Structure-guided Rational Design Research Articles

Discovery of the selective and nanomolar inhibitor of DPP-4 more potent than sitagliptin by structure-guided rational design

Various therapeutic targets and approaches are commonly employed in the management of Type 2 Diabetes. These encompass diverse groups of drugs that target different mechanisms involved in glucose regulation. Inhibition of the DPP-4 enzyme has been proven an excellent target for antidiabetic drug design. Our previous work on discovering multitarget antidiabetic drugs led to the identification of a gallic acid-thiazolidinedione hybrid as a potent DPP4 inhibitor (IC50 = 36 nM). In current research, our efforts resulted in a new dihydropyrimidine-based scaffold with enhanced DPP4 inhibition potential. After virtual evaluation, the designed molecules with excellent interaction patterns and binding energy values were synthesized in the wet laboratory. The inhibition potential of synthesized compounds was assessed against the DPP-4 enzyme. Compound 46 with single digit IC50 value 2 nM exhibited 4-fold and 18-fold higher activity than Sitagliptin and our previously reported hybrid respectively. Moreover, compounds 46, 47 and 50 have shown manyfold selectivity against DPP8 and DPP9. Further pretreatment with compounds 43, 45–47 and 50 (at doses of 10 and 20 mg/kg) in OGTT conducted on rats resulted in a significant decrease in the serum glucose levels compared to the control group. In the long-term STZ-induced diabetic rats, tested compound 50 performed similarly to the reference drug. Molecular dynamics simulations and in-silico molecular docking studies were employed to elucidate the time-dependent interactions of inhibitors within the active sites of DPP4. The compounds examined in this work might serve as a possible lead in the development of effective diabetic mellitus treatments.

Engineering an Ancestral Glycosyltransferase for Biosynthesis of 2-Phenylethyl-β-d-Glucopyranoside and Salidroside.

Phenylethanoid glycosides (PhGs) are naturally occurring glycosides derived from plants with various biological activities. Glycosyltransferases catalyze the production of PhGs from phenylethanols via a transglycosylation reaction. The low activity and stability of glycosyltransferase limit its industrial application. An ancestral glycosyltransferase, UGTAn85, with heat resistance, alkali resistance, and high stability was resurrected using ancestral sequence reconstruction technology. This enzyme can efficiently convert phenylethanols to PhGs. The optimal reaction temperature and pH for UGTAn85 were found to be 70 °C and pH 10.0, respectively. This study employed a combination of structure-guided rational design and co-evolution analysis to enhance its catalytic activity. Potential mutation sites were identified through computer-aided design, including homology modeling, molecular docking, Rosetta dock design, molecular dynamics simulation, and co-evolution analysis. By targeted mutagenesis, the UGTAn85 mutant Q23E/N65D exhibited a 2.2-fold increase in enzyme activity (11.85 U/mg) and elevated affinity (Km = 0.11 mM) for 2-phenylethanol compared to UGTAn85. Following a fed-batch reaction, 36.16 g/L 2-phenylethyl-β-d-glucopyranoside and 51.49 g/L salidroside could be produced within 24 h, respectively. The findings in this study provide a new perspective on enhancing the stability and activity of glycosyltransferases, as well as a potential biocatalyst for the industrial production of PhGs.

Functional characterization and structural basis of an efficient ochratoxin A-degrading amidohydrolase

Ochratoxin A (OTA) contamination in various agro-products poses a serious threat to the global food safety and human health, leading to enormous economic losses. Enzyme-mediated OTA degradation is an appealing strategy, and the search for more efficient enzymes is a prerequisite for achieving this goal. Here, a novel amidohydrolase, termed PwADH, was demonstrated to exhibit 7.3-fold higher activity than that of the most efficient OTA-degrading ADH3 previously reported. Cryo-electron microscopy structure analysis indicated that additional hydrogen-bond interactions among OTA and the adjacent residue H163, the more compact substrate-binding pocket, and the wider entry to the substrate-access cavity might account for the more efficient OTA-degrading activity of PwADH compared with that of ADH3. We conducted a structure-guided rational design of PwADH and obtained an upgraded variant, G88D, whose OTA-degrading activity was elevated by 1.2-fold. In addition, PwADH and the upgraded G88D were successfully expressed in the industrial yeast Pichia pastoris, and their catalytic activities were compared to those of their counterparts produced in E. coli, revealing the feasibility of producing PwADH and its variants in industrial yeast strains. These results illustrate the structural basis of a novel, efficient OTA-degrading amidohydrolase and will be beneficial for the development of high-efficiency OTA-degrading approaches.

Functional characterization, structural basis, and protein engineering of a rare flavonoid 2′-O-glycosyltransferase from Scutellaria baicalensis

Glycosylation is an important post-modification reaction in plant secondary metabolism, and contributes to structural diversity of bioactive natural products. In plants, glycosylation is usually catalyzed by UDP-glycosyltransferases. Flavonoid 2′-O-glycosides are rare glycosides. However, no UGTs have been reported, thus far, to specifically catalyze 2′-O-glycosylation of flavonoids. In this work, UGT71AP2 was identified from the medicinal plant Scutellaria baicalensis as the first flavonoid 2′-O-glycosyltransferase. It could preferentially transfer a glycosyl moiety to 2′-hydroxy of at least nine flavonoids to yield six new compounds. Some of the 2′-O-glycosides showed noticeable inhibitory activities against cyclooxygenase 2. The crystal structure of UGT71AP2 (2.15 Å) was solved, and mechanisms of its regio-selectivity was interpreted by pKa calculations, molecular docking, MD simulation, MM/GBSA binding free energy, QM/MM, and hydrogen‒deuterium exchange mass spectrometry analysis. Through structure-guided rational design, we obtained the L138T/V179D/M180T mutant with remarkably enhanced regio-selectivity (the ratio of 7-O-glycosylation byproducts decreased from 48% to 4%) and catalytic efficiency of 2′-O-glycosylation (kcat/Km, 0.23 L/(s·μmol), 12-fold higher than the native). Moreover, UGT71AP2 also possesses moderate UDP-dependent de-glycosylation activity, and is a dual function glycosyltransferase. This work provides an efficient biocatalyst and sets a good example for protein engineering to optimize enzyme catalytic features through rational design.

Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

Semi-rational enzyme engineering is a powerful method to develop industrial biocatalysts. Profiting from advances in molecular biology and bioinformatics, semi-rational approaches can effectively accelerate enzyme engineering campaigns. Here, we present the optimization of a ketoreductase from Sporidiobolus salmonicolor for the chemo-enzymatic synthesis of ipatasertib, a potent protein kinase B inhibitor. Harnessing the power of mutational scanning and structure-guided rational design, we created a 10-amino acid substituted variant exhibiting a 64-fold higher apparent kcat and improved robustness under process conditions compared to the wild-type enzyme. In addition, the benefit of algorithm-aided enzyme engineering was studied to derive correlations in protein sequence-function data, and it was found that the applied Gaussian processes allowed us to reduce enzyme library size. The final scalable and high performing biocatalytic process yielded the alcohol intermediate with ≥ 98% conversion and a diastereomeric excess of 99.7% (R,R-trans) from 100 g L−1 ketone after 30 h. Modelling and kinetic studies shed light on the mechanistic factors governing the improved reaction outcome, with mutations T134V, A238K, M242W and Q245S exerting the most beneficial effect on reduction activity towards the target ketone.

Growth-Coupled Evolutionary Pressure Improving Epimerases for D-Allulose Biosynthesis Using a Biosensor-Assisted In Vivo Selection Platform.

Fast screening strategies that enable high-throughput evaluation and identification of desired variants from diversified enzyme libraries are crucial to tailoring biocatalysts for the synthesis of D-allulose, which is currently limited by the poor catalytic performance of ketose 3-epimerases (KEases). Here, the study designs a minimally equipment-dependent, high-throughput, and growth-coupled in vivo screening platform founded on a redesigned D-allulose-dependent biosensor system. The genetic elements modulating regulator PsiR expression levels undergo systematic optimization to improve the growth-responsive dynamic range of the biosensor, which presents ≈30-fold facilitated growth optical density with a high signal-to-noise ratio (1.52 to 0.05) toward D-allulose concentrations from 0 to 100mm. Structural analysisand evolutionary conservation analysis of Agrobacterium sp. SUL3 D-allulose 3-epimerase (ADAE) reveal a highly conserved catalytic active site and variable hydrophobic pocket, which together regulate substrate recognition. Structure-guided rational design and directed evolution are implemented using the growth-coupled in vivo screening platform to reprogram ADAE, in which a mutant M42 (P38N/V102A/Y201L/S207N/I251R) is identified with a 6.28-fold enhancement of catalytic activity and significantly improved thermostability with a 2.5-fold increase of the half-life at 60 °C. The research demonstrates that biosensor-assisted growth-coupled evolutionary pressure combined with structure-guided rational design provides a universal route for engineering KEases.

