Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

Mass Activated Droplet Sorting (MADS) Enables High-Throughput Screening of Enzymatic Reactions at Nanoliter Scale.

Microfluidic droplet sorting enables the high-throughput screening and selection of water-in-oil microreactors at speeds and volumes unparalleled by traditional well-plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high-throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI-MS). Droplets are split, one portion is analyzed by ESI-MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s-1 is achieved with 98 % accuracy using a self-correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label-free ESI-MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.

Identification of sirtuin 5 inhibitors by ultrafast microchip electrophoresis using nanoliter volume samples.

Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that catalyzes removal of post-translational modifications, such as succinyl and malonyl moieties, on lysine residues. In light of SIRT5's roles in regulating metabolism, and its reported oncogenic functions, SIRT5 modulators would be valuable tools for basic biological research and perhaps clinically. Several fluorescence assays for sirtuin modulators have been developed; however, the use of fluorogenic substrates has the potential to cause false positive results due to interactions of engineered substrates with enzyme or test compounds. Therefore, development of high-throughput screening (HTS) assays based on other methods is valuable. In this study, we report the development of a SIRT5 assay using microchip electrophoresis (MCE) for identification of SIRT5 modulators. A novel SIRT5 substrate based on succinate dehydrogenase (SDH) was developed to allow rapid and efficient separation of substrate and product peptide. To achieve high throughput, samples were injected onto the microchip using a droplet-based scheme. By coupling this approach to existing HTS sample preparation workflows, 1408 samples were analyzed at 0.5 Hz in 46 min. Using a 250 ms separation time, eight MCE injections could be made from each sample generating >11,000 electropherograms during analysis. Of the 1280 chemicals tested, eight were identified as inhibiting SIRT5 activity by at least 70% and verified by dose-response analysis.

An Inexpensive, Open-Source USB Arduino Data Acquisition Device for Chemical Instrumentation.

Many research and teaching labs rely on USB data acquisition devices to collect voltage signals from instrumentation. However, these devices can be cost-prohibitive (especially when large numbers are needed for teaching labs) and require software to be developed for operation. In this article, we describe the development and use of an open-source USB data acquisition device (with 16-bit acquisition resolution) built using simple electronic components and an Arduino Uno that costs under $50. Additionally, open-source software written in Python is included so that data can be acquired using nearly any PC or Mac computer with a simple USB connection. Use of the device was demonstrated for a sophomore-level analytical experiment using GC and a CE-UV separation on an instrument used for research purposes.

Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators.

High-throughput screening (HTS) using multiwell plates and fluorescence plate readers is a powerful tool for drug discovery and evaluation by allowing tens of thousands of assays to be completed in 1 day. Although this method has been successful, electrophoresis-based methods for screening are also of interest to avoid difficulties associated fluorescence assays such as requirements to engineer fluorogenic reactions and false positives. We have developed a method using droplet microfluidics to couple multiwell plate-based assays to microchip electrophoresis (MCE) to screen enzyme modulators. Samples contained in multiwell plates are reformatted in to plugs with a sample volume of 8 nL segmented by an immiscible oil. The segmented flow sample streams are coupled to a hybrid polydimethylsiloxane-glass microfluidic device capable of selectively extracting the aqueous samples from the droplet stream and rapidly analyzing by MCE with laser-induced fluorescence detection. This system was demonstrated by screening a test library of 140 compounds against using protein kinase A. For each sample in the screen, two droplets are generated, allowing approximately 6 MCE injections per sample. Using a 1 s separation at 2000 V/cm, we are able to analyze 96 samples in 12 min. Separation resolution between the internal standard, substrate, and product is 1.2 and average separation efficiency is 16,000 plates/s using real samples. Twenty-five compounds were identified as modulators during primary screening and verified using dose-response curves.

Detection of prolactin inducible protein mRNA, a biomarker for breast cancer metastasis, using a molecular beacon-based assay.

Mortality due to breast cancer is increasingly linked to early, undetected metastasis, making methods for earlier detection acutely necessary. We describe the development of an assay based on molecular beacon (MB) chemistry with fluorescence detection to monitor a breast cancer biomarker for the analysis of breast cancer metastasis. The MB assay is based on the complementary base-pairing interactions of the MB nucleic acid with mRNA indicative of breast cancer metastasis. The presence of mRNA is characterized by an increase in the fluorescence intensity of the molecular beacon. The assay gives a linear, reproducible response to prolactin inducible protein mRNA, with a limit of detection in the high picomolar range. This method sensitively and specifically identifies a biomarker directly in serum samples in minimal time and with a straightforward procedure, dramatically reducing the total time for sample analysis over current methods from days to hours. The potential impact of this work in detection and understanding of breast cancer metastasis lies in improvements in simplicity, accuracy, and speed over current methods, which could allow for improved patient treatment and prognoses. Ultimately, additional sample throughput will result in better understanding of disease progression.

