Browsing by Author "Haystead, Timothy A J"

Anticoagulant agents are commonly used drugs to reduce blood coagulation in acute and chronic clinical settings. Many of these drugs target the common pathway of coagulation because it is critical for thrombin generation and disruption of this portion of the pathway has profound effects on the hemostatic process. Currently available drugs for these indications struggle with balancing desired activity with immunogenicity and poor reversibility or irreversibility in the event of hemorrhage. While improvements are being made with the current drugs, new drugs with better therapeutic indices are needed for surgical intervention and chronic indications to prevent thrombosis from occurring.

A class of therapeutics known as aptamers may be able to meet the need for safer anticoagulant agents. Aptamer are short single-stranded RNA oligonucleotides that adopt specific secondary and tertiary structures based upon their sequence. They can be generated to both enzymes and cofactors because they derive their inhibitory activity by blocking protein-protein interactions, rather than active site inhibition. They inhibit their target proteins with a high level of specificity and bind with high affinity to their target. Additionally, they can be reversed using two different antidote approaches, specific oligonucleotide antidotes, or with cationic, “universal” antidotes. The reversal of their activity is both rapid and durable.

The ability of aptamers to be generated to cofactors has been conclusively proven by generating an aptamer targeting the common pathway coagulation cofactor, Factor V (FV). We developed two aptamers with anticoagulant ability that bind to both FV and FVa, the active cofactor. Both aptamers were truncated to smaller functional sizes and had specific point mutant aptamers developed for use as controls. The anticoagulant activity of both aptamer-mutant pairs was characterized using plasma-based clotting assays and whole blood assays. The mechanism of action resulting in anticoagulant activity was assessed for one aptamer. The aptamer was found to block FVa docking to membrane surfaces, a mechanism not previously observed in any of our other anticoagulant aptamers.

To explore development of aptamers as anticoagulant agents targeting the common pathway for surgical interventions, we fused two anticoagulant aptamers targeting Factor X and prothrombin into a single molecule. The bivalent aptamer was truncated to a minimal size while maintaining robust anticoagulant activity. Characterization of the bivalent aptamer in plasma-based clotting assays indicated we had generated a very robust anticoagulant therapeutic. Furthermore, we were able to simultaneously reverse the activity of both aptamers with a single oligonucleotide antidote. This rapid and complete reversal of anticoagulant activity is not available in the antithrombotic agents currently used in surgery.

During oncogenesis, cancer cells go through metabolic reprogramming to maintain their high growth rates and adapt to changes in the microenvironment and the lack of essential nutrients. Several types of cancer are dependent on de novo fatty acid synthesis to sustain their growth rates by providing precursors to construct membranes and produce vital signaling lipids. Fatty acid synthase (FASN) catalyze the terminal step of de novo fatty acid synthesis and it is highly expressed in many types of cancers where it’s up-regulation is correlated with cancer aggressiveness and low therapeutic outcome. Many FASN inhibitors were developed and showed potent anticancer activity however, only one inhibitor advanced to early stage clinical trials with some dose limiting toxicities. Using a modified fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, we identified HS-106, a thiophenopyrimiden FASN inhibitor that has anti-neoplastic activity against breast cancer in vitro and in vivo. HS-106 was able to inhibit both; purified human FASN activity and cellular fatty acid synthesis activity as evaluated by radioactive tracers incorporation into lipids experiments. In proliferation and apoptosis assays, HS-106 was able to block proliferation and induce apoptosis in several breast cancer cell lines. Several rescue experiment and global lipidome analysis were performed to probe the mechanism by which HS-106 induces apoptosis. HS-106 was found to induce several changes in lipids metabolism: (i) inhibit fatty acids synthesis. (ii) Inhibit fatty acids oxidation as indicated by the ability of inhibiting Malonyl CoA accumulation to block HS-106 induced apoptosis and the increase in the abundance of ceramides. (iii) Increase fatty acids uptake and neutral lipids formation as confirmed 14C Palmitate uptake assay and neutral lipids staining. (iv)Inhibit the formation of phospholipids by inhibiting de novo fatty acid synthesis and diverting exogenous fatty acids to neutral lipids. All of these events would lead to disruption in membranes structure and function. HS-106 was also tested in Lapatinib resistant cell lines and it was able to induce apoptosis and synergizes Lapatinib activity in these cell lines. This may be due the disruption of lipid rafts based on the observation that HS-106 reduces the expression of both HER2 and HER3. HS-106 was found to be well tolerated and bioavailable in mice with high elimination rate. HS-106 efficacy was tested in MMTV neu mouse model. Although did not significantly reduced tumor size (alone), HS-106 was able to double the median survival of the mice and showed potent antitumor activity when combined with Carboplatin. Similar results were obtained when same combinations and dosing schedule was used in C3Tag mouse model except for the inability of HS-106 affect mice survival.

