Scientific evidence suggests that quercetin (QUR) has anxiolytic-like effects in experimental animals. However, the mechanism of action responsible for its anxiolytic-like effects is yet to be ...discovered. The goal of this research is to assess QUR’s anxiolytic effects in mouse models to explicate the possible mechanism of action. After acute intraperitoneal (i.p.) treatment with QUR at a dose of 50 mg/kg (i.p.), behavioral models of open-field, hole board, swing box, and light–dark tests were performed. QUR was combined with a GABAergic agonist (diazepam) and/or antagonist (flumazenil) group. Furthermore, in silico analysis was also conducted to observe the interaction of QUR and GABA (α5), GABA (β1), and GABA (β2) receptors. In the experimental animal model, QUR had an anxiolytic-like effect. QUR, when combined with diazepam (2 mg/kg, i.p.), drastically potentiated an anxiolytic effect of diazepam. QUR is a more highly competitive ligand for the benzodiazepine recognition site that can displace flumazenil (2.5 mg/kg, i.p.). In all the test models, QUR acted similar to diazepam, with enhanced effects of the standard anxiolytic drug, which were reversed by pre-treatment with flumazenil. QUR showed the best interaction with the GABA (α5) receptor compared to the GABA (β1) and GABA (β2) receptors. In conclusion, QUR may exert an anxiolytic-like effect on mice, probably through the GABA-receptor-interacting pathway.
Purpose
Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the ...highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of
GABRA5
, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABA
A
R). We are advancing a therapeutic approach for group 3 based on GABA
A
R modulation using benzodiazepine-derivatives.
Methods
We performed analysis of
GABR
and
MYC
expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABA
A
R in group 3 cells.
Results
Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of
GABR
genes, which code for GABA
A
R subunits α5, β3 and γ2 and 3. There are ~ 1000 functional α5-GABA
A
Rs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 10
9
ions/s. Benzodiazepines, designed to prefer α5-GABA
A
R, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABA
A
R (0.8 µM EC
50
) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response,
TP53
upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization.
Conclusion
GABRA5
expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABA
A
R is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.
GABAARs (gamma-aminobutyric acid type A receptors) are transmembrane pentameric ligand-gated chloride ion channels that respond to GABA, the central nervous system's principal inhibitory ...neurotransmitter (CNS). The benzodiazepines (BZDs) bind between the GABAAR α+γ2-subunits at their extracellular interface. The binding of ligands to distinct subunits of GABAA receptors, notably the α1-6β2/3γ2 ion channels, can have a wide range of effects on brain activities. The sedative, ataxic, amnesic, anticonvulsant, and addictive actions of GABAARs' α1-subtype selective ion channels should be avoided, except for the anticonvulsant and anxiolytic effects, while creating ligands for this BZ allosteric modulatory site. Many studies have linked the α2/3-containing GABAARs to anxiolytic, anticonvulsant, and antinociceptive properties. Muscle relaxation may be mediated by interaction of α3 subtypes at higher doses. GABAARs that include the α5 subtype are known to play a role in cognition, learning, and memory. GABA activity disruption at α5 GABAAR subtypes plays a role in the pathophysiology of CNS illnesses such schizophrenia, major depressive disorder (MDD), bipolar disorder, and some anxiety disorders such as OCD.Medulloblastoma is the most common pediatric brain tumor. There are four subgroups of medulloblastoma: WNT, SHH, Group 3 and Group 4. Group 3 has the highest morbidity rate, relapse and metastasis rate. The standard treatment of medulloblastoma includes surgical removal of the tumor followed by radiation and chemotherapy which cause unwanted side effects such hearing impairment, permanent damage to the endocrine system and neurocognitive functions and secondary tumors. Better treatments of medulloblastoma are needed. In group 3 medulloblastoma tumors they show a high expression of GABRA5 receptors, which is the α5 subunit of ligand gated ionotropic γ-aminobutyric acid type A receptors. In recent times, there is a lot of evidence published on the role of ion channel activity on brain cancer progression. Collaborating with Dr. Sengupta’s research group at Emory University it was shown that by using positive