The PARTNER trial of neoadjuvant olaparib with chemotherapy in triple-negative breast cancer.

PARTNER is a prospective, phase II-III, randomized controlled clinical trial that recruited patients with triple-negative breast cancer1,2, who were germline BRCA1 and BRCA2 wild type3. Here we report the results of the trial. Patients (n = 559) were randomized on a 1:1 basis to receive neoadjuvant carboplatin-paclitaxel with or without 150 mg olaparib twice daily, on days 3 to 14, of each of four cycles (gap schedule olaparib, research arm) followed by three cycles of anthracycline-based chemotherapy before surgery. The primary end point was pathologic complete response (pCR)4, and secondary end points included event-free survival (EFS) and overall survival (OS)5. pCR was achieved in 51% of patients in the research arm and 52% in the control arm (P = 0.753). Estimated EFS at 36 months in the research and control arms was 80% and 79% (log-rank P > 0.9), respectively; OS was 90% and 87.2% (log-rank P = 0.8), respectively. In patients with pCR, estimated EFS at 36 months was 90%, and in those with non-pCR it was 70% (log-rank P < 0.001), and OS was 96% and 83% (log-rank P < 0.001), respectively. Neoadjuvant olaparib did not improve pCR rates, EFS or OS when added to carboplatin-paclitaxel and anthracycline-based chemotherapy in patients with triple-negative breast cancer who were germline BRCA1 and BRCA2 wild type. ClinicalTrials.gov ID: NCT03150576 .

Fibrolamellar carcinoma: Challenging the challenge.

Fibrolamellar carcinoma (FLC) is a rare and poorly understood malignancy, which seems to be more prevalent in young patients compared with conventional hepatocellular carcinoma (HCC). Performing prospective clinical trials recruiting patients diagnosed with FLC has proven challenging with scarce data available guiding clinical management. The use of a number of chemotherapy compounds in these patients, including cisplatin, epirubicin, 5-fluorouracil (5-FU) and recombinant interferon α-2B (IFN-α-2B), has been reported in the literature, mainly in the form of case reports. The most promising systemic therapy tested so far is the combination of 5-FU infusion and 3-weekly IFN-α-2B, based on results from a phase II clinical trial. This article provides an overview of our own experience with this treatment schedule for patients with FLC, confirming its activity and treatment-derived benefit in the real world. Current challenges being faced by healthcare professionals treating patients with advanced FLC are discussed, especially the increasingly limited access to IFN-α-2B, which could compromise the access to an active therapy in the coming future, and the difficulties in the development of new treatment options for advanced FLC.

Systematically Scrutinizing the Impact of Substitution Sites on Thermostability and Detergent Tolerance for Bacillus subtilis Lipase A.

Improving an enzyme's (thermo-)stability or tolerance against solvents and detergents is highly relevant in protein engineering and biotechnology. Recent developments have tended toward data-driven approaches, where available knowledge about the protein is used to identify substitution sites with high potential to yield protein variants with improved stability, and subsequently, substitutions are engineered by site-directed or site-saturation (SSM) mutagenesis. However, the development and validation of algorithms for data-driven approaches have been hampered by the lack of availability of large-scale data measured in a uniform way and being unbiased with respect to substitution types and locations. Here, we extend our knowledge on guidelines for protein engineering following a data-driven approach by scrutinizing the impact of substitution sites on thermostability or/and detergent tolerance for Bacillus subtilis lipase A (BsLipA) at very large scale. We systematically analyze a complete experimental SSM library of BsLipA containing all 3439 possible single variants, which was evaluated as to thermostability and tolerances against four detergents under respectively uniform conditions. Our results provide systematic and unbiased reference data at unprecedented scale for a biotechnologically important protein, identify consistently defined hot spot types for evaluating the performance of data-driven protein-engineering approaches, and show that the rigidity theory and ensemble-based approach Constraint Network Analysis yields hot spot predictions with an up to ninefold gain in precision over random classification.

Identification of patients with pancreatic adenocarcinoma due to inheritable mutation: Challenges of daily clinical practice.