GalNAc-Lipid nanoparticles enable non-LDLR dependent hepatic delivery of a CRISPR base editing therapy

Lipid nanoparticles have demonstrated utility in hepatic delivery of a range of therapeutic modalities and typically deliver their cargo via low-density lipoprotein receptor-mediated endocytosis. For patients lacking sufficient low-density lipoprotein receptor activity, such as those with homozygous familial hypercholesterolemia, an alternate strategy is needed. Here we show the use of structure-guided rational design in a series of mouse and non-human primate studies to optimize a GalNAc-Lipid nanoparticle that allows for low-density lipoprotein receptor independent delivery. In low-density lipoprotein receptor-deficient non-human primates administered a CRISPR base editing therapy targeting the ANGPTL3 gene, the introduction of an optimized GalNAc-based asialoglycoprotein receptor ligand to the nanoparticle surface increased liver editing from 5% to 61% with minimal editing in nontargeted tissues. Similar editing was noted in wild-type monkeys, with durable blood ANGPTL3 protein reduction up to 89% six months post dosing. These results suggest that GalNAc-Lipid nanoparticles may effectively deliver to both patients with intact low-density lipoprotein receptor activity as well as those afflicted by homozygous familial hypercholesterolemia.

Improved 2α-Hydroxylation Efficiency of Steroids by CYP154C2 Using Structure-Guided Rational Design.

Cytochrome P450 enzymes are promising biocatalysts for industrial use because they catalyze site-selective C-H oxidation and have diverse catalytic reactions and a broad substrate range. In this study, the 2α-hydroxylation activity of CYP154C2 from Streptomyces avermitilis MA-4680T toward androstenedione (ASD) was identified by an in vitro conversion assay. The testosterone (TES)-bound structure of CYP154C2 was solved at 1.42 Å, and this structure was used to design eight mutants, including single, double, and triple mutants, to improve the conversion efficiency. Mutants L88F/M191F and M191F/V285L were found to enhance the conversion rates significantly (i.e., 8.9-fold and 7.4-fold for TES, 46.5-fold and 19.5-fold for ASD, respectively) compared with the wild-type (WT) enzyme while retaining high 2α-position selectivity. The substrate binding affinity of the L88F/M191F mutant toward TES and ASD was enhanced compared with that of WT CYP154C2, supporting the measured increase in the conversion efficiencies. Moreover, the total turnover number and kcat/Km of the L88F/M191F and M191F/V285L mutants increased significantly. Interestingly, all mutants containing L88F generated 16α-hydroxylation products, suggesting that L88 in CYP154C2 plays a vital role in substrate selectivity and that the amino acid corresponding to L88 in the 154C subfamily affects the orientation of steroid binding and substrate selectivity. IMPORTANCE Hydroxylated derivatives of steroids play essential roles in medicine. Cytochrome P450 enzymes selectively hydroxylate methyne groups on steroids, which can dramatically change their polarity, biological activity and toxicity. There is a paucity of reports on the 2α-hydroxylation of steroids, and documented 2α-hydroxylate P450s show extremely low conversion efficiency and/or low regio- and stereoselectivity. This study conducted crystal structure analysis and structure-guided rational engineering of CYP154C2 and efficiently enhanced the conversion efficiency of TES and ASD with high regio- and stereoselectivity. Our results provide an effective strategy and theoretical basis for the 2α-hydroxylation of steroids, and the structure-guided rational design of P450s should facilitate P450 applications in the biosynthesis of steroid drugs.

Substantial Improvement of an Epimerase for the Synthesis of D-Allulose byBiosensor-Based High-Throughput Microdroplet Screening.