From the above, HS-106 represent a novel FASN inhibitor that has anticancer activity both in vivo and in vitro. Being a chemically tractable molecule, the synthetic route to HS-106 is readily adaptable for the preparation of analogs that are similar in structure, suggesting that, the pharmacological properties of HS-106 can be improved.

Despite its ubiquitous abundance, Hsp90 inhibitors have shown promise in anti-cancer clinical trials, suggesting that Hsp90 inhibitors selectively target tumor cells while exhibiting minimal effects in normal cells. Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is strongly correlated with malignancy. Development of small molecule probes that can specifically detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a fluorescent cell impermeable Hsp90 inhibitor, HS-131, to target eHsp90 in vivo. HS-131 was characterized biochemically to ensure specificity for eHsp90, and an inactive analog was also synthesized to be used as an in vivo control.

Through confocal microscopy, eHsp90 can be visualized with cell impermeable, fluorophore-tagged Hsp90 inhibitors. High resolution confocal and real time lattice light sheet microscopy showed that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is subsequently actively internalized. This internalization occurs in the presence and absence of inhibitors. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by over-expressing p110HER2, leading to malignant transformation of these cells. Internalization of eHsp90 is also increased after inhibition of Hsp70, suggesting that overcompensation of the heat shock response can also upregulate the eHsp90 trafficking mechanism. Whole body 3D cryo fluorescence imaging and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression is a unique phenomenon in vivo.

Taken together, these results suggest that active and differential internalization of eHsp90 in aggressive cancer cells contributes to the selectivity observed upon Hsp90 inhibitor treatment and may provide a novel metastatic biomarker for solid tumors and may lead to the development of a tumor-specific drug delivery system.

Diseases caused by smooth muscle dysfunction such as hypertension and asthma are major public health concerns, a better understanding of the signaling pathways that regulate smooth muscle contraction could identify new drug targets. The opposing effects of two enzymes; calcium/calmodulin regulated myosin light chain kinase (MLCK) and smooth muscle myosin phosphatase (SMPP-1M) determine the amount of force generated by smooth muscle. The calcium-independent signaling mediated by myosin phosphatase is regulated by several kinases which include zipper interacting protein kinase (ZIPK). Our laboratory has shown that ZIPK is able to phosphorylate and inhibit SMPP-1M which results in increased smooth muscle contraction. Additional studies demonstrated that ZIPK is also regulated by phosphorylation. The goal of this study is to identify kinases in the context of smooth muscle that regulate ZIPK and to define the events required for ZIPK activation.

A proteomic approach which employed ATP-affinity chromatography coupled with mass spectrometry isolated discreet kinase activities towards ZIPK, these activities were attributed to integrin-linked kinase (ILK) and Rho kinase 1 (ROCK1). ILK phosphorylates ZIPK at Thr180 while ROCK1 phosphorylates ZIPK at Thr265 and Thr299.

Additionally the ATP-affinity media used for kinase enrichment in the proteomic screen was used as a tool to measure ZIPK activation. Pre-incubating ZIPK with ROCK before the assay resulted in increased binding which suggests phosphorylation of ZIPK by ROCK is activating. Increasing the substrate concentration in the assay resulted in increased ZIPK binding, this result was only observed when the assay was performed with the full-length protein. Phosphorylation of residues in the kinase domain along with substrate binding relieves inhibition and results in kinase activation.