allosteric modulators of GABRA5 such as benzodiazepines like KRM-II-08 and QH-II-66 the cell viability of group 3 medulloblastoma can be impaired in vivo and in vitro with better specificity and potency than the standard of care treatments in the clinic. In this research several analogues of KRM-II-08 were designed, synthesized, and assayed and the most potent analogue binds with native tumor receptors with EC50 and IC50 values of ~0.8 micromolar. As a result of this binding, there is a 2 x 109 ions.sec-1 chloride flux which morphologically evokes mitochondrial membrane depolarization, nuclei distention, and cellular blebbing. This is correlated with the localization of pro-apoptotic Bcl-2-associated death promoter (BAD) protein. Thus, this potent, non-toxic benzodiazepine may serve as an efficient anti-cancer drug for group 3 type medulloblastoma. This study was published in 2019.Melanoma is the deadliest form of skin cancer. More than 100,000 people are expected to be diagnosed in the USA in 2021. Current treatment with radiotherapy and immune checkpoint inhibitors does not show significant improvement in patients. Therapy combined with QH-II-66, radiation, and an immune checkpoint inhibitor shows improved results in controlling the metastasis by lowering the mass of the tumor. By gene expression analysis it was seen that these cancer cells show high expression of GABAA receptors, includes α5 GABAA subunits. Electrophysiology shows these receptors are functional. This sensitization to melanoma cells is benzodiazepine exclusive and does not impair normal cells. In a syngeneic mouse model of melanoma QH-II-66 showed increase in the depolarization in mitochondria which initiates programmed cell death of cancer cells. Combined therapy with QH II 66 and radiation show even better results. Lymphocyte and CD8+T cell counts were also increased after the treatment. Large-scale synthesis here was developed for QH II 66. It was important to point out the related benzodiazepine was either very weak or not active at all in these cancers. Thus, this potent, non-toxic benzodiazepine may serve as an efficient anti-cancer drug for melanoma. This study produced one publication in 2021 and two patent application submission in 2022.After these studies a large scale synthesis was required. An efficient large scale synthetic route was developed for KRM II 08 and QH II 66. In the new route there was a 40% overall yield for QH II 66 and a 35% overall yield for KRM II 08. It was achieved by purification of steps by a crystallization process. This synthetic route helped reduce the time and money, as well as made the procedure more efficient to synthesize these two compounds. More than 30 grams of each of these two compounds were synthesized during this study.These two-lead compounds were also tested in H1792 lung cancer cell lines. Both compounds were active in H1792 lung cancer cell lines. At least 20 analogues were synthesized and analyzed on H1792 cancer cell lines in collaboration of Dr. Krummel’s and Dr. Sengupta’s lab at University of Cincinnati. A new novel compound TA II 73 was discovered during this process which was two times more efficacious than the previous lead compound QH II 66 in H1792 non-small cell lung cancer cell lines, melanoma cell lines and glioblastoma cell lines. This TA II 73 developed a whole new series of anticancer benzodiazepines which have a 2’CH3 in the pendent phenyl ring. TA II 73 is non sedative on the rotorod assay at 40 mg/Kg dose. During this process the synthetic route for TA II 73 was also improved. Experiment also showed that it is also active in LN 18 cell line which was derived from glioblastoma cancer patients.There are important consequences of the development of the anticancer drugs contained in a privileged benzodiazepine skeleton. QH II 66, KRM II 08, TA II 73 and many other anticancer drugs have gone through the blood brain barrier in 20 to 30 minutes, as opposed to many anticancer drugs which do not have to be injected directly into the brain. While QH II 66 and analogs greatly enhance the anticancer activity of radiation and/or immune checkpoint inhibitor when they are given together, the combination of all three is even more potent, synergistically in mouse cancer models. In addition, these anticancer compounds are non-toxic for normal cells.Furthermore, the anxiolytic activity and slight sedative activity of these agents will be important to patients who are undergoing dual or combination therapy because some stress will be decreased. It is felt, especially in combination therapy, anticancer drugs developed here will have clinical significance.