Biosynthesis of D-allulose has been achieved using ketose 3-epimerases (KEases), but its application is limited by poor catalytic performance. Here, we redesigned a genetically encoded biosensor based on a D-allulose-responsive transcriptional regulator for real-time monitoring of D-allulose. An ultrahigh-throughput droplet-based microfluidic screening platform was further constructed by coupling with this D-allulose-detecting biosensor for the directed evolution of the KEases. Structural analysis of Sinorhizobium fredii D-allulose 3-epimerase (SfDAE) revealed that a highly flexible helix/loop region exposes or occludes the catalytic center as an essential lid conformation regulating substrate recognition. We reprogrammed SfDAE using structure-guided rational design and directed evolution, in which a mutant M3-2 was identified with 17-fold enhanced catalytic efficiency. Our research offers a paradigm for the design and optimization of a biosensor-based microdroplet screening platform.

Development of an enzymatic process for the synthesis of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol, the key intermediate of ticagrelor

Asymmetric bioreductions catalyzed by ketoreductases have shown considerable potential in the synthesis of chiral alcohols. However, the synthesis routes of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL), a key chiral intermediate in the synthesis of ticagrelor, still pose significant challenges in term of unsatisfactory substrate loading and production cycle. In this study, a structure-guided rational design was applied to a ketoreductase from Candida glabrata (CgKR1) for the asymmetric reduction of the ketone precursor 2-chloro-1-(3, 4-difluorophenyl) ethanone (CFPO). A number of mutants were obtained with remarkably improved activity and inversed stereoselectivity compared with CgKR1-WT. In particular, F92V/Y208T mutant and F92T/Y208V mutant exhibited strict S-stereoselectivity and extremely high catalytic activity for CFPO. In addition, CFPO with the concentration of 200 g/L can be completely converted into (S)-CFPL on a preparative scale (5.0 g, 92.8% isolated yield) in 4 h through optimizing some parameters in the reduction process, delivering a high space-time yield (STY) of 1200 g L−1 day−1. This study not only provides insight into the relationship between the structure and function of CgKR1, but also establishes an efficient enzymatic process for the biocatalytic synthesis of crucial chiral intermediate (S)-CFPL of ticagrelor.

H2O2-Driven Hydroxylation of Steroids Catalyzed by Cytochrome P450 CYP105D18: Exploration of the Substrate Access Channel.

CYP105D18 supports H2O2 as an oxygen surrogate for catalysis well and shows high H2O2 resistance capacity. We report the hydroxylation of different steroids using H2O2 as a cosubstrate. Testosterone was regiospecifically hydroxylated to 2β-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (kcat/Km) toward testosterone was increased 1.36-fold in the F184A mutant over that in the wild-type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications. IMPORTANCE The structural modification of steroids is a challenging chemical reaction. Modifying the core ring and the side chain improves the biological activity of steroids. In particular, bacterial cytochrome P450s are used as promiscuous enzymes for the activation of nonreactive carbons of steroids. In the present work, we reported the H2O2-mediated hydroxylation of steroids by CYP105D18, which also overcomes the use of expensive cofactors. Further, exploring the substrate access channel and modifying the bulky amino acid F184A increase substrate conversion while modifying the substrate recognizing amino acid S63 markedly decreases product formation. Exploring the substrate access channel and the rational design of CYP105D18 can improve the substrate conversion, which facilitates the engineering of P450s for industrial application.

Photoinduced Promiscuity of Cyclohexanone Monooxygenase for the Enantioselective Synthesis of α-Fluoroketones.

The development of mild, efficient, and enantioselective methods for preparing chiral fluorinated compounds has been a long-standing challenge. Herein, we report a promiscuous cyclohexanone monooxygenase (CHMO) for the photoinduced synthesis of chiral α-fluoroketones via enantioselective reductive dehalogenation of α,α-halofluoroketones. Wild-type CHMO from Acinetobacter sp. possesses this promiscuous ability innately; however, the yield and stereoselectivity are low. A structure-guided rational design of CHMO improved the yield and stereoselectivity remarkably. Mechanistic studies and molecular simulations demonstrated that this photoinduced CHMO catalyzes the reductive dehalogenation via a novel electron transfer (ET)/proton transfer (PT) mechanism, distinct from that of previously reported reductases with similar promiscuity. This methodology was expanded to various substrates, and desirable chiral α-fluoroketones were obtained in high yields (up to 99 %) and e.r. values (up to 99:1).

