Binding of the human nucleotide excision repair proteins XPA and XPC/HR23B to the 5R-thymine glycol lesion and structure of the cis-(5R,6S) thymine glycol epimer in the 5′-GTgG-3′ sequence: destabilization of two base pairs at the lesion site

The 5R thymine glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5′-GTgG-3′ sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson–Crick alignment. The Tg N3H imine and A N6 amine protons undergo increased solvent exchange. Stacking between Tg and the 3′-neighbor G•C base pair is disrupted. The solvent accessible surface and T2 relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH3 group is favored; propeller twisting of the Tg•A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3′-neighbor guanine alleviate steric clash with the 5′-neighbor base pair. Tg also destabilizes the 5′-neighbor G•C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA

Development of Alkyne-Containing Pyrazolopyrimidines To Overcome Drug Resistance of Bcr-Abl Kinase

Despite the success of imatinib at inhibiting Bcr-Abl and treating chronic myelogenous leukemia (CML), resistance to the therapy occurs over time in patients. In particular, the resistance to imatinib caused by the gatekeeper mutation T315I in Bcr-Abl remains a challenge in the clinic. Inspired by the successful development of ponatinib to curb drug resistance, we hypothesize that the incorporation of an alkyne linker in other heterocyclic scaffolds can also achieve potent inhibition of Bcr-Abl^T315I by allowing for simultaneous occupancy of both the active site and the allosteric pocket in the Abl kinase domain. Herein, we describe the design, synthesis, and characterization of a series of alkyne-containing pyrazolopyrimidines as Bcr-Abl inhibitors. Our results demonstrate that some alkyne-containing pyrazolopyrimidines potently inhibit not only Abl^T315I in vitro but also Bcr-Abl^T315I in cells. These pyrazolopyrimidines can serve as lead compounds for future development of novel targeted therapy to overcome drug resistance of CML

Development of Alkyne-Containing Pyrazolopyrimidines To Overcome Drug Resistance of Bcr-Abl Kinase

Despite the success of imatinib at inhibiting Bcr-Abl and treating chronic myelogenous leukemia (CML), resistance to the therapy occurs over time in patients. In particular, the resistance to imatinib caused by the gatekeeper mutation T315I in Bcr-Abl remains a challenge in the clinic. Inspired by the successful development of ponatinib to curb drug resistance, we hypothesize that the incorporation of an alkyne linker in other heterocyclic scaffolds can also achieve potent inhibition of Bcr-Abl^T315I by allowing for simultaneous occupancy of both the active site and the allosteric pocket in the Abl kinase domain. Herein, we describe the design, synthesis, and characterization of a series of alkyne-containing pyrazolopyrimidines as Bcr-Abl inhibitors. Our results demonstrate that some alkyne-containing pyrazolopyrimidines potently inhibit not only Abl^T315I in vitro but also Bcr-Abl^T315I in cells. These pyrazolopyrimidines can serve as lead compounds for future development of novel targeted therapy to overcome drug resistance of CML

5′-β,γ-CHF-ATP Diastereomers: Synthesis and Fluorine-Mediated Selective Binding by c‑Src Protein Kinase

The first preparation of the individual β,γ-CHF-ATP stereoisomers <b>12a</b> and <b>12b</b> is reported. Configurationally differing solely by the orientation of the C–F fluorine, <b>12a</b> and <b>12b</b> have discrete ³¹P (202 MHz, pH 10.9, Δδ_Pα 6 Hz, Δδ_Pβ 4 Hz) and ¹⁹F NMR (470 MHz, pH 9.8, Δδ_F 25 Hz) spectral signatures and exhibit a 6-fold difference in IC₅₀ values for c-Src kinase, attributed to a unique interaction of the (<i>S</i>)-fluorine of bound <b>12b</b> with R388 in the active site

A Chemical-Genetic Approach to Generate Selective Covalent Inhibitors of Protein Kinases

Although a previously developed bump-hole approach has proven powerful in generating specific inhibitors for mapping functions of protein kinases, its application is limited by the intolerance of the large-to-small mutation by certain kinases and the inability to control two kinases separately in the same cells. Herein, we describe the development of an alternative chemical-genetic approach to overcome these limitations. Our approach features the use of an engineered <u>cys</u>teine residue at a particular position as a reactive feature to sensitize a kinase of interest to selective covalent blockade by <u>ele</u>ctrophilic inhibitors and is thus termed the <i>Ele-Cys</i> approach. We successfully applied the <i>Ele-Cys</i> approach to identify selective covalent inhibitors of a receptor tyrosine kinase EphB1 and solved cocrystal structures to determine the mode of covalent binding. Importantly, the <i>Ele-Cys</i> and bump-hole approaches afforded orthogonal inhibition of two distinct kinases in the cell, opening the door to their combined use in the study of multikinase signaling pathways

Design of a Vaccine Passport Validation System Using Blockchain-based Architecture: Development Study

BackgroundCOVID-19 is an ongoing global pandemic caused by SARS-CoV-2. As of June 2021, 5 emergency vaccines were available for COVID-19 prevention, and with the improvement of vaccination rates and the resumption of activities in each country, verification of vaccination has become an important issue. Currently, in most areas, vaccination and reverse transcription polymerase chain reaction (RT-PCR) test results are certified and validated on paper. This leads to the problem of counterfeit documents. Therefore, a global vaccination record is needed. ObjectiveThe main objective of this study is to design a vaccine passport (VP) validation system based on a general blockchain architecture for international use in a simulated environment. With decentralized characteristics, the system is expected to have the advantages of low cost, high interoperability, effectiveness, security, and verifiability through blockchain architecture. MethodsThe blockchain decentralized mechanism was used to build an open and anticounterfeiting information platform for VPs. The contents of a vaccination card are recorded according to international Fast Healthcare Interoperability Resource (FHIR) standards, and blockchain smart contracts (SCs) are used for authorization and authentication to achieve hierarchical management of various international hospitals and people receiving injections. The blockchain stores an encrypted vaccination path managed by the user who manages the private key. The blockchain uses the proof-of-authority (PoA) public chain and can access all information through the specified chain. This will achieve the goal of keeping development costs low and streamlining vaccine transit management so that countries in different economies can use them. ResultsThe openness of the blockchain helps to create transparency and data accuracy. This blockchain architecture contains a total of 3 entities. All approvals are published on Open Ledger. Smart certificates enable authorization and authentication, and encryption and decryption mechanisms guarantee data protection. This proof of concept demonstrates the design of blockchain architecture, which can achieve accurate global VP verification at an affordable price. In this study, an actual VP case was established and demonstrated. An open blockchain, an individually approved certification mechanism, and an international standard vaccination record were introduced. ConclusionsBlockchain architecture can be used to build a viable international VP authentication process with the advantages of low cost, high interoperability, effectiveness, security, and verifiability

Development of Specific, Irreversible Inhibitors for a Receptor Tyrosine Kinase EphB3

Erythropoietin-producing human hepatocellular carcinoma (Eph) receptor tyrosine kinases (RTKs) regulate a variety of dynamic cellular events, including cell protrusion, migration, proliferation, and cell-fate determination. Small-molecule inhibitors of Eph kinases are valuable tools for dissecting the physiological and pathological roles of Eph. However, there is a lack of small-molecule inhibitors that are selective for individual Eph isoforms due to the high homology within the family. Herein, we report the development of the first potent and specific inhibitors of a single Eph isoform, EphB3. Through structural bioinformatic analysis, we identified a cysteine in the hinge region of the EphB3 kinase domain, a feature that is not shared with any other human kinases. We synthesized and characterized a series of electrophilic quinazolines to target this unique, reactive feature in EphB3. Some of the electrophilic quinazolines selectively and potently inhibited EphB3 both in vitro and in cells. Cocrystal structures of EphB3 in complex with two quinazolines confirmed the covalent linkage between the protein and the inhibitors. A “clickable” version of an optimized inhibitor was created and employed to verify specific target engagement in the whole proteome and to probe the extent and kinetics of target engagement of existing EphB3 inhibitors. Furthermore, we demonstrate that the autophosphorylation of EphB3 within the juxtamembrane region occurs in <i>trans</i> using a specific inhibitor. These exquisitely specific inhibitors will facilitate the dissection of EphB3’s role in various biological processes and disease contribution

Remarkably Stereospecific Utilization of ATP α,β-Halomethylene Analogues by Protein Kinases

ATP analogues containing a CXY group in place of the α,β-bridging oxygen atom are powerful chemical probes for studying ATP-dependent enzymes. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y). We report here a novel method based on derivatization of a bisphosphonate precursor with a d-phenylglycine chiral auxiliary that enables preparation of the individual diastereomers of α,β-CHF-ATP and α,β-CHCl-ATP, which differ only in the configuration at the CHX carbon. When tested on a dozen divergent protein kinases, these individual diastereomers exhibit remarkable diastereospecificity (up to over 1000-fold) in utilization by the enzymes. This high selectivity can be exploited in an enzymatic approach to obtain the otherwise inaccessible diastereomers of α,β-CHBr-ATP. The crystal structure of a tyrosine kinase Src bound to α,β-CHX-ADP establishes the absolute configuration of the CHX carbon and helps clarify the origin of the remarkable diastereospecificity observed. We further synthesized the individual diastereomers of α,β-CHF-γ-thiol-ATP and demonstrated their utility in labeling a wide spectrum of kinase substrates. The novel ATP substrate analogues afforded by these two complementary strategies should have broad application in the study of the structure and function of ATP-dependent enzymes