Abstract Background: Although oxidative phosphorylation (OXPHOS) is the main ATP source in primary prostate cancer, as the tumor progresses to castration resistance and/or metastatic disease, glycolysis becomes increasingly prevalent. Dependence on glycolysis can further increase under hypoxic conditions found in advanced cancers. We previously demonstrated that a novel small molecule, BKIDC-1553, inhibits proliferation and tumor growth of prostate cancer cell lines and xenografts through a hexokinase-2 (HK2) dependent inhibition of glycolysis. HK2 is a glucose 6-phosphorylating enzyme that works in conjunction with glucose transporter-1 (GLUT1) to drive glycolysis in cells. While our previous work suggests that BKIDC-1553 works primarily through a HK2 dependent mechanism, the objective of this current project is to further examine how expression and function of HK2 and GLUT1 are altered in response to BKIDC-1553 treatment. Methods: Hypoxic conditions are common in late-stage prostate cancer. Effects of hypoxia on cell proliferation and expression levels of HK1, HK2, and GLUT1 were examined with and without BKIDC-1553 treatment. To examine role of HK1, HK2, and GLUT1 in response to BKIDC-1553 treatment, we created CRISPR knockouts and shRNA knockdowns of HK1 and HK2 in the LNCaP prostate cancer cell line. GLUT1 was inhibited using a commercially available inhibitor, BAY-876. PCR and Western blots were used to confirm expression levels of HK1, HK2, and GLUT1. MTS proliferation assays were used to examine effect of KO and KD on response to BKIDC-1553 and BAY-876. Results: GLUT1 levels, but not HK1 or 2, increased at 72 hrs under hypoxic conditions. HK1, HK2, and GLUT1 levels all increased in cells in response to 144 hrs of BKIDC-1553 compared with 72 hrs treatment in both normoxic and hypoxic conditions. The KO and KD studies were all conducted in normoxic conditions. Westerns blots demonstrated that HK2 KO, but not HK1 KO, results in increased levels of GLUT1. HK1 or HK2 KD, however, did not alter GLUT1 levels. HK2 KD cells, with GLUT1 levels remaining the same, displayed less growth inhibition than control cells in response to BKIDC-1553 treatment, while HK2 KO cells, with increased levels of GLUT1, remained sensitive to BKIDC-1553. Because GLUT1 levels increase in response to HK2 KO, we wanted to see if inhibiting GLUT1 in these cells could decrease their proliferation. We found that KO cells were indeed more sensitive to growth inhibition of BAY-876. Conclusion/Summary: These studies indicate that KO of HK2 increases levels of GLUT1. These cells remain sensitive to BKIDC-1553 treatment, which could in part be due to the cells' continued reliance on glycolysis through increased GLUT1. A continued reliance on glycolysis allows BKIDC-1553 to continue to inhibit cell growth. Future studies examining how combination treatment with BKIDC-1553 and BAY-876 affects growth of advanced prostate cancer cells is underway. Citation Format: Cynthia Sprenger, Mika Munari, Shihua Sun, Kathryn Soriano Epilepsia, Stephen Plymate. Alterations in glycolytic pathway enzymes and metabolic plasticity in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 448.

