Date of Award

May 2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

James JC Cook

Committee Members

Alan AS Schwabacher, Arsenio AP Pacheco, Xiaohua XP Peng, Alexander AA Arnold

Keywords

Benzodiazepines, Cancer, Medulloblastoma, Melanoma, New series of benzodiazepine, Non small cell lung cancer

Abstract

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.

Download

Share

COinS