BACKGROUND: Identification of germ-line mutations in pancreatic ductal adenocarcinoma (PDAC) could impact on patient/family. AIM: To assess the referral pathways for genetic consultations in PDAC. METHODS: Electronic records of PDAC patients were reviewed retrospectively. Patients eligible for genetic consultation referral were identified following the European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer (EUROPAC) criteria. RESULTS: Four-hundred patients were eligible. Of 113 patients (28.3%) meeting EUROPAC criteria, 8.8% were referred for genetic opinion. Germ-line mutations were identified in 0.75% of the whole population. CONCLUSION: Earlier referrals and increased awareness may be able to overcome the low rate of successful genetic appointments.

Unraveling the effects of amino acid substitutions enhancing lipase resistance to an ionic liquid: a molecular dynamics study.

Understanding of the structural and dynamic properties of enzymes in non-aqueous media (e.g., ionic liquids, ILs) is highly attractive for protein engineers and synthetic biochemists. Despite a growing number of molecular dynamics (MD) simulation studies on the influence of different ILs on wild-type enzymes, the effects of various amino acid substitutions on the stability and activity of enzymes in ILs remain to be unraveled at the molecular level. Herein, we selected fifty previously reported Bacillus subtilis lipase A (BSLA) variants with increased resistance towards an IL (15 vol% 1-butyl-3-methylimidazolium trifluoromethanesulfonate; [Bmim][TfO]), and also ten non-resistant BSLA variants for a MD simulation study to identify the underlying molecular principles. Some important properties differentiating resistant and non-resistant BSLA variants from wild-type were elucidated. Results show that, in 15 vol% [Bmim][TfO] aqueous solution, 40% and 60% of non-resistant variants have lower and equal probabilities to form a catalytically important hydrogen bond between S77 and H156 compared to wild-type, whereas 36% and 56% of resistant variants show increased and equal probabilities, respectively. Introducing positively charged amino acids close to the substrate-binding cleft for instance I12R is beneficial for the BSLA resistance towards 15 vol% [Bmim][TfO], likely due to the reduced probability of [Bmim]+ cations clustering near the cleft. In contrast, substitution with a large hydrophobic residue like I12F can block the cleft through hydrophobic interaction with a neighboring nonpolar loop 134-137 or/and an attractive π-π interaction with [Bmim]+ cations. In addition, the resistant variants having polar substitutions on the surface show higher ability to stabilize the surface water molecule network in comparison to non-resistant variants. This study can guide experimentalists to rationally design promising IL-resistant enzymes, and contribute to a deeper understanding of protein-IL interactions at the molecular level.

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains.

Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7-RNA polymerase-dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315-327, 2018.

High-Throughput Screening Assays for Lipolytic Enzymes.

Screening is defined as the identification of hits within a large library of variants of an enzyme or protein with a predefined property. In theory, each variant present in the respective library needs to be assayed; however, to save time and consumables, many screening regimes involve a primary round to identify clones producing active enzymes. Such primary or prescreenings for lipolytic enzyme activity are often carried out on agar plates containing pH indicators or substrates as triolein or tributyrin. Subsequently, high-throughput screening assays are usually performed in microtiter plate (MTP) format using chromogenic or fluorogenic substrates and, if available, automated liquid handling robotics. Here, we describe different assay systems to determine the activity and enantioselectivity of lipases and esterases as well as the synthesis of several substrates. We also report on the construction of a complete site saturation library derived from lipase A of Bacillus subtilis and its testing for detergent tolerance. This approach allows for the identification of amino acids affecting sensitivity or resistance against different detergents.

Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis.

BACKGROUND: Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. RESULTS: In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. CONCLUSIONS: Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.

A combination of mutational and computational scanning guides the design of an artificial ligand-binding controlled lipase.

Allostery, i.e. the control of enzyme activity by a small molecule at a location distant from the enzyme's active site, represents a mechanism essential for sustaining life. The rational design of allostery is a non-trivial task but can be achieved by fusion of a sensory domain, which responds to environmental stimuli with a change in its structure. Hereby, the site of domain fusion is difficult to predict. We here explore the possibility to rationally engineer allostery into the naturally not allosterically regulated Bacillus subtilis lipase A, by fusion of the citrate-binding sensor-domain of the CitA sensory-kinase of Klebsiella pneumoniae. The site of domain fusion was rationally determined based on whole-protein site-saturation mutagenesis data, complemented by computational evolutionary-coupling analyses. Functional assays, combined with biochemical and biophysical studies suggest a mechanism for control, similar but distinct to the one of the parent CitA protein, with citrate acting as an indirect modulator of Triton-X100 inhibition of the fusion protein. Our study demonstrates that the introduction of ligand-dependent regulatory control by domain fusion is surprisingly facile, suggesting that the catalytic mechanism of some enzymes may be evolutionary optimized in a way that it can easily be perturbed by small conformational changes.

