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Structural and Kinetic Factors Governing Drug-Target Specificity and Resistance.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Structural and Kinetic Factors Governing Drug-Target Specificity and Resistance.
Author:	Rangwala`, Aziz Mohammedi.
Published:	Ann Arbor : ProQuest Dissertations & Theses, 2023
Description:	186 p.
Notes:	Source: Dissertations Abstracts International, Volume: 84-12, Section: B.
Notes:	Advisor: Seeliger, Markus A.
Contained By:	Dissertations Abstracts International84-12B.
Subject:	Biochemistry.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=30313801
ISBN:	9798379637729

Structural and Kinetic Factors Governing Drug-Target Specificity and Resistance.
Rangwala`, Aziz Mohammedi.

Structural and Kinetic Factors Governing Drug-Target Specificity and Resistance. - Ann Arbor : ProQuest Dissertations & Theses, 2023 - 186 p.

Source: Dissertations Abstracts International, Volume: 84-12, Section: B.

Thesis (Ph.D.)--State University of New York at Stony Brook, 2023.

This item must not be sold to any third party vendors.

My PhD research focuses on how to develop specific drugs and how mutations in a drug receptor can cause drug resistance. Protein kinase inhibitors are potent anticancer therapeutics. Imatinib, a selective inhibitor of BCR-ABL1 kinase, reduced mortality in chronic myeloid leukemia by 80%, but 22-41% of patients acquire imatinib resistance. Mutations within the BCRABL1 kinase domain are the most common cause for relapse. While some mutations directly alter or abrogate imatinib binding, many resistance mutations are located distal to the drug-binding site, and it remains unclear how these mutations confer resistance. Earlier investigations of small sets of patient-derived imatinib resistance mutations indicated that some mutant proteins were still in fact sensitive to imatinib in biochemical assays; however, these assays were performed at constant drug concentrations, which differ from the physiological fluctuations of plasma drug concentrations in patients. Residence time describes the lifetime of the drug-target complex and is a superior predictor of in vivo potency than the measurement of equilibrium binding affinity in several systems.We hypothesized that mutations could cause resistance not by altering the equilibrium binding affinity Kd. but rather by increasing drug dissociation rates and decreasing imatinib’s residence time, ultimately translating to reduced target inhibition. We tested how 94 patient-derived Abl mutants affected imatinib affinity and binding kinetics in live cells using NanoBRET Target Engagement. We found that two-thirds of the clinically observed imatinib resistance mutations in Abl kinase significantly reduce drug affinity, whereas one-third bind imatinib with similar or tighter affinity than Abl wild-type (WT). We identified three imatinib resistance mutations, N368S, V299L, and G251E, that have similar imatinib affinity to Abl WT but increased imatinib dissociation rates. I biochemically characterized the stability binding kinetics, and ATP and drug affinity using novel fluorescence, fluorescence anisotropy, and luminescence techniques. We further validated the observed effect of the N368S mutation in vitro and proposed a mechanism of its increased dissociation rate using molecular dynamics simulations. This workflow represents the first broad-spectrum analysis of the characteristics governing target engagement using the full range of mutations in a clinically-relevant kinase. Mutations at these sites are present ubiquitously throughout the kinome, which suggests that kinetic resistance to structurally-selective kinase inhibitors may be a widespread mechanism. We envision that this study will serve as a paradigm for further investigation of analogous mutations in other kinases such as the epidermal growth factor receptor (EGFR) and uncover a broader role for binding kinetics in drug resistance.I addressed the question of how to develop specific inhibitors against the challenging cyclophilin family in collaboration with Alexander Peterson and David Liu (Harvard). The cyclophilin family consists of 17 proteins containing a structurally-conserved peptidyl-prolylisomerase (PPIase) domain. Cyclophilin D (CypD) is a unique mitochondrial cyclophilin which serves as the key regulator of the mitochondrial permeability transition pore (mPTP), a transient channel on the inner mitochondrial membrane that opens under oxidative stress and causes mitochondrial swelling and rupture. CypD-trigged mPTP opening is the driving pathophysiological force behind ischemia-reperfusion injury and necrosis in cerebral stroke and myocardial infarction. Cyclophilins are also essential in the life cycle of multiple viruses, including hepatitis C and SARS-CoV-2. Therefore, there has been great interest in developing specific cyclophilin inhibitors. However, previous efforts have been stymied by the high sequence identity (61-86%) and structural conservation of human PPIase domains. Using a DNA-templated macrocycle library and iterated structural biology and small-molecule engineering, we developed potent and isoform-selective CypD and CypE inhibitors. These findings reveal a generalized strategy to generate in vitro isoform-selective small-molecule cyclophilin modulators, advancing their suitability as targets for biological investigation and therapeutic intervention. We extended this study in collaboration with Brookhaven National Laboratory to create a high-throughput fragment-based crystallography pipeline for small molecule development, which will serve as a critical resource for future structure-guided small-molecule discovery efforts in the scientific community. Our strategy for the development of selective Cyp inhibitors paves the way for the development of subtype-selective inhibitors and fluorescent probes, which we envision will open a new field of cyclophilin-focused inquiry.

