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Biocatalytic Synthesis of Chiral 1,2,3,4-Tetrahydroquinolines
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Yongzheng Chen, Liu Song, Zhongqiang Wang
In 2014, Zhu and co-workers reported the application of the CHAO variants in the deracemization of 2-substituted 1,2,3,4-tetrahydroquinolines by the genetic improvement of a previously cloned FAD-dependent cyclohexylamine oxidase (CHAO) (Li et al., 2014). In their study, 11 amino acid residues (F88, T198, L199, M226, Q233, Y321, F351, L353, F368, P422, and Y459) were selected for site-saturation mutagenesis. After screening of ∼4500 clones from the 11 libraries, T198F, L199T, M226F, and Y459T were identified with specific activity toward 2-methyl-THQ compared to wild type CHAO which had no or trace activity. Further efforts led to the identification of a triple mutant (T198FL199SM226F) revealing the best activity of all variants. For other substrates with substitutes such as allyl, benzyl, phenyl on 2-position of THQs, the reactivity toward 2-substituted THQs turned out to be of the following order: methyl > allyl > benzyl > phenyl substituents. This ordering suggested that the enzyme activity was significantly influenced by the steric factor of substrates.
Role of Engineered Proteins as Therapeutic Formulations
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Khushboo Gulati, Krishna Mohan Poluri
In contrast to random mutagenesis, focused mutagenesis involves generation of small libraries that can be readily screened. Smaller libraries are the result of mutations that are focused only to protein hot spot residues. Hot spots correspond to the residues which have phenomenal functional/structural role. Site saturation mutagenesis (SSM) is the most commonly employed focused mutagenesis method which tries all the 20 amino acids against the targeted amino acid in a single shot. The two most commonly employed SSM methods are: (1) site directed mutagenesis (SDM) that uses complementary primer pairs containing the mutation site (Matsumura and Rowe, 2005; Alcalde et al., 2006). (2) Overlap extension PCR that employs two pairs of complementary primers with two rounds of PCR to introduce the mutation at the targeted site (Gratz and Jose, 2008). Other focused mutagenesis methods such as cassette mutagenesis (Reidhaar-Olson and Sauer, 1988), sequence saturation mutagenesis (SeSAM) (Wong et al., 2008), single-primer reactions in parallel (SPRINP) (Edelheit et al., 2009), megaprimed and ligase-Free (Tseng et al., 2008), Ω-PCR (Chen et al., 2013), PFunkel (Firnberg and Ostermeier, 2012), ominchange (Dennig et al., 2011), OSCARR (Hidalgo et al., 2008), trimer-dimer mutagenesis (Gaytan et al., 2009), synthetic saturation mutagenesis (Patwardhan et al., 2009), Amber codon saturation mutagenesis have also been developed and are being employed for the creation of variant libraries (Shozen et al., 2012).
Dasatinib: A Dual ABL and SRC Inhibitor
Published in Jorge Cortes, Michael Deininger, Chronic Myeloid Leukemia, 2006
Alfonso Quintás-Cardama, Hagop Kantarjian, Jorge Cortes
In cellular assays, dasatinib inhibited the proliferation of BCR-ABL– transfected Ba/F3 cells and human BCR-AbL–expressing K562 cells with IC50 values of 1.3 and <1 nM, respectively, and demonstrated high potency against 14 of 15 clinically relevant imatinib-resistant Abl mutations (14–16), supporting the less stringent conformational requirements of dasatinib on Abl for kinase inhibition compared to imatinib (15). Of note, the T315I retained kinase activity even in the presence of μM concentrations of dasatinib, suggesting that this residue acts as a gatekeeper for ATP-competitive small-molecule kinase inhibitors. In a model of imatinib-resistant, BCR-ABL–mediated disease, severe combined immunodeficient (SCID) mice were injected intravenously with Ba/F3 cells expressing different Bcr-Abl isoforms as well as the firefly luciferase gene. Administration of dasatinib 10 mg/kg twice daily by oral gavage for two weeks resulted in mice showing greater than 1-log lower levels of bioluminescent activity and prolonged survival compared with untreated controls. Mice with the T315I mutation did not respond. Notably, dasatinib markedly inhibited the growth of bone marrow progenitors isolated from patients with CML with imatinib-sensitive or -resistant (M351T) disease, but not marrow progenitors obtained from healthy volunteers (15). In a saturation mutagenesis screening, significantly fewer mutations were induced with dasatinib compared to imatinib. Still, 10 BCR-AbL mutants were generated, with the most frequent being F317V > T315A > T315I > F317L. All these mutants represent points of contact between dasatinib and the Abl kinase. These mutants could potentially account for clinical resistance to dasatinib in clinical practice. In fact, instances of dasatinib failure associated with one of these mutations, T315A, have been described (17). Interestingly, this mutation is effectively inhibited by imatinib. Of note, the combination of dasatinib with imatinib greatly reduced the recovery of drug-resistant clones (17). Similar results were observed in another cell-line based mutagenesis assay (18).