Application of Rigidity Theory to the Thermostabilization of Lipase A from Bacillus subtilis.

Protein thermostability is a crucial factor for biotechnological enzyme applications. Protein engineering studies aimed at improving thermostability have successfully applied both directed evolution and rational design. However, for rational approaches, the major challenge remains the prediction of mutation sites and optimal amino acid substitutions. Recently, we showed that such mutation sites can be identified as structural weak spots by rigidity theory-based thermal unfolding simulations of proteins. Here, we describe and validate a unique, ensemble-based, yet highly efficient strategy to predict optimal amino acid substitutions at structural weak spots for improving a protein's thermostability. For this, we exploit the fact that in the majority of cases an increased structural rigidity of the folded state has been found as the cause for thermostability. When applied prospectively to lipase A from Bacillus subtilis, we achieved both a high success rate (25% over all experimentally tested mutations, which raises to 60% if small-to-large residue mutations and mutations in the active site are excluded) in predicting significantly thermostabilized lipase variants and a remarkably large increase in those variants' thermostability (up to 6.6°C) based on single amino acid mutations. When considering negative controls in addition and evaluating the performance of our approach as a binary classifier, the accuracy is 63% and increases to 83% if small-to-large residue mutations and mutations in the active site are excluded. The gain in precision (predictive value for increased thermostability) over random classification is 1.6-fold (2.4-fold). Furthermore, an increase in thermostability predicted by our approach significantly points to increased experimental thermostability (p < 0.05). These results suggest that our strategy is a valuable complement to existing methods for rational protein design aimed at improving thermostability.

A particular silent codon exchange in a recombinant gene greatly influences host cell metabolic activity.

BACKGROUND: Recombinant protein production using Escherichia coli as expression host is highly efficient, however, it also induces strong host cell metabolic burden. Energy and biomass precursors are withdrawn from the host's metabolism as they are required for plasmid replication, heterologous gene expression and protein production. Rare codons in a heterologous gene may be a further drawback. This study aims to investigate the influence of particular silent codon exchanges within a heterologous gene on host cell metabolic activity. Silent mutations were introduced into the coding sequence of a model protein to introduce all synonymous arginine or leucine codons at two randomly defined positions, as well as substitutions leading to identical amino acid exchanges with different synonymous codons. The respective E. coli clones were compared during cultivation in a mineral autoinduction medium using specialized online and offline measuring techniques to quantitatively analyze effects on respiration, biomass and protein production, as well as on carbon source consumption, plasmid copy number, intracellular nucleobases and mRNA content of each clone. RESULTS: Host stain metabolic burden correlates with recombinant protein production. Upon heterologous gene expression, tremendous differences in respiration, biomass and protein production were observed. According to their different respiration activity the E. coli clones could be classified into two groups, Type A and Type B. Type A clones tended to higher product formation, Type B clones showed stronger biomass formation. Whereas codon usage and intracellular nucleobases had no influence on the Type-A-Type-B-behavior, plasmid copy number, mRNA content and carbon source consumption strongly differed between the two groups. CONCLUSIONS: Particular silent codon exchanges in a heterologous gene sequence led to differences in initial growth of Type A and Type B clones. Thus, the biomass concentration at the time point of induction varied. In consequence, not only plasmid copy number and expression levels differed between the two groups, but also the kinetics of lactose and glycerol consumption. Even though the underlying molecular mechanisms are not yet identified we observed the astonishing phenomenon that particular silent codon exchanges within a heterologous gene tremendously affect host cell metabolism and recombinant protein production. This could have great impact on codon optimization of heterologous genes, screening procedures for improved variants, and biotechnological protein production processes.

Exploring the protein stability landscape: Bacillus subtilis lipase A as a model for detergent tolerance.

