abstract

The Human Genome Project ultimately aimed to translate DNA sequence into drugs. With the draft in hand, the Molecular Libraries Program set out to prosecute all genome-encoded proteins for drug discovery with automated high-throughput screening (HTS). This ambitious vision remains unfulfilled, even while innovations in sequencing technology have fully democratized access to genome-scale sequencing. Why? While the central dogma of biology allows us to chart the entirety of cellular metabolism through sequencing, there is no direct coding for chemistry. The rules of base pairing that relate DNA gene to RNA transcript and amino acid sequence do not exist for relating small molecule structure with macromolecular binding partner and subsequently cellular function. Obtaining such relationships genome-wide is unapproachable via state-of-the-art HTS, akin to attempting genome-wide association studies using turn-of-the-millennium Sanger DNA sequencing.

Our laboratory has been engaged in a multi-pronged technology development campaign to revolutionize molecular screening through miniaturization in pursuit of genome-scale drug discovery capabilities. We employed DNA-encoded library (DEL) synthesis principles in the development of solid-phase DELs prepared on microscopic beads, each harboring 100 fmol of a single library member and a DNA tag whose sequence describes the structure of the library member. Loading these DEL beads into 100-pL microfluidic droplets followed by on-line photocleavage, incubation, fluorescence-activated droplet sorting, and DNA sequencing of the sorted DEL beads reveals the chemical structures of bioactive compounds. This scalable library synthesis and screening platform has proven useful in several proof-of-concept projects involving current clinical targets. Moving forward, we face the problem of druggability and proteome-scale assay development. Developing biochemical or cellular assays for all genome-encoded targets is not scalable and likely impossible as most proteins have ill-defined or unknown activity and may not function outside of their native context.

In this dissertation I describe the utility of selective translation modulation as a modality that circumvents canonical druggability constraints while also raising the prospect of in vitro transcription translation (IVTT) as a universal biochemical assay for screening. In Chapter 2, I present the development of an IVTT activity assay by fusing a GFP reporter to various gene sequences that are known to be selectively inhibited by compound PF846. Wheat germ IVTT was miniaturized to microfluidic droplets and demonstrated to exhibit excellent assay quality both in response to positive control PF846 and beads displaying a photocleavable broad-spectrum translation inhibitor, hygromycin B. Chapter 3 describes the screening of a 5,348-member DEL of custom translation inhibitor analogs. Screening a proof-of-concept PCSK9-GFP reporter yielded several hits that selectively inhibited PCSK9-GFP IVTT. Chapter 4 details the screening of an unbiased 55,296-member DEL to discover novel translation inhibitor chemotypes. These studies set the stage for scaling microfluidic IVTT DEL screening to the human proteome, irrespective of mature protein function, to fulfill the Genome Project's vision of proteome-wide control over cellular pharmacology.

Recorded ground motions are traditionally used to represent future earthquakes for the seismic performance assessment and design of structures. However, there is a shortage of recorded ground motions that are able to represent a variety of possible scenarios (specifically large magnitude and short distance events). Structural engineers are hesitant in using simulated ground motions as surrogates for missing natural recordings, believing they are not equivalent to recorded ground motions in accurately estimating seismic demand in structures. This research recommends a validation test for simulated ground motions to increase the engineering community’s confidence in using them in engineering applications.

This dissertation first introduces a novel statistical approach that can be used in current seismic performance assessment methods. Gaussian copulas are used to characterize the dependence structure of demand used for the assessment and calculation of loss in buildings due to earthquakes. Using Gaussian copulas is shown to increase the overall accuracy in seismic performance assessment methodology compared to the procedure that is currently being used, outlined by FEMA P-58 (2015). Following the proposal of a more accurate method to characterize structural demand used in seismic performance assessment, a three step methodology, titled Vector-Based Intensity-measure Method (VBIM), for ground motion simulation validation at the structural response level is proposed. Three case study structures are used for the application of VBIM: 2- and 12-story special steel moment-resisting frame (SMRF) buildings and a two-span, cast-in-place concrete bridge. Results indicate that models of simulated and recorded ground motions that predict structural response based on waveform parameters are similar. The results of this study provide recommendations for ground motion simulators regarding the required accuracy of these key parameters in order for simulated ground motions to accurately predict structural response while also providing several steps of validation that show similarities between recorded and simulated ground motions.

Diffuse reflectance infrared Fourier transform spectrometry (DRIFTS) is a powerful technique for analyzing solid powders and for following their reactions in real time. We demonstrate that it can also be applied to studying the uptake and reactions of gases in liquid films. Within the DRIFTS cell, a 10%(w/w) mixture of MgCl(2) x 6H(2)O in NaCl was equilibrated with air at 50% RH, which is above the deliquescence point of the magnesium salt but below that of NaCl. This mixture of NaCl coated with an aqueous magnesium chloride solution was then reacted with gas phase OH to generate hydroxide ions via a previously identified interface reaction. This treatment, hereafter referred to as OH-processing, was sufficient to convert some of the magnesium chloride to Mg(OH)(2) and Mg(2)(OH)(3)Cl x 4H(2)O, making the aqueous film basic and providing a reservoir of alkalinity. Subsequent addition of SO(2) to the basic processed mixture resulted in its uptake and conversion to sulfite which was measured by FTIR. The sulfite was simultaneously oxidized to sulfate by HOCl/OCl(-) that was formed in the initial OH-processing of the salt. Further uptake and oxidation of SO(2) in the aqueous film took place when the salt was subsequently exposed to O(3). These studies demonstrate that DRIFTS can be used to study the chemistry in liquid films in real time, and are consistent with the hypothesis that the reaction of gaseous OH with chloride ions generates alkalinity that enhances the uptake and oxidation of SO(2) under these laboratory conditions.

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