Bacteriophage-derived lysin proteins are potentially effective antimicrobials that would benefit from engineered improvements to their bioavailability and specific activity. Here, the catalytic domain of LysEFm5, a lysin with activity against vancomycin-resistant Enterococcus faecium (VRE), was subjected to site-saturation mutagenesis at positions whose selection was guided by sequence and structural information from homologous proteins. A second-order Potts model with parameters inferred from large sets of homologous sequence information was used to predict the average change in the statistical fitness for mutant libraries with diversity at pairs of sites within the secondary catalytic shell. Guided by the statistical fitness, nine double mutant saturation libraries were created and plated on agar containing autoclaved VRE to quickly identify and segregate catalytically active (halo-forming) and inactive (non-halo-forming) variants. High-throughput DNA sequencing of 873 unique variants showed that the statistical fitness was predictive of the retention or loss of catalytic activity (area under the curve [AUC], 0.840 to 0.894), with the inclusion of more diverse sequences in the starting multiple-sequence alignment improving the classification accuracy when pairwise amino acid couplings (epistasis) were considered. Of eight random halo-forming variants selected for more sensitive testing, one showed a 1.8 (±0.4)-fold improvement in specific activity and an 11.5 ± 0.8°C increase in melting temperature compared to those of the wild type. Our results demonstrate that a computationally informed approach employing homologous protein information coupled with a mid-throughput screening assay allows for the expedited discovery of lysin variants with improved properties. IMPORTANCE Broad-spectrum antibiotics can indiscriminately kill most bacteria, including commensal species that are a part of the normal human flora. This can potentially lead to the proliferation of drug-resistant bacteria upon elimination of competing species and to unwanted autoimmune effects in patients. Bacteriophage-derived lysin proteins are an alternative to conventional antibiotics that have coevolved alongside specific bacterial hosts. Lysins are capable of targeting conserved substrates in the bacterial cell wall essential for its viability. To engineer these proteins to exhibit improved therapeutically relevant properties, homology-guided statistical approaches can be used to identify compelling sites for mutation and to quantify the functional constraints acting on these sites to direct mutagenic library creation. The platform described herein couples this informed approach with a visual plate assay that can be used to simultaneously screen hundreds of mutants for catalytic activity, allowing for the streamlined identification of improved lysin variants.

Poor medication adherence is a pervasive issue with considerable health and socioeconomic consequences. Although the underlying reasons are generally understood, traditional intervention strategies rooted in patient-centric education and empowerment have proved to be prohibitively complex and/or ineffective. Formulating a pharmaceutical in a drug delivery system (DDS) is a promising alternative that can directly mitigate many common impediments to adherence, including frequent dosing, adverse effects and a delayed onset of action. Existing DDSs have already positively influenced patient acceptability and improved rates of adherence across various disease and intervention types. The next generation of systems have the potential to instate an even more radical paradigm shift by, for example, permitting oral delivery of biomacromolecules, allowing for autonomous dose regulation and enabling several doses to be mimicked with a single administration. Their success, however, is contingent on their ability to address the problems that have made DDSs unsuccessful in the past.

Despite the success of therapeutics and prophylactics in prolonging life and improving quality of life, these benefits are limited by poor patient adherence, which can be as low as 50% in patients with chronic conditions. [3][4][5] This lack of patient adherence contributes to negative outcomes, including death, and results in an additional $289 billion in healthcare costs each year in the United States alone. [6][7][8] Reducing drug dosing frequency has been identified as one of the most effective means to increase patient adherence. [9,10] However, many diseases including diabetes, cancer, human immunodeficiency virus infection, depression, and autoimmune disorders, are typically treated with frequent, repeated, and longterm administration of therapeutics, often as frequently as multiple times a day, to maintain drug levels that are both safe and effective. Controlled drug delivery systems represent a promising solution to mitigate compliance issues. By releasing drugs over an extended period of time, these systems can be administered less frequently, thereby improving adherence and patient outcomes. For example, the FDA-approved Lupron Depot, composed of drug-loaded biodegradable microspheres, has been shown to improve patient adherence and convenience by reducing administration frequency from a once-daily injection to one injection every one to six months. [11,12] Oral delivery systems are convenient, but their rapid passage through the gastrointestinal tract limits their duration of action, often requiring frequent re-dosing that can lead to lower levels of patient adherence compared to less frequent parenteral injection(s). [13] Unfortunately, most injectable controlled-release systems generate an initial burst release followed by first-order release kinetics in which drug is released at a perpetually lower rate over time. [14,15] Although these devices extend the duration of drug activity, their front-loaded and slowing rate of release limits their ability to maintain therapeutic efficacy over a long period of time, especially when the biological half-life of the drug is short or the therapeutic window is small. Increasing initial drug loading can extend the duration of release in these Pulsatile drug delivery systems have the potential to improve patient adherence and therapeutic efficacy by providing a sequence of doses in a single injection. Herein, a novel platform, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED) is developed, which enables the high-throughput fabrication of microparticles exhibiting pulsatile release. In PULSED, biodegradable polymeric microstructures with an open cavity are formed using high-resolution 3D printing and soft lithography, filled with drug, and sealed using a contactless heating step in which the polymer flows over the orifice to form a complete shell around a drug-loaded core. Poly(lactic-co-glycolic acid) particles with this structure can rapidly release encapsulated material after delays of 10 ± 1, 15 ± 1, 17 ± 2, or 36 ± 1 days in vivo, ...

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