Characterization of a new L-carnosine synthase mined from deep-sea sediment metagenome

L-Carnosine is a natural biologically active dipeptide with critical physiological functions, such as antioxidant, antiglycation, and cytoplasmic buffering properties. Direct enzymatic synthesis is a promising way for L-carnosine production. In this study, a new aminopeptidase (gene_236976) with synthetic activity toward L-carnosine was identified by a metagenome mining approach from deep-sea sediment and functionally expressed in Escherichia coli. The enzyme shared a low identity of 14.3% with reported L-carnosine dipeptidase (SmPepD) from Serratia marcescens. β-Alanine methyl ester was proven to be the best substrate for the synthesis, and no ATP was needed for the enzymatic reaction. The enzyme activity was increased by structure-guided rational design. Only the mutant of G310 site gave positive results, and G310A mutant showed the best performance among the site-direct saturation mutagenesis, indicating that the additional CH3 group of mutant G310A was the main factor affecting the enzymatic activity. The engineered enzyme produced about 10 mM L-carnosine was produced from substrates of 50 mM β-alanine methyl ester and 50 mM L-histidine, under a tentatively optimized condition. This study enriched the enzyme resources for developing the microbial synthesis process of L-carnosine production.

Structure-guided rational design of the Geobacillus thermoglucosidasius feruloyl esterase GthFAE to improve its thermostability

Feruloyl esterases are indispensable biocatalysts catalyzing the cleavage of ester bonds between polysaccharides and their hydroxycinnamoyl cross-links. GthFAE from Geobacillus thermoglucosidasius was identified as a thermophilic alkaline feruloyl esterase with potential applications in paper manufacturing. To improve the enzymatic properties rationally and efficiently, the structure of GthFAE was solved at 1.9 Å, revealing a core domain of classical α/β hydrolase fold and an inserted α/β cap domain. In silico analysis based on it helped us to investigate whether the residues at the active center have positive effects on the stability, and how. Several site-directed mutations were conducted, of which substitutions at residues T41 and T150 apparently improved the thermostability. The combination mutant T41N/T150R exhibited an optimal temperature of 65 °C, a 6.4 °C higher Tm compared to wild type by 80 °C, and a 35-fold longer in half-life (201 min) at 70 °C. Molecular dynamics simulations further illustrated that the structure of T41N/T150R was more stable than the wild type and T150R stabilized the cap domain by introducing salt bridges to the region with E154 and D164. This study not only highlighted residues within the active center on their thermostability improving effects, but also contributed to the prospective industrial application of GthFAE.

Improving the catalytic performance of xylanase from Bacillus circulans through structure-based rational design

Endo-1,4-β-xylanase is one of the most important enzymes employed in biorefineries for obtaining fermentable sugars from hemicellulosic components. Herein, we aimed to improve the catalytic performance of Bacillus circulans xylanase (Bcx) using a structure-guided rational design. A systematic analysis of flexible motions revealed that the R49 component of Bcx (i) constrains the global conformational changes essential for substrate binding and (ii) is involved in modulating flexible motion. Site-saturated mutagenesis of the R49 residue led to the engineering of the active mutants with the trade-off between flexibility and rigidity. The most active mutant R49N improved the catalytic performance, including its catalytic efficiency (7.51-fold), conformational stability (0.7 °C improvement), and production of xylose oligomers (2.18-fold higher xylobiose and 1.72-fold higher xylotriose). The results discussed herein can be applied to enhance the catalytic performance of industrially important enzymes by controlling flexibility.

Rational design of geranylgeranyl diphosphate synthase enhances carotenoid production and improves photosynthetic efficiency in Nicotiana tabacum

Restricted genetic diversity can supply only a limited number of elite genes for modern plant cultivation and transgenesis. In this study, we demonstrate that rational design enables the engineering of geranylgeranyl diphosphate synthase (NtGGPPS), an enzyme of the methylerythritol phosphate pathway (MEP) in the model plant Nicotiana tabacum. As the crucial bottleneck in carotenoid biosynthesis, NtGGPPS1 interacts with phytoene synthase (NtPSY1) to channel GGPP into the production of carotenoids. Loss of this enzyme in the ntggpps1 mutant leads to decreased carotenoid accumulation. With the aim of enhancing NtGGPPS1 activity, we undertook structure-guided rational redesign of its substrate binding pocket in combination with sequence alignment. The activity of the designed NtGGPPS1 (a pentuple mutant of five sites V154A/I161L/F218Y/I209S/V233E, d-NtGGPPS1) was measured by a high-throughput colorimetric assay. d-NtGGPPS1 exhibited significantly higher conversion of IPP and each co-substrate (DMAPP ~1995.5-fold, GPP ~25.9-fold, and FPP ~16.7-fold) for GGPP synthesis compared with wild-type NtGGPPS1. Importantly, the transient and stable expression of d-NtGGPPS1 in the ntggpps1 mutant increased carotenoid levels in leaves, improved photosynthetic efficiency, and increased biomass relative to NtGGPPS1. These findings provide a firm basis for the engineering of GGPPS and will facilitate the development of quality and yield traits. Our results open the door for the structure-guided rational design of elite genes in higher plants.

Structure-Guided Rational Design of a Mono- and Diacylglycerol Lipase from Aspergillus oryzae: A Single Residue Mutant Increases the Hydrolysis Ability.

Engineering of enzymes on the basis of protein structures are rational and efficient approaches to acquire biocatalysts of desired performances. In this study, we focused on a special mono- and diacylglycerol lipase (MDGL) isolated from the lipolytic enzyme-enriched fungus Aspergillus oryzae and discovered improved variants based on its crystal structure. We first solved the crystal structure of Aspergillus oryzae lipase (AOL) at 1.7 Å resolution. Structure analysis and sequence alignment of AOL and other MDGLs revealed that the residue V269 is of vital importance for catalysis. Replacement of the V269 in AOL with the corresponding residues in other MDGLs has led to noticeable changes in hydrolysis without sacrificing the thermostability and substrate specificity. Among the investigated variants, V269D exhibited about a six-fold higher hydrolysis activity compared to the wild type. Molecular dynamics simulations and protein-ligand interaction frequency analyses revealed that the Asp substitution enhanced the substrate affinity of AOL. Our work sheds light on understanding the catalytic process of AOL and helps tailoring MDGLs with desired catalytic performance to fulfill the demand for biotechnological applications.

Improved thermostability of proteinase K and recognizing the synergistic effect of Rosetta and FoldX approaches

AbstractProteinase K (PRK) is a proteolytic enzyme that has been widely used in industrial applications. However, poor stability has severely limited the uses of PRK. In this work, we used two structure-guided rational design methods, Rosetta and FoldX, to modify PRK thermostability. Fifty-two single amino acid conversion mutants were constructed based on software predictions of residues that could affect protein stability. Experimental characterization revealed that 46% (21 mutants) exhibited enhanced thermostability. The top four variants, D260V, T4Y, S216Q, and S219Q, showed improved half-lives at 69°C by 12.4-, 2.6-, 2.3-, and 2.2-fold that of the parent enzyme, respectively. We also found that selecting mutations predicted by both methods could increase the predictive accuracy over that of either method alone, with 73% of the shared predicted mutations resulting in higher thermostability. In addition to providing promising new variants of PRK in industrial applications, our findings also show that combining these programs may synergistically improve their predictive accuracy.

Rational Design of a Humanized Antibody Inhibitor of Cathepsin B.

Cathepsin B (CTSB) is an abundant cysteine protease that functions in both endolysosomal compartments and extracellular regions. A considerable number of preclinical and clinical studies indicate that CTSB is implicated in many human diseases. Expression levels and activity of CTSB significantly correlate with disease progression and severity. Current inhibitors of CTSB are lack of adequate specificity and pharmacological activities. Through structure-guided rational design, we hereby designed and generated a humanized antibody inhibitor targeting human CTSB. This was achieved by genetically fusing the propeptide of procathepsin B, a naturally occurring inhibitor of CTSB, into heavy chain complementarity-determining region 3 (CDR3H) of Herceptin that is used in the clinic for the treatment of breast cancer. The resulting antibody-propeptide fusion displayed high specificity for inhibiting CTSB proteolytic activity at nanomolar levels. Pharmacokinetic studies in mice revealed a plasma half-life of approximately 42 h for this anti-CTSB antibody inhibitor, comparable to that of the parental Herceptin scaffold. This study demonstrates a new approach for the efficient generation of humanized antibody inhibitors with high potency and specificity for human CTSB, which may be extended to develop antibody inhibitors against other disease relevant cathepsin proteases.

Structure-guided evolution of carbonyl reductase for efficient biosynthesis of ethyl (R)-2-hydroxy-4-phenylbutyrate

This study reported an attractive engineered carbonyl reductase fromGluconobacter oxydansthrough a structure-guided rational design to catalyze the synthesis of high concentration (R)-HPBE.