Cellular damage and deregulated apoptotic cell death lead to functional impairment, and a main consequence of these events is aging. Cellular damage is initiated by different stress/risk factors such as oxidative stress, inflammation, and heavy metals. These stress/risk factors affect the cellular homeostasis by altering methylation status of several aging and Alzheimer’s disease associated genes; these effects can be manifested immediately after exposure to stress and at later stages of life. However, when cellular damage exceeds certain threshold levels apoptosis is initiated. This review discusses the stress factors involved in cellular damage and the role and potential of TSPO-mediated cell death in aging as well as in Alzheimer’s disease, which is also characterized by extensive cell death. Mitochondrial-mediated apoptotic death through the release of cytochrome c is regulated by TSPO, and increased expression of this protein is observed in both elderly people and in patients with Alzheimer’s disease. TSPO forms and mediates opening of the mitochondrial membrane pore, mPTP and oxidizes cardiolipin, and these events lead to the leakage of apoptotic death mediators, such as cytochrome c, resulting in cell death. However, TSPO has many proposed functions and can also increase steroid synthesis, which leads to inhibition of inflammation and inhibition of the release of apoptotic factors, thereby decreasing cell damage and promoting cell survival. Thus, TSPO mediates apoptosis and decreases the cell damage, which in turn dictates the process of aging as well as the functionality of organs such as the brain. TSPO modulation with ligands in the Alzheimer’s disease mouse model showed improvement in behavioral symptoms, and studies in Drosophila species showed increased cell survival and prolonged lifespan in flies after TSPO inhibition. These data suggest that since effects/signs of stress can manifest at any time, prevention through change in lifestyle and TSPO modulation could be potential strategies for altering both the aging process and the progression of Alzheimer’s disease.

Alzheimer’s disease, the most common type of dementia, is a progressive brain disease that destroys cognitive function and eventually leads to death. In patients with Alzheimer’s disease, beta amyloids and tau proteins form plaques/oligomers and oligomers/tangles that affect the ability of neurons to function properly. Heat shock protein 70 (HSP70) has the ability to prevent aggregation/oligomerization of beta amyloid/tau proteins, making it a potential drug target. To determine this potential, it is essential that we have appropriate in vitro and cell-based assays that help identify specific molecules that affect this aggregation or oligomerization through HSP70. Potential drug candidates could be identified through a series of assays, starting with ATPase assays, followed by aggregation assays with enzymes/proteins and cell-based systems. ATPase assays are effective in identification of ATPase modulators but do not determine the effect of the molecule on beta amyloid and tau proteins. Molecules identified through ATPase assays are validated by thioflavin T aggregation assays in the presence of HSP70. These assays help uncover if a molecule affects beta amyloid and tau through HSP70, but are limited by their in vitro nature. Potential drug candidates are further validated through cell-based assays using mammalian, yeast, or bacterial cultures. However, while these assays are able to determine the effect of a specific molecule on beta amyloid and tau, they fail to determine whether the action is HSP70-dependent. The creation of a novel, direct assay that can demonstrate the antiaggregation effect of a molecule as well as its action through HSP70 would reduce the number of false-positive drug candidates and be more cost-effective and time-effective.

Thymidylate synthase (TS) is a well-validated cancer target that undergoes conformational switching between active and inactive states. Two mutant human TS (hTS) proteins are predicted from crystal structures to be stabilized in an inactive conformation to differing extents, with M190K populating the inactive conformation to a greater extent than A191K. Studies of intrinsic fluorescence and circular dichroism revealed that the structures of the mutants differ from those of hTS. Inclusion of the substrate dUMP was without effect on M190K but induced structural changes in A191K that are unique, relative to hTS. The effect of strong stabilization in an inactive conformation on protein phosphorylation by casein kinase 2 (CK2) was investigated. M190K was highly phosphorylated by CK2 relative to an active-stabilized mutant, R163K hTS. dUMP had no detectable effect on phosphorylation of M190K; however, dUMP inhibited phosphorylation of hTS and R163K. Studies of temperature dependence of catalysis revealed that the E act and temperature optimum are higher for A191K than hTS. The potency of the active-site inhibitor, raltitrexed, was lower for A191K than hTS. The response of A191K to the allosteric inhibitor, propylene diphosphonate (PDPA) was concentration dependent. Mixed inhibition was observed at low concentrations; at higher concentrations, A191K exhibited nonhyperbolic behavior with respect to dUMP and inhibition of catalysis was reversed by substrate saturation. In summary, inactive-stabilized mutants differ from hTS in thermal stability and response to substrates and PDPA. Importantly, phosphorylation of hTS by CK2 is selective for the inactive conformation, providing the first indication of physiological relevance for conformational switching.

Inhibitors of the enzyme thymidylate synthase (TS) are typically used as anti-cancer drugs to limit the growth of cancer cells. These inhibitors deplete TTP which is essential for DNA replication and repair. The enzyme in humans is postulated to undergo conformational switching between active and inactive forms. We are testing the hypothesis that stabilization of the inactive conformation will lead to increased turnover of hTS. Stabilization of hTS to degradation is observed after exposure to TS inhibitors in clinical use, which are active site-targeted. X-ray crystallographic studies of 1,3-propanediphosphonic acid (PDPA) bound to hTS led to the prediction that PDPA stabilizes the inactive conformation. Furthermore, PDPA exhibited competitive-noncompetitive behavior in TS catalytic reactions, consistent with allosteric inhibition. A cell-permeable isostere of PDPA inhibited hTS in catalytic assays, inhibited cell proliferation in a thymidine-dependent manner, and induced down regulation of hTS in cultivated cells. This is the first TS inhibitor to decrease the stability of hTS to degradation. To identify lead compounds that bind to the inactive conformation, molecular docking and structure-based virtual screening techniques were applied. Putative interaction sites were obtained from crystal structures of liganded hTS (with PDPA) and docked with 10,000 compounds using Ligand Fit (Drug Discovery Studio). Lead molecules with highest binding scores were selected and their efficacy analyzed in in vitro catalytic assays with hTS. Several compounds showed 30% – 50% inhibition of TS activity at 10 μM concentration. These derivatives were more potent than PDPA and inhibited the growth of cells expressing hTS, with IC50s of 100 µM – 200 µM. Data derived from computational and in vitro experiments are being employed in iterative cycles in the design of more potent, allosteric TS inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1348. doi:10.1158/1538-7445.AM2011-1348

The nucleotide stress response (NSR) is defined as activation of nucleoside salvage pathways to rescue cells from the detrimental consequences caused by a deficiency of or increased demand for nucleotides. Thymidylate depletion due to loss of function of the enzyme, thymidylate synthase (TS) or exposure to TS inhibitors disrupts the balance in nucleotide pools and damages DNA leading to stress-induced cell death. In order to replete nucleotide pools, cells activate nucleoside (ENT 1) transporters to increase the influx of extracellular nucleosides. TS catalyzes the de-novo synthesis of thymidylate for DNA replication. The enzyme in humans undergoes conformational switching between active and inactive forms. We are testing the hypothesis that stabilization of the inactive conformation will not induce the resistance mechanisms that are observed after exposure to active site-targeted TS inhibitors. To identify lead compounds that bind to the inactive conformation, a NSR assay has been developed, which measures a stress response to TMP depletion. TS inhibitors that bind to either the active or inactive conformations will induce TMP stress which is measured as induction of uptake of a fluorescent probe, N2, N3-etheno-6-thiomethylpurine riboside (ETMPR). In order to differentiate TS inhibitors that are selective for active and inactive conformations, we conducted an experiment on paired isogenic cell lines that express either wild-type hTS (undergoes conformational switching) or a mutant hTS (R163K - stabilized in the active conformation). Cells exposed to the potent active site TS inhibitor, raltitrexed (RTX) exhibited a similar level of uptake of ETMPR. When cells are exposed to glutarate (inactive site inhibitor predicted by structure-based drug design), fluorescent nucleoside uptake was preferentially induced in cells expressing wild-type hTS, consistent with the prediction that glutarate is an inactive site targeted drug. Protein turnover studies revealed that glutarate decreases TS expression, which is opposite to the effect of active TS inhibitors, which stabilize TS to degradation. Collectively, the data indicate that the NSR assay will be useful in identifying lead inactive site-targeted drugs and that these drugs will not stabilize TS, a mechanism thought to cause resistance to active state TS inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5508.