ISBN: 9798379637729Subjects--Topical Terms:

188609
Biochemistry.
Subjects--Index Terms:

Cyclophilin

Structural and Kinetic Factors Governing Drug-Target Specificity and Resistance.
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245 1 0 $a Structural and Kinetic Factors Governing Drug-Target Specificity and Resistance.
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300 $a 186 p.
500 $a Source: Dissertations Abstracts International, Volume: 84-12, Section: B.
500 $a Advisor: Seeliger, Markus A.
502 $a Thesis (Ph.D.)--State University of New York at Stony Brook, 2023.
506 $a This item must not be sold to any third party vendors.
520 $a My PhD research focuses on how to develop specific drugs and how mutations in a drug receptor can cause drug resistance. Protein kinase inhibitors are potent anticancer therapeutics. Imatinib, a selective inhibitor of BCR-ABL1 kinase, reduced mortality in chronic myeloid leukemia by 80%, but 22-41% of patients acquire imatinib resistance. Mutations within the BCRABL1 kinase domain are the most common cause for relapse. While some mutations directly alter or abrogate imatinib binding, many resistance mutations are located distal to the drug-binding site, and it remains unclear how these mutations confer resistance. Earlier investigations of small sets of patient-derived imatinib resistance mutations indicated that some mutant proteins were still in fact sensitive to imatinib in biochemical assays; however, these assays were performed at constant drug concentrations, which differ from the physiological fluctuations of plasma drug concentrations in patients. Residence time describes the lifetime of the drug-target complex and is a superior predictor of in vivo potency than the measurement of equilibrium binding affinity in several systems.We hypothesized that mutations could cause resistance not by altering the equilibrium binding affinity Kd. but rather by increasing drug dissociation rates and decreasing imatinib’s residence time, ultimately translating to reduced target inhibition. We tested how 94 patient-derived Abl mutants affected imatinib affinity and binding kinetics in live cells using NanoBRET Target Engagement. We found that two-thirds of the clinically observed imatinib resistance mutations in Abl kinase significantly reduce drug affinity, whereas one-third bind imatinib with similar or tighter affinity than Abl wild-type (WT). We identified three imatinib resistance mutations, N368S, V299L, and G251E, that have similar imatinib affinity to Abl WT but increased imatinib dissociation rates. I biochemically characterized the stability binding kinetics, and ATP and drug affinity using novel fluorescence, fluorescence anisotropy, and luminescence techniques. We further validated the observed effect of the N368S mutation in vitro and proposed a mechanism of its increased dissociation rate using molecular dynamics simulations. This workflow represents the first broad-spectrum analysis of the characteristics governing target engagement using the full range of mutations in a clinically-relevant kinase. Mutations at these sites are present ubiquitously throughout the kinome, which suggests that kinetic resistance to structurally-selective kinase inhibitors may be a widespread mechanism. We envision that this study will serve as a paradigm for further investigation of analogous mutations in other kinases such as the epidermal growth factor receptor (EGFR) and uncover a broader role for binding kinetics in drug resistance.I addressed the question of how to develop specific inhibitors against the challenging cyclophilin family in collaboration with Alexander Peterson and David Liu (Harvard). The cyclophilin family consists of 17 proteins containing a structurally-conserved peptidyl-prolylisomerase (PPIase) domain. Cyclophilin D (CypD) is a unique mitochondrial cyclophilin which serves as the key regulator of the mitochondrial permeability transition pore (mPTP), a transient channel on the inner mitochondrial membrane that opens under oxidative stress and causes mitochondrial swelling and rupture. CypD-trigged mPTP opening is the driving pathophysiological force behind ischemia-reperfusion injury and necrosis in cerebral stroke and myocardial infarction. Cyclophilins are also essential in the life cycle of multiple viruses, including hepatitis C and SARS-CoV-2. Therefore, there has been great interest in developing specific cyclophilin inhibitors. However, previous efforts have been stymied by the high sequence identity (61-86%) and structural conservation of human PPIase domains. Using a DNA-templated macrocycle library and iterated structural biology and small-molecule engineering, we developed potent and isoform-selective CypD and CypE inhibitors. These findings reveal a generalized strategy to generate in vitro isoform-selective small-molecule cyclophilin modulators, advancing their suitability as targets for biological investigation and therapeutic intervention. We extended this study in collaboration with Brookhaven National Laboratory to create a high-throughput fragment-based crystallography pipeline for small molecule development, which will serve as a critical resource for future structure-guided small-molecule discovery efforts in the scientific community. Our strategy for the development of selective Cyp inhibitors paves the way for the development of subtype-selective inhibitors and fluorescent probes, which we envision will open a new field of cyclophilin-focused inquiry.
590 $a School code: 0771.
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650 4 $a Molecular biology. $3 188737
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653 $a Luminescence
653 $a X-ray crystallography
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