Finally fluorescence microscopy along with targeted mutations of ZIPK was used to determine the mechanism of cellular transport. This was done to address the difference in cellular localization between human and murine cells. The localization of human ZIPK is dictated by nuclear localization sequence 2 (NLS2) and the phosphorylation state of Thr299; the mechanism is not shared by the murine form of ZIPK.

Completion of this work has provided additional information about the signaling interactions that take place in smooth muscle; the results suggest that ZIPK is a convergence point for multiple signaling pathways that lead to SMPP-1 inhibition and subsequently smooth muscle contraction. This study also contributes significantly to our knowledge of the molecular dynamics that lead to active full length ZIPK. Future research that employs animal modeling as a tool to investigate ZIPK will be informed by the experiments that address the cellular localization of ZIPK.

Chronic pain is a prevalent health concern, affecting up to 100 million people in the US alone, yet treatment options remain limited. The origins of pain have been tightly linked to inflammation and in specific tumor necrosis factor (TNF) a pro-inflammatory cytokine, has been identified as a key driver of pain and inflammation. TNF can bind to its TNFR1 receptor located on the terminals of primary afferent nociceptors to directly increase their activity. In addition, TNF can stimulate pro-inflammatory cytokine production and immune cell activation. Patients with chronic pain exhibit increased levels of TNF, with higher levels in those with greater pain. TNF antibodies (eg, Remicade, Humira and Cimzia) are currently available for the treatment of inflammatory pain conditions such as rheumatoid arthritis. However, 40% of patients fail to respond to TNF antibodies, leading for the need to develop novel small molecule TNF inhibitors. TGFβ-activated kinase (TAK1) inhibition has been previously shown to potently reduce TNF signaling. Takinib, a potent inhibitor of TAK1 represents a novel method of regulating TNF production and pro-inflammatory signaling. Here, I show that TAK1 inhibition prevents inflammatory, neuropathic and functional pain by modulating immune responses and nociceptor activity. Overall, our findings support the therapeutic potential of a TAK1 inhibitor as a novel therapeutic to reduce inflammation and chronic pain.

On the surface heat shock protein 90 (Hsp90) is an unlikely drug target for the treatment of any disease, let alone cancer. Hsp90 is highly conserved and ubiquitously expressed in all cells. There are four major isoforms encoded by distinct genes and together they may constitute 1-3% of the cellular protein. Genetic deletion results in nonviable phenotypes in some organisms, and there are no recognized polymorphisms suggesting an association or causal relationship with any human disease. With respect to cancer, the proteins absence from some recent high profile articles underlines the perception that it is an unlikely bona fide target to treat this disease. Yet, to date, there are 17 distinct Hsp90 inhibitors in clinical trials for multiple indications in cancer. The protein has been championed for over 20 years by the National Cancer Institute as a cancer target since the discovery of the antitumor activity of geldanamycin. Rather than focus on the intracellular inhibition of Hsp90, we have shifted our aim to the differences of Hsp90 between cancer and normal tissue, namely its extracellular expression.

My graduate thesis work has focused on the characterization of a series of novel small molecule imaging agents (fluor-tethered Hsp90 inhibitors) that enable the specific detection of ectopically expressed Hsp90 on tumor cells. We believe that these molecules will have a large impact in the near future on the diagnosis and treatment of metastatic breast cancer as well as other cancers. This hypothesis is based on recent findings in the clinical literature that have linked upregulation of Hsp90 with poor outcomes in multiple subtypes of breast cancer. Additionally, several papers have also reported an association of the expression of extracellular Hsp90 and metastatic progression in several human cancers. Hsp90 is currently considered by some as a cutting edge cancer drug target. The Haystead lab synthesized a series of tethered Hsp90 inhibitors that were modified with fluorophores and other imaging moieties in such a way as to preserve the binding to Hsp90 and enable detection through non-invasive imaging techniques. In a series of cell-based, live animal and biochemical studies we demonstrated that these molecules are highly selective for Hsp90 and can be used to specifically recognize intact tumor cells expressing ectopic Hsp90. Furthermore, we also observed that once bound to ectopic Hsp90, our tethered-inhibitors are actively internalized and this process can be blocked with Hsp90 antibodies. These findings have two implications; first, Hsp90 is undergoing active cycling at the plasma membrane; second, the finding that once bound to surface Hsp90 our fluor-tethered inhibitors can be internalized despite their polar nature. These results suggest a new therapeutic strategy that will enable specific delivery of tumor killing agents (e.g. 131I or metabolic poisons) to metastatic cells. This is unique because the use of small molecule inhibitors and not antibody- or nanoparticle-based payload delivery strategies offers advantages in formulation, cost and reproducibility.

In addition to payload delivery possibilities, we also show the utility of the tethered-inhibitors diagnostically by demonstrating their use in the detection of tumors in mouse models of human breast cancer. As a result of our animal studies, we believe our molecules in their present form could be used to address a currently unmet need in the early diagnosis of aggressive breast cancer and discriminating this from more indolent forms.

Furthermore, the tethered Hsp90 inhibitors have been used to make ligand affinity chromatography resins that have facilitated the discovery of other unique Hsp90 expressions and functions associated with cancer. We have found a pool of Hsp90 that is misfolded as determined by affinity chromatography depletion and a leftward thermal stability shift in the population of Hsp90 that flows through the ligand affinity resins. Differential trypsin digest patterns detected by mass spectrometry reveal also that the native protein has sites that are more accessible to trypsinization. This could have further implications in treating and detecting differences between cancerous tissues and normal tissues by designing an antibody that recognizes the exposed portions of the misfolded Hsp90. Together this body of work illustrates that not only is Hsp90 different in total expression levels in cancers, but is ectopically expressed and misfolded so as to provide other opportunities for therapeutic intervention that improve the safety for more clinical applications.

Inducible Heat shock protein (Hsp70i) is a protein chaperone that is utilized during tumorigenesis and viral infections for efficient propagation. Overexpression of Hsp70i is observed in a wide spectrum of human tumors, and this overexpression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Hsp70i aids in cancer cell propagation through regulation of anti-apoptotic and cell survival pathways. Furthermore, Hsp70i is induced following infection for several viruses and aids viral propagation, in part through regulation of anti-apoptotic pathways as well as promoting the folding of newly synthesized proteins. Due to the parallel role of Hsp70i in both cancer and viral pathogenesis, identification of small-molecule inhibitors selective for Hsp70i could provide tools for the development of novel therapeutics and further elucidate the role of Hsp70i in both cancer and viral infections.

To date, few Hsp70 inhibitors have been identified and characterized, and their efficacy in clinical settings is unknown. Through the fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, an allosteric inhibitor selective for Hsp70i was identified, called HS-72. We show that HS-72 is highly selective for Hsp70i, over the broader purinome and other Hsp70 family members, in particular the closely related constitutively active Hsp70 family member, Hsc70. Additionally, HS-72 acts as an allosteric inhibitor to induce a conformational change and inhibit Hsp70i activity. HS-72 displays hallmarks of Hsp70i inhibition in vitro by promoting Hsp70i substrate protein degradation, protein aggregation, and selective growth inhibition of cancer cells. In wild type mice HS-72 is well tolerated and a limited PK study shows HS-72 is bioavailable. Furthermore, in a MMTV-neu breast cancer mouse model, HS-72 shows efficacy to inhibit tumor growth and promote survival.

Due to the similar utilization of Hsp70i in cancer and viral pathogenesis, this suggests the potential for HS-72 as an antiviral agent. Dengue virus (DENV) is of great public health importance due to estimates of up to 400 million infections per year, coupled with the geographic distribution of the virus, which is now endemic in over 100 countries worldwide. There is also a pressing need for DENV interventions, owing to the lack of approved vaccines or antiviral therapies. DENV is reliant on host factors throughout the viral life cycle and Hsp70i has been implicated as a host factor in DENV pathogenesis. Additionally, the complete role of Hsp70i in DENV pathogenesis remains to be elucidated, highlighting a unique opportunity to use HS-72 as a tool to specifically probe Hsp70i function. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Furthermore, inducing Hsp70i expression prior to infection, correlates with an increase in DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained, through inhibiting the entry stage of the viral life cycle. Following infection, Hsp70i localizes to the cell surface and interacts with the DENV receptor complex to mediate viral entry. While, HS-72 treatment results in a disruption of the interaction of Hsp70i with the DENV receptor complex, yielding a reduction in infected cells.

Collectively this work further supports Hsp70i as an anticancer and anti-dengue virus target, and identifies HS-72, a chemical scaffold that is amenable to resynthesis and iteration, as an ideal starting point for a new generation of therapeutics targeting Hsp70i.

Tumor necrosis factor  (TNF) has positive and negative roles in human disease. In certain cancers, TNF is infused locally to promote tumor regression, but dose-limiting inflammatory effects limit broader utility. In autoimmune disease, anti-TNF antibodies control inflammation in most patients, but these benefits are offset by cost and tachyphylaxis that develops during chronic treatment. Transforming growth factor beta-activated kinase 1 (TAK1) acts as a key mediator between survival and cell death in TNF-mediated signaling, uniquely providing a drug development opportunity for cancer and autoimmunity. Takinib is a potent and selective TAK1 inhibitor (IC50 9.5nM) that induces apoptosis in a TNF-dependent manner in cell models of metastatic breast cancer and rheumatoid arthritis. The mechanisms underlying this specificity were revealed in enzymatic and co-crystallization studies. These data show that Takinib targets the kinase in the DFG-in conformation and forms direct and water-mediated hydrogen bonds with catalytic residues. Mechanistic studies of TAK1 autophosphorylation demonstrated a substrate-like intermolecular mechanism, during which Takinib treatment slows down the rate-limiting step. Overall, our data show Takinib is an attractive starting point for the development of inhibitors that greatly sensitize cells to TNF-induced cell death, broadening the therapeutic efficacy of TNF for cancer and autoimmune disease.

Dengue viruses (DENV) are mosquito‐borne flaviviruses that pose a continued and growing threat to global health. There are estimated to be 390 million DENV infections each year, and because there is no vaccine or approved therapeutic treatment, developing a small‐molecule treatment is imperative. Possible small‐molecule drug therapies for DENV could be immune system modulators, inhibitors of DENV‐required host factor, or inhibitors of a viral gene product. In this study, we chose to take the latter approach and focused our drug discovery efforts on the most highly conserved flaviviral protein, non‐structural protein 5 (NS5). NS5 contains two major domains, each with different enzymatic activities. The N‐terminus has methyltransferase activity, and the C terminus, an RNA‐dependent RNA polymerase (RdRp). The activities of both domains are purine‐dependent, and therefore both domains contribute to the purine‐binding properties of NS5. Inhibition of either of these domains in NS5 results in inadequate propagation of DENV, and the purine‐binding domains present ideal drug targets for disrupting these activities. These factors make NS5 protein an ideal candidate target for our small‐molecule library screen.

A high‐throughput fluorescence‐based screen was employed to identify anti‐DENV compounds based on their ability to competitively bind NS5. The screen was performed by binding green fluorescent protein NS5 fusion protein (GFP‐NS5) to immobilized ATP resin, and then performing parallel elutions using over 3,000 distinct compounds. One compound in particular, HS‐205020, was able to competitively elute GFP‐NS5 from the ATP resin and also exhibited antiviral activity in both the U937+DCSIGN human monocyte cell line and BHK‐21 cells. Additionally, HS‐205020 was able to inhibit DENV NS5 RNA polymerase activity in vitro. HS‐205020 is chemically distinct from the majority of previously reported NS5 inhibitors, which are nucleoside analogs that can cause severe toxicity in animal studies. In contrast, over the concentration range that produced anti‐DENV effects, HS‐205020 showed comparable viabilities to ribavirin, an FDA approved hepatitis C virus (HCV) therapeutic. These findings support HS‐205020 as a potential dengue antiviral candidate, and its chemical scaffold represents as an ideal starting compound for future structure‐activity relationship studies.