The androgen receptor (AR) is the major driver of prostate cancer (PCa) adenocarcinoma. For PCa cells to proliferate, metabolic programs e.g. glycolysis, lipogenesis, oxidative phosphorylation need to be activated to supply the proliferating cells with key anabolic elements. It has recently been shown that glycolysis and enzymes supportive of this pathway including GLUT 1, hexokinase 2 (HK2), and phosphofructokinase (PFK2) are directly upregulated by the AR in castrate resistant prostate cancer (CRPC). HK2 is of particular interest as a target in PCa since, although it is present in fetal tissues, it is suppressed in adult tissues and its function in normal tissues (formation of glucose 6-phosphate) is maintained by hexokinase 1(HK1); however, HK2 expression is markedly increased in AR-driven CRPC in part by an increase in hypoxia-associated HIF1a. Thus HK2 may be an important target in CRPC. In this study we present results of a small molecules, SGI-1553 and SGI-1676 based on a pyrrolopyrimidine-background developed at the University of Washington, that inhibit growth of AR positive cell lines including LNCaP, LNCaP95, VCaP, and LaPC4 but have no effect on PC-3 and DU-145 AR-negative lines. We show that the SGIs specifically inhibit HK2 activity but do not affect HK1. The effects on glycolysis were demonstrated by the suppression of the ECAR using a SeaHorseFX96 instrument. Metabolic flux assay with D-[U-13C] glucose demonstrated reduction in levels of downstream metabolites dihydroxyacetone phosphate (DHAP), phosphoglycerate (PG), phosphoenolpyruvate (PEP), and pyruvate. Indicating a block at either glucose uptake (GLUT1) or HK. We subsequently used two different radiolabeled glucose analogs [3H] 2DG and [3H] 3-O-methyl D-glucose (3-OMG) to demonstrate SGIs inhibit HK. CRISPr KO assays demonstrated that HK2 not HK1 activity on ATP generation as a measure of activity was suppressed by our SGIs. Since the effects of the SGIs were most marked in AR-positive lines and were demonstrated not to interact directly with AR, we examined downstream AR signaling with a probasin-luciferase reporter assay and showed marked suppression of AR R1881 induced activity in LNCaP cells, p <0.001. RNA seq studies also demonstrated suppression of downstream AR-genes. CHiP-seq studies were done to identify effects on AREs. Since SGI-1553 is orally bioavailable in mice with >24 hr half -life and no toxicity at therapeutic doses (20mg/kg three time a week), we treated mice bearing the LuCaP 35 AR-driven PDX model, or PC-3 and DU-145 AR-negative xenografts. There was marked suppression of LuCaP 35 growth (p<0.001) but no effect on PC-3 orDU-145 growth compared to control. Conclusion: SGIs-1553 and 1676 target glycolysis and suppress AR signaling in AR-driven PCa cell lines and PDX models, in part, by inhibition of inhibition of HK2 activity. SGI-1553 is orally bioavailable with an excellent PK profile and no observed toxicity at therapeutic doses in mice, rats and dogs and thus represents the first in-class therapy to target glycolysis in PCa. Citation Format: Takuma Uo, Cynthia Sprenger, Ian Sweet, Dustin Maly, Ilsa Coleman, Shihua Sun, Kayode Ojo, Mika Munari, Soojin Kim, Kathryn Epilepsia, Gayani Perera, Wanting Han, Peter Nelson, Ryan Choi, Matt Nguyen, Matt Hulverson, Wesley van Voorhis, Stephen Plymate. Inhibition of androgen receptor signaling in castrate resistant prostate cancer in association with inhibition of glycolysis by targeting hexokinase 2 activity with pyrrolopyrimidine-based small molecules [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR015.

Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in bothE. coliand mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.

Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co(2+) bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.

Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compounds found in garlic, onion, mustard and cinnamon extracts, activation of TRPV1 by these extracts remains controversial. Here we establish that TRPV1 is activated by pungent extracts from onion and garlic, as well as by allicin, the active compound in these preparations, and participates together with TRPA1 in the pain-related behavior induced by this compound. We found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects we observed. Our findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the molecular basis of noxious stimuli detection.

TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159-212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487-517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature. 389:816-824). When activated, they produce depolarization through the influx of Na+, but their high Ca2+ permeability is also important for mediating the response to pain. In particular, Ca2+ influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Neuroscience. 55:1015-1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525-3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, Ca2+/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg2+, reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by Ca2+ alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca2+. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca2+-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH2-terminal domain demonstrated that a stretch of approximately 30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca2+-dependent manner. The physiological response to pain involves an influx of Ca2+ through TRPV1. Our results indicate that this Ca2+ influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin.

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca2+. Not surprisingly, they are blocked by a number of Ca2+ channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca2+-activated K+ channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.