Abstract
Most advanced-stage non-small cell lung cancer (NSCLC) patients have brain metastases that render a dismal prognosis. Treatment of metastatic brain lesions from NSCLC and other tumor types ...include radiation as part of a multimodal treatment regimen. Challenges in the application of radiotherapy include overcoming radiation resistance and reducing associated co-morbidities. Non-toxic therapeutics capable of sensitizing tumors to radiation are needed to improve survival and mitigate radiation side-effects. Many CNS and solid systemic tumors express ligand-gated ion channels, which may contribute to tumor growth. Leveraging ion channels is therefore a potential way of diminishing the spread of cancer. We find that NSCLC and its brain metastases express subunits of the type-A GABA-gated chloride channel or GABAA receptor. Importantly, patient-derived NSCLC cells have functional GABAA receptors. We identified a brain penetrant, small molecule activator of GABAA receptors (AMLAL-101), which alone impairs the viability of both primary NSCLC cells and brain metastatic cells. In addition, AMLAL-101 combined with radiation is a highly potent inducer of NSCLC cell death and clonogenic arrest. Using a human ex vivo model of NSCLC-on-chip, we assessed the efficacy and toxicity of AMLAL-101 relative to Docetaxel, an antimicrotubular agent used in treating advanced NSCLC. AMLAL-101 is as potent as Docetaxel but does not exhibit its toxic side effects. AMLAL-101 also potentiates radiation in vivo, significantly reducing lung adenocarcinoma xenograft tumor growth in mice, equivalent to docetaxel plus radiation. Mechanistically, AMLAL-101 activates GABAA receptors in NSCLC and synergizes with radiation by inducing an autophagic response that includes: (i) stabilization of Beclin-1, BNIP3L/NIX, and GABARAP; (ii) ATG7 upregulation; and (iii) utilization of ubiquitin-binding protein p62. Activating GABAA receptors in NSCLC and other tumor types may improve radiation efficacy and mitigate its toxic side effects in treating brain metastases.
Abstract
Standard-of-care for melanoma brain metastases is moving to stereotactic radiosurgery (SRS) with immunotherapy. Although this combined approach shows improved patient survival, the responses ...are not durable. SRS can also cause adverse reactions such as radiation necrosis, which can result in significant morbidity and even mortality. Lowering SRS dose without negatively impacting its effectiveness would therefore be highly beneficial clinically. Our published data suggests that when treating syngeneic mouse model B16F10 melanoma tumors, the radiation dose can be reduced and its effectiveness can be improved, by activating intrinsic melanoma GABAA receptor (GABAAR) activity using a novel agent, AMLAL-101. This agent acts to enhance GABAAR mediated chloride transport in melanoma cells, which in turn triggers mitochondrial dysregulation associated with tumor regression in mice. While AMLAL-101 alone reduces tumor growth, combining it with radiation results in near complete regression of the irradiated tumor, as well as shrinking of tumor not in the radiation field due to an immune response, i.e., a potent abscopal effect. AMLAL-101 may also function to regulate immune cell infiltration into the tumor microenvironment. Analysis of AMLAL-101 treated tumors show increased infiltration into the tumor microenvironment of: (1) polyfunctional (TNFα/IFN-γ) CD8+ T cells; and (2) tumor-associated macrophages (TAMs) of the tumor inhibitory M1-phenotype, possibly due to IFN-γ stimulation. We posit that activating GABAAR with AMLAL-101 promotes both a ‘direct’ anti-tumor response and an ‘indirect’, immune-driven anti-tumor activity in metastatic melanoma tumors. AMLAL-101 may address an urgent unmet need for a non-toxic therapeutic that can potentiate radiation and mitigate its toxicity profile to positively impact clinical outcomes of patients with brain metastatic disease treated with radiotherapy.