Computational design of a neutralizing antibody with picomolar binding affinity for all concerning SARS-CoV-2 variants
Published in mAbs, 2022
Bo-Seong Jeong, Jeong Seok Cha, Insu Hwang, Uijin Kim, Jared Adolf-Bryfogle, Brian Coventry, Hyun-Soo Cho, Kyun-Do Kim, Byung-Ha Oh
The computational sequence design of CDRs involving the Monte Carlo search for amino acid sequence is analogous to the experimental phage display biopanning. It is notable that recent experimental approach led to the discovery of two nAbs against the SARS-CoV-2 RBD by using a ~ 109-diversity phage display library constructed based on the anti-SARS-CoV-1 mAbs (m396, 80 R, CR3022).42 In this study, experimental affinity enhancement based on structural validation at high-resolution increased the binding affinity for the wild-type RBD by more than 20-fold, while computational affinity maturation for the N501Y mutant RBD increased affinity much further. This was all accomplished while also enhancing the antibody’s affinity for the wild-type RBD. Whereas D27LEY exhibits ultrapotent binding affinity for SARS-CoV-2 variants with the N501Y mutation, its binding affinities for other variants lacking this mutation are lower, but still potent. It is likely that saturation mutagenesis at other selected sites could further enhance binding affinity for all variants.
Early flowering, good grain quality mutants through gamma rays and EMS for enhancing per day productivity in rice (Oryza sativa L.)
Published in International Journal of Radiation Biology, 2021
Vinithashri Gautam, Manonmani Swaminathan, Manoharan Akilan, Anand Gurusamy, Meena Suresh, Bhuvaneswari Kaithamalai, A. John Joel
Mutation breeding mainly involves physical mutagens like gamma rays, electron beam, and chemical mutagens like Ethyl Methane Sulfonate (EMS), Sodium Azide (SA) and Methyl nitroso urea for generating variability in crop plants. The effects of mutation range from single nucleotide changes to large deletions or chromosomal rearrangements. For the past 80 years, the creation of hereditary aberrations and the development of more than 70 percent of mutant varieties were achieved using ionizing radiations (Mba 2013). Gamma rays have a shorter wavelength and penetrate deeper into the plant tissues. The mutagenic effect is reflected because of Deoxyribonucleic acid (DNA) double-strand breaks. Point mutations created by the chemical mutagens leads to both losses of function and gain of function phenotypes as in the cases of tolerance to the herbicide glyphosate (Bradshaw et al. 1997) and sulfonyl urea in the legume group Medicago truncatula (Oldach et al. 2008). In case of rice, EMS mutagenesis was employed to isolate Herbicide Tolerant Mutant-22 (HTM-22) which confers tolerance against broad-spectrum herbicide Imazethapyr (Shoba et al. 2017). Any genotype can be mutagenized and the distribution of mutation is random in the genome. Genome wide saturation mutagenesis can be attained by using a small mutant population because of high concentration of mutation. Response of plants to mutagens is influenced by genotypes. Frequency of mutations tends to differ across physical, chemical and combination treatments across different genotypes.
Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life
Published in mAbs, 2019
Brian C. Mackness, Julie A. Jaworski, Ekaterina Boudanova, Anna Park, Delphine Valente, Christine Mauriac, Olivier Pasquier, Thorsten Schmidt, Mostafa Kabiri, Abdullah Kandira, Katarina Radošević, Huawei Qiu
A number of lead mutations with improved FcRn off rates identified in this study have overlap with previously reported variants for half-life extension, including M252Y and T256E in YTE,4 T307A in AAA3 and N434F in the NHance platform.16 Some of other mutations have been previously reported, including T307Q and N434Y, but generally in combination with a host (3+) of other mutations.11,16 The creation of unique combinations from the lead saturation mutations with optimal properties is the major strength of the saturation mutagenesis approach. Almost 90% of the combinations identified in our study surpassed the FcRn binding affinity of the YTE and LS benchmarks at pH 6.0 by orders of magnitude. However, a large portion of these variants would have limited application towards antibody half-life extension due to the simultaneous affinity improvement at pH 7.4.17,28 The N434F or N434Y mutations are responsible for the dramatic enhancement in the affinity at pH 7.4 and, as a result, are not present in any of the final lead variants.