Ank105 co-immunoprecipitated with a number of high and low molecular weight proteins in the absence of PDGF stimulation. Identification of Ank105 binding partners would provide further insight in the mechanism of Ank105-mediated constitutive PDGFR degradation. In summary, Ank105 promoted the attenuation of PDGFR signaling via alteration of constitutive PDGFR endocytosis and targeting of constitutive PDGFR for degradation, potentially through interaction with ubiquitin and ubiquitinated proteins. Reduction of constitutive PDGFR levels in cancers with PDGFR driver mutations, acquired PDGF responsiveness and stromal expression of PDGFR, could significantly reduce tumor proliferation, tumorigenesis and increase effectiveness of chemotherapeutics.

Pleckstrin homology (PH) domains are structurally conserved domains, which generally bind to phosphatidylinositol phosphate (PtdInsP) lipids. They are present in a variety of proteins, including those that are upregulated in cancer growth and metastasis, and represent a crucial component of intracellular signaling cascades and membrane translocation. Thus, they may be considered as attractive targets for cancer drug therapy. AKT (protein kinase B), a pleckstrin homology lipid binding domain and a serine/threonine kinase-containing protein, is a key component of the phophatidylinositol-3-kinase (PI3K)/AKT cell survival signaling pathway which is activated in a variety of cancers, including prostate, pancreatic, and skin cancers. In this study, I report the finding of a novel inhibitor of AKT; PH-427. I describe its effects on binding to the PH domain of AKT thus preventing its binding to PtdIns3-P at the plasma membrane and subsequent activation. In vivo testing of the drug led to reduction of tumor size and numbers in a mouse pancreatic cancer model. Additional testing of PH-427 on squamous cell carcinomas revealed that the drug is able to reduce tumor burden and multiplicity in vivo when topically applied. Thus, we demonstrate proof-of-principle in targeting PH domains as a viable cancer drug therapy option. The effects of PH-427 raised the intriguing possibility that targeting PH domains may have beneficial effects in other signaling pathways with PH domain-containing proteins. Guanine exchange factors (GEFs) contain a Dbl homology (DH) domain and a PH domain and have been shown to be involved in the process of metastasis. More specifically, RacGEFs activate Rac1 GTPase by facilitating the exchange of GDP to GTP. Over-expression of certain GEFs has been shown to contribute to increased malignancy in a variety of cancers. T-lymphoma invasion and metastasis-inducing protein-1 (Tiam1) is a highly conserved GEF and contains an N-terminal pleckstrin homology domain (nPH) and a DH/C-terminal PH domain (cPH). Tiam1 has been found to be over-expressed in several cancers, including breast, colon and prostate cancers. In this study, I describe the identification, development, experimental testing, and potential mechanism of action of novel small molecule inhibitors targeting the RacGEF Tiam1 to inhibit prostate cancer bone metastasis.

Heme oxygenases (HO) catalyze the degradation of heme into biliverdin, carbon monoxide (CO) and free iron. The two major isoforms, HO-2 (constitutive) and HO-1 (inducible by various stressors such as heavy metals and reactive oxygen species) are involved in a variety of physiological functions, including anti-inflammation, antiapoptosis, neuromodulation, and vascular regulation. Major tools used in exploring these actions have been metalloporphyrin analogs of heme that inhibit the HOs. However, these tools are limited by their lack of selectivity; they affect other heme-dependent enzymes, such as cytochromes P450 (CYPs), soluble guanylyl cyclase (sGC), and nitric oxide synthase (NOS). Our laboratory has been able to successfully synthesize a series of small molecule non-porphyrin HO inhibitors (QC-xx) that have had little or no effect against sGC and NOS; however, their effects on various CYP isoforms has yet to be fully elucidated. In order to determine the effects on CYP enzyme activity, microsomal preparations of two CYP isoforms (2E1 and 3A1/3A2) were incubated with varying concentrations of HO inhibitor and the activity was determined via spectrophotometric analysis. Results indicated that some QC compounds demonstrated little to no inhibition of CYP2E1 and/or CYP3A1/2, while some others did inhibit these CYP isoforms. Four regions of interest were analyzed further and several structural changes were identified as conferring increased HO inhibition and decreased effect on both CYP2E1 and 3A1/2. Based on the information obtained, three putative compounds were designed and it is hypothesized that these compounds will be selective inhibitors for HO-1 over HO-2 and will display little effect on either CYP2E1 or 3A1/2 activities. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-11-20 11:19:48.841

Rheumatoid arthritis (RA) is a debilitating chronic inflammatory disease causing bone and cartilage degradation. Macrophages are known to play a role in RA pathology by producing pro-inflammatory cytokines and chemokines, which activates immune cells, drives inflammation and facilitates the degradation of bone and cartilage. Alterations in epigenetic mechanisms, processes that regulate gene expression, have been implicated in the regulation of pro-inflammatory cytokines in RA. Therefore, the aim of this thesis was to determine specific epigenetic variation between RA patient blood and synovial fluid (SF)-derived macrophages (SF MLS). Granulocyte and macrophages colony stimulating factor (GM-CSF) was used to differentiate healthy donor and RA patient blood monocytes into macrophages. Lipopolysaccharide (LPS) was used to stimulate blood and SF-derived macrophages to initiate inflammatory cytokine production. A library of small molecule inhibitors was used to identify key epigenetic regulators of pro-inflammatory cytokine production. Bromodomain and extra-terminal (BET) protein inhibitors (JQ1, I-BET151, PFI-1) were the only class of inhibitor to show consistent down regulation of pro-inflammatory cytokines in both healthy and RA patient-derived macrophages. However, only JQ1 was shown to reduce TNFα production significantly in SF MLS. Transcriptional profiling of RA patient SF MLS indicated a preference for a pro-inflammatory phenotype, and a resistance to steroids (a trait found in 30% of RA patients); SF MLS production of chemokines and cytokines were not downregulated by glucocorticoids in comparison to corresponding blood-derived macrophages. However, JQ1 treatment successfully suppressed these genes. In addition, silencing of BRD4 in blood-derived macrophages from healthy donors reduced pro-inflammatory cytokine production. Chromatin immunoprecipitation studies showed BRD4 was localised to pro-inflammatory promoter regions upon LPS stimulation and displaced in the presence of JQ1. These studies identified BET proteins BRD2, 3 and 4, as essential epigenetic regulators of pro-inflammatory cytokine and chemokine production in both healthy donors and RA patient macrophages. Furthermore, the observation that BET inhibitors can regulate genes that are steroid resistant in RA patient SF MLS, highlights their therapeutic potential in RA.

<p>A successful flaviviral life cycle involves several coordinated events between viral proteins and host factors. The polyprotein processing at the surface of the ER membrane results in the formation of several replication proteins that bring about changes in the ER membrane making it permissive for viral genome amplification. Non-structural proteins 4A (NS4A) and non-structural protein 4B (NS4B) are two of the most important integral membrane proteins of DENV that are essential part of the viral replicase complex. The cleavage at NS4A-2K-NS4B is temporally and spatially regulated. The cleavage at the N-terminal of 2K is carried out by viral NS2B/3 protease while host signalase cleaves on the C-terminal side at the ER lumen to give rise to a mature NS4B protein. This thesis primarily focuses on demonstrating the function of 2K as an independent peptide rather than simply a signal sequence, and the role 2K plays, when present as 2K-NS4B vs NS4B. Moreover, this thesis has attempted to explore the function of transmembrane domains (TMDs) in replication separating them from their membrane anchor function. This thesis will also describe the development of a ZIKV replicon and its use in screening small molecule inhibitors in the last chapter.</p><p>In Chapter 2 of the thesis, we established 2K as an independent, information carrying peptide rather than just a signal peptide. A strategy involving chimeric virus generation and mutational analysis supported the notion that 2K is rather unique and important for viral replication and infectious particle production. Using an interserotypic 2K chimeric virus, it was established that the 2Ks of DENV are serotype specific, however, they are interchangeable with a huge fitness cost in infectious particle production. We further showed that individual amino acid residues towards then end of h-region and C-terminus of the 2K peptide affect viral replication and infectious particle production. Moreover, it was shown that the 2K peptide consists of a highly conserved ‘DNQL’ region at its N-terminal that plays an important role in viral replication.</p><p>Chapter 3 details the mechanistic aspect of the effects observed in interserotypic 2K chimeric viruses. The interserotypic chimeric viruses were comparable to wild type in replication, however, they were deficient in infectious particle production early in the life cycle. The major change to be noted in the chimeric viruses was the absence of signalase cleavage at the 2K-NS4B junction. We demonstrated that in a virus infected system, 2K-NS4B and NS4B populations are always present which led us to look for any specific functions of the cleaved vs uncleaved 2K-NS4B protein. Using a transcomplementation system where NS4B was presented in the absence of 2K, we showed that particle production can be rescued in the interserotypic 2K chimeric viruses. It was further concluded using NS4B truncations that the property of NS4B to rescue particle production was concentrated in the ER luminal loop. Further, alanine scanning mutagenesis of the conserved residues of ER loop resulted in pinpointing T198 and its involvement in the early stages of viral packaging.</p><p>Chapter 4 examined the role of TMDs of NS4A and NS4B and attempted to define their roles separately from their membrane anchoring functions. Several interserotypic TMD chimeric viruses were generated to address the function of these domains. We concluded that TMD1 and TMD3 of NS4A could be replaced with partial success across the DENV serotypes, whereas, TMD2 was serotype specific. The specificity of TMD2 of NS4A is not contributed by a single amino acid and should be a function of the secondary structure formed by TMD2 as it sits on the inner leaflet of the ER membrane. We demonstrated the variable roles different TMDs of NS4B play in viral replication using a similar strategy of reverse genetics of chimeric viruses. TMD1 of NS4B was replaceable with no to minimal effect, whereas, the remaining four showed variable effect upon substitution. More importantly, we demonstrated how the reorientation of TMD5 of NS4B post NS2B/3 cleavage might vary in different serotypes of DENV using revertant virus obtained from the TMD5 interserotypic chimera. Analysis of interserotypic cytosolic and ER luminal loop chimeras of NS4B pointed to functional conservation of the cytosolic loop between DENV-2 and DENV-3, whereas, the remaining cytosolic loops and the ER loops showed variable level of defects upon substitution, suggesting their functions in serotype-dependent manner.</p><p>Chapter 5 describes the construction and characterization of a ZIKV replicon system and use of it to screen several small molecule inhibitors of the flaviviruses MTase. Several small molecule inhibitors of flavivirus N-7-MTase were designed/synthesized in Dr. Arun K Ghosh’s lab which would target the extra pocket unique to the flavivirus SAM-binding site. We analyzed the docking of a set of these compounds into MTase domain of NS5 of ZIKV, DENV and YFV and screened them for their ability to inhibit replication of ZIKV, DENV and YFV. A huge variation in the activity profile of these compounds were observed against different flaviviruses even though these compounds were targeted against the highly conserved MTase domain of flavivirus NS5. GRL-002- and GRL-004-16-MT specifically inhibited ZIKV replication with low micromolar IC₅₀ value, while these compounds showed little to no effect on DENV and YFV.<b> </b>On the other hand, compounds GRL-007-, GRL-0012- and GRL-0015-16-MT demonstrated a dual inhibitory effect against DENV and YFV albeit the CC₅₀ values of the GRL-012 and GRL-015 were concerning. Compounds GRL-007-16-MT showed broad spectrum activity against ZIKV, DENV and YFV even though it was slightly cytotoxic to Vero cells. Moreover, GRL-002-16 was inhibitory to YFV while ineffective against DENV, whereas, GRL-016-16 had the opposite effect. Our results reveal the differential efficacies of the small molecule inhibitors targeting N-7-MTase. The experimental data suggests these compounds have different cytotoxicities in different cell lines and the compounds act in a virus-specific way. Nonetheless, we were able to shortlist some potent compounds for future modifications.</p>