A systematic study was conducted with Bacillus subtilis lipase A (BSLA) to determine the effect of every single amino acid substitution on detergent tolerance. BSLA is a minimal α/ß-hydrolase of 181 amino acids with a known crystal structure. It can be expressed in Escherichia coli and is biochemically well characterized. Site saturation mutagenesis resulted in a library of 3439 variants, each with a single amino acid exchange as confirmed by DNA sequencing. The library was tested against four detergents, namely SDS, CTAB, Tween 80, and sulfobetaine. Surface remodeling emerged as an effective engineering strategy to increase tolerance towards detergents. Amino acid residues that significantly affect the tolerance for each of the four detergents were identified. In summary, this systematic analysis provides an experimental dataset to help derive novel protein engineering strategies as well as to direct modeling efforts.

Towards understanding directed evolution: more than half of all amino acid positions contribute to ionic liquid resistance of Bacillus subtilis lipase A.

Ionic liquids (ILs) are attractive (co-)solvents for biocatalysis. However, in high concentration (>10 % IL), enzymes usually show decreased activity. No general principles have been discovered to improve IL resistance of enzymes by protein engineering. We present a systematic study to elucidate general engineering principles by site saturation mutagenesis on the complete gene bsla. Screening in presence of four [BMIM]-based ILs revealed two unexpected lessons on directed evolution: 1) resistance improvement was obtainable at 50-69 % of all amino acid positions, thus explaining the success of small sized random mutant libraries; 2) 6-13 % of substitutions led to improved resistance. Among these, 66-95 % were substitutions by chemically different amino acids (e.g., aromatic to polar/aliphatic/charged amino acids), thus indicating that mutagenesis methods introducing such changes should, at least for lipases like BSLA, be favored to improve IL resistance.

Exchange of single amino acids at different positions of a recombinant protein affects metabolic burden in Escherichia coli.

BACKGROUND: Escherichia coli is commonly used in academia and industry for expressing recombinant proteins because of its well-characterized molecular genetics and the availability of numerous expression vectors and strains. One important issue during recombinant protein production is the so-called 'metabolic burden': the material and energy normally reserved for microbial metabolism which is sapped from the bacterium to produce the recombinant protein. This material and energy drain harms biomass formation and modifies respiration. To the best of our knowledge, no research has investigated so far whether a single amino acid exchange in a recombinant protein affects the metabolic burden phenomenon. Thus, in this study, 15 E. coli BL21(DE3) clones expressing either the fusion tags, a recombinant wild type lipase, or 13 different lipase variants are investigated to quantitatively analyze the respective effects of single amino acid exchanges at different positions on respiration, biomass and protein production of each clone. Therefore, two small-scale online monitoring systems, namely a Respiration Activity MOnitoring System (RAMOS) and a microtiter plate based cultivation system (BioLector) are applied. RESULTS: Upon expression of all enzyme variants, strong variations were found in the Oxygen Transfer Rate (OTR), biomass and protein (lipase) production of the respective E. coli clones. Two distinct patterns of respiration behavior were observed and, so, the clones could be classified into two groups (Type A and B). Potential factors to explain these patterns were evaluated (e.g. plasmid copy number, inclusion body formation). However, no decisive factor could yet be identified. Five distinct cultivation phases could be determined from OTR curves which give real-time information about carbon source consumption, biomass and protein production. In general, it was found that the quantity of product increased with the duration of active respiration. CONCLUSIONS: This work demonstrates that single amino acid exchanges in a recombinant protein influence the metabolic burden during protein production. The small-scale online monitoring devices RAMOS and BioLector enable the real-time detection of even smallest differences in respiration behavior, biomass and protein production in the E. coli clones investigated. Hence, this study underscores the importance of parallel online monitoring systems to unveil the relevance of single amino acid exchanges for the recombinant protein production.

Optimal scanning of all single-point mutants of a protein.

In protein engineering, useful information may be gained from systematically generating and screening all single-point mutants of a given protein. We model and analyze an iterative two-stage procedure to generate all these mutants. At each position, L variants are generated in the first stage via saturation mutagenesis and are sequenced. In the second stage, the missing variants (out of the 19 possible single-point substitutions) are produced via site-directed mutagenesis. We study the economic tradeoff associated with varying L, and derive its optimal value given the experimental parameters.