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The pharmacological actions exerted by benzodiazepines are dependent on the discrete α protein subunits of the γ-aminobutyric acid type A receptor (GABAA R). Recent developments via a ...cryo-EM structure of the α1β3γ2L GABAA R ion channel provide crucial insights into ligand efficacy and binding affinity at this subtype. We investigated the molecular interactions of diazepam and alprazolam bound GABAA R structures (6HUP and 6HUO) to determine key binding interaction domains. A halogen bond between the chlorine atoms of diazepam and alprazolam with the group on the backbone of the α1 histidine amino acid 102 is important to the positive allosteric modulatory actions of diazepam and alprazolam in the α1β3γ2L GABAA R ion channel. In order to gain insight into α subtype selectivity we designed and synthesized close structural analogs of diazepam and alprazolam. These compounds were then docked into the recently publish cryo-EM structures of GABAA Rs (6HUP and 6HUO). This modeling along with radio-ligand binding data resulted in the conclusion that the non-classical bioisosteric replacement of the chlorine atom at C7 with an ethinyl group (compound 5) resulted in an 11-fold gain in α5 binding selectivity over the α1 subtype. Moreover, the potency of compound 5 resulted in a ligand with less sedation than diazepam, while still maintaining the same anxiolytic potency. These modeling data extend our understanding of the structural requirements for α-subtype-selective compounds that can be utilized to achieve improved medical treatments. It is clear that the ethinyl group in place of a halogen atom decreases the affinity and efficacy of benzodiazepines and imidazodiazepines at α1 subtypes, which results in less sedation and ataxia.
Abstract
PURPOSE
AMLAL-101 is a novel agent which preferentially targets α3, α5 subtypes of ɣ-amino butyric acid receptors and shows anti-tumor activity against disparate cancer types. AMLAL-101 is ...being advanced as an ‘add-on’ to potentiate treatment of primary and metastatic brain cancers. However, AMLAL-101 must penetrate the blood-brain barrier (BBB) and show sufficient brain retention. The primary purpose of this study was to determine the plasma pharmacokinetics (PK) and quantitative estimate of the BBB permeability of AMLAL-101.
METHODS
We performed intracranial microdialysis, employing jugular vein cannulated Sprague-Dawley rats which facilitated simultaneous serial blood and brain extracellular fluid (ECF) sampling. AMLAL-101 was injected i.p. at 5 mg/kg and serial blood and brain ECF samples collected up to 10 h post-dosing. Plasma and ECF samples were analyzed by LC/MS-MS and plasma and ECF concentration vs time PK profiles determined. In vivo recovery analysis was performed using retrodialysis and rapid equilibrium dialysis employed to determine the extent of protein binding.
RESULTS
AMLAL-101 plasma protein binding was 85% and in vivo recovery from ECF was 25%. AMLAL-101 peak concentration (Cmax) in plasma and brain ECF were 15 µM and 13.8 µM, respectively. The plasma and brain ECF area under the concentration (AUC0-10) were 27.5 h.µg/mL and 24.10 h.µg/mL, respectively. The brain partitioning of unbound AMLAL-101 (Kp,uu; determined either as a ratio of brain ECF Cmax:unbound plasma Cmax or brain ECF AUC: unbound plasma AUC), were 6.13 and 4.13, respectively. The elimination half-life of AMLAL-101 was 3 h for both brain ECF and plasma.
CONCLUSIONS
These results suggest that AMLAL-101 has the requisite BBB permeability required for brain cancer therapeutics. AMLAL-101 shows significant brain retention when compared to a chemically similar agent that does not show anti-cancer activity, which may contribute to efficacy of AMLAL-101 as an anti-tumor agent for treatment of brain cancers.
Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest ...relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABA
R). We are advancing a therapeutic approach for group 3 based on GABA
R modulation using benzodiazepine-derivatives.
We performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional 'druggable' GABA
R in group 3 cells.
Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABA
R subunits α5, β3 and γ2 and 3. There are ~ 1000 functional α5-GABA
Rs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 10
ions/s. Benzodiazepines, designed to prefer α5-GABA
R, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine ('KRM-II-08') binds to the α5-GABA
R (0.8 µM EC
) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization.
GABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABA
R is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.
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