Quantitation and speciation of residual protein within active pharmaceutical ingredients using image analysis with SDS-PAGE.

Recent advances in biocatalysis and directed enzyme evolution has led to a variety of enzymatically-driven, elegant processes for active pharmaceutical ingredient (API) production. For biocatalytic processes, quantitation of any residual protein within a given API is of great importance to ensure process robustness and quality, pure pharmaceutical products. Typical analytical methods for analyzing residual enzymes within an API, such as enzyme-linked immunosorbent assays (ELISA), colorimetric assays, and liquid chromatographic techniques, are limited for determining only the concentration of known proteins and require harsh solvents with high API levels for analysis. For the first time, total residual protein content in a small molecule API was quantitated using image analysis applied to SDS-PAGE. Herein, a proposed methodology for residual protein detection, quantitation, and size-based speciation is presented, in which an orthogonal technique is offered to traditional analysis methods, such as ELISA. Results indicate that our application of the analytical methodology is able to reliably quantitate both protein standards and the total residual protein present within a final API, with good agreement as compared to traditional ELISA results. Further, speciation of the residual protein within the API provides key information concerning the individual residual proteins present, including their molecular weight, which can lead to improved process development efforts for residual protein rejection and control. This analytical methodology thus offers an alternative tool for easily identifying, quantitating, and speciating residual protein content in the presence of small molecule APIs, with potential for wide applicability across industry for biocatalytic or directed enzyme evolution efforts within process development.

The effect of co-surfactant-modified micelles on chiral separations in EKC.

The use of chiral pseudostationary phases in EKC provides high efficiencies and excellent resolution for enantiomeric separations. The chiral pseudostationary phases of interest in this study are alcohol-modified ("swollen") micelles, in which a co-surfactant (medium chain-length alcohol) is added with the surfactant. In this study, the chiral surfactant, dodecoxycarbonylvaline (DDCV), along with the co-surfactant, 2-hexanol, has been prepared as swollen micelle in order to investigate the chiral separation of enantiomeric pairs. Three sets of experiments were investigated in which swollen micelle systems contained: chiral surfactant and racemic co-surfactant; chiral surfactant and chiral co-surfactant; and phase ratio increases, in which both chiral surfactant and chiral co-surfactant were employed. In the first two sets of experiments, co-surfactant concentration was held constant and the surfactant concentration was increased. In the third set of experiments, both surfactant and chiral surfactant concentrations were increased proportionally. The chromatographic figures of merit for each enantiomeric pair were investigated and compared with various chiral aggregate systems. In swollen micelle compositions using constant racemic 2-hexanol concentration, when DDCV concentration increased, enantioselectivity and resolution increased; whereas, efficiency remained constant for most of the test compounds. Compositions using constant S-2-hexanol concentration reached a maximum in all chromatographic figures of merit when DDCV concentration was increased from 2 to 3%. An increase in both surfactant and co-surfactant concentrations led to noisy baselines and chiral aggregates that were generally unstable in solution.

Combination of circular dichroism spectroscopy and size-exclusion chromatography coupled with HDX-MS for studying global conformational structures of peptides in solution.

Misfolding of therapeutic peptides and proteins can lead to numerous issues, ranging in severity, including loss of function, aggregation, immunogenicity, and cytotoxicity. A primary component of protein folding is secondary structure, including α-helices and ß-sheets. Many native peptides and proteins are predominately α-helical therefore, it is of critical importance to develop robust and reliable analytical tools to investigate protein higher order structure, including the percentage of α-helix under various conditions, to evaluate protein folding and prevent the negative effects of misfolding. However, given the complexity of protein folding and higher order structure, it is unlikely that one technique will provide a comprehensive analysis. To bridge this gap, this study presents the combination of two orthogonal techniques - circular dichroism (CD) and size-exclusion chromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS) to investigate global peptide and protein conformations. Also, the incorporation of trifluoroethanol (TFE), a known stabilizer of α-helical structures, into the analyses, aims to enhance the discrimination power of these two techniques by increasing the alpha helical stability range of study. CD data was used to estimate the percent of α-helix content and its thermal stability while online SEC-HDX-MS screening compared global conformational changes of each peptide based on a difference in the number of deuterons exchanged to protons, ΔHDX. The workflow described in this report can be very beneficial in pharmaceutical development. The model peptides were chosen to demonstrate the workflow with commercially available compounds. The goal of this study was to show a proof-of-concept for direct correlation of these methodologies and to estimate the percentage of α-helix content at a particular ΔHDX, which is indicative of the state of protein folding.

Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry.

Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chemistry development where solutions are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet-MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)-MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10-50 nL) by tuning the sheath flow within the CE-MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (r2 ∼ 0.95) were found between the droplet-MS and LC-MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription-translation inside the droplets and directly analyzing the intact reaction mixture droplets by MS. The synthesized protein attained comparable activity to the protein standard, and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS analysis of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries.