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STIP1 homology and U-Box containing protein 1 (STUB1) plays a key role in maintaining cell health during stress and aging. Recent evidence suggested STUB1 also helps regulate immunity with the potential of clearing malignant cells. Indeed, we and others have shown that STUB1 is a pivotal negative regulator of interferon gamma sensing – a process critical to the immunosurveillance of tumors and pathogens. Thus far, investigation of STUB1’s role relies mostly on genetic approaches as pharmacological inhibitors of this protein are lacking. Identification of a STUB1 tool compound is important as it would allow therapeutically relevant target validation in a broader sense. Accordingly, we leveraged phage display and computational modeling to identify and refine STUB1 binders. Screening of >10E9 macrocyclic peptides resulted in several conserved motifs as well as structurally diverse leads. Co-crystal structure of the peptide hit and STUB1 has enabled us to employ structure-based in silico design for further optimization. Of the modifications employed, replacing the hydrophilic solvent-exposed region of the macrocyclic peptides with a hydrophobic scaffold improved cellular permeability, while the binding conformation was maintained. Further substitution of the permeability-limiting terminal aspartic acid with a tetrazole bioisostere retained the binding to certain extent while improving permeability, suggesting a path forward. The current lead, although not optimal for cellular study, provides a valuable template for further development into selective tool compounds for STUB1 to enable target validation.

Stapled α-helical peptides can bind to and modulate historically intractable targets while addressing the traditional liabilities associated with peptide therapeutics. However, their pipeline advancement has been impeded by the challenges of identifying peptides with sufficient cellular uptake to engage the target protein while lacking off-target toxicities. Here, we advance the field to arrive at a workflow for identifying advanced stapled peptide lead molecules with on-target in vivo activity with no off-target cell proliferation effects. Specifically, we generated a >350-member library based on ATSP-7041, a stapled peptide Mdm2(X) antagonist with validated on-target cellular effects but with significant off-target activity. Key insights from library analysis include 1) a clear correlation between lipophilicity and permeability, 2) removal of positive charge to avoid off-target toxicities, 3) judicious placement of anionic residues to enhance peptide solubility/behavior, 4) optimization of C-terminal length and helicity to enhance cell activity, 5) optimization of staple type/number to avoid polypharmacology. Incorporation of one or more of these attributes led to molecules with improved in vitro and in vivo activities (up to a >292x improved cell proliferation EC50). A subset of peptides were devoid of off-target cell proliferation effects in cell lines lacking wild-type p53 protein (up to a >3800x on-target index). This latter improvement contrasted with clinical Mdm2 antagonistic molecules. Application of these ‘design rules’ to a distinct Mdm2(X) peptide series resulted in rapid improvement in cellular activity (>150x) and removal of off-target toxicities. Overall, the detailed workflow outlined here should help researchers identify stapled α-helical peptides for therapeutic impact.

Macrocyclic peptides have the potential to address intracellular protein–protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure–activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight – the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry – an approach that has yet to succeed in the clinic despite a long history of attempts – carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers – some of the most treatment-resistant human malignancies., Targeting undruggable intracellular proteins with peptides: novel on-target macrocyclic peptide inhibitors of KRAS with broad inhibition of proliferation of multiple KRAS-dependent cancer cell lines.

Mutations to RAS proteins (H-, N-, and K-RAS) are among the most common oncogenic drivers, and tumors harboring these lesions are some of the most difficult to treat. Although covalent small molecules against KRASG12C have shown promising efficacy against lung cancers, traditional barriers remain for drugging the more prevalent KRASG12D and KRASG12V mutants. Targeted degradation has emerged as an attractive alternative approach, but for KRAS, identification of the required high-affinity ligands continues to be a challenge. Another significant hurdle is the discovery of a hybrid molecule that appends an E3 ligase-recruiting moiety in a manner that satisfies the precise geometries required for productive polyubiquitin transfer while maintaining favorable druglike properties. To gain insights into the advantages and feasibility of KRAS targeted degradation, we applied a protein-based degrader (biodegrader) approach. This workflow centers on the intracellular expression of a chimeric protein consisting of a high-affinity target-binding domain fused to an engineered E3 ligase adapter. A series of anti-RAS biodegraders spanning different RAS isoform/nucleotide-state specificities and leveraging different E3 ligases provided definitive evidence for RAS degradability. Further, these established that the functional consequences of KRAS degradation are context dependent. Of broader significance, using the exquisite degradation specificity that biodegraders can possess, we demonstrated how this technology can be applied to answer questions that other approaches cannot. Specifically, application of the GDP-state specific degrader uncovered the relative prevalence of the “off-state” of WT and various KRAS mutants in the cellular context. Finally, if delivery challenges can be addressed, anti-RAS biodegraders will be exciting candidates for clinical development., Biodegraders, engineered proteins that fuse a high-affinity binder (e.g., RBD, K27, R11.1.6, and NS1) to an E3 ligase (e.g., SPOP), prompt degradation of RAS and reveal novel biological insights.

Pulmonary diseases and injury lead to structural and functional changes in the lung parenchyma and airways, often resulting in measurable sound transmission changes on the chest wall surface. Additionally, noninvasive imaging of externally driven mechanical wave motion in the chest (e.g., using magnetic resonance elastography) can provide information about lung stiffness and other structural property changes which may be of diagnostic value. In the present study, a comprehensive computational simulation (in silico) model was developed to simulate sound wave propagation in the airways, parenchyma, and chest wall under normal and pathological conditions that create distributed structural (e.g., pneumothoraces) and diffuse material (e.g., fibrosis) changes, as well as a localized structural and material changes as may be seen with a neoplasm. Experiments were carried out in normal subjects to validate the baseline model. Sound waves with frequency content from 50 to 600 Hz were introduced into the airways of three healthy human subjects through the mouth, and transthoracic transmitted waves were measured by scanning laser Doppler vibrometry at the chest wall surface. The computational model predictions of a frequency-dependent decreased sound transmission due to pneumothorax were consistent with experimental measurements reported in previous work. Predictions for the case of fibrosis show that while shear wave motion is altered, changes to compression wave propagation are negligible, and thus insonification, which primarily drives compression waves, is not ideal to detect the presence of fibrosis. Results from the numerical simulation of a tumor show an increase in the wavelength of propagating waves in the immediate vicinity of the tumor region., Graphical Abstract

Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. mRNA optimization is indispensable for reducing immunogenicity, ensuring stability, and maximizing protein output. We present mRNAid, an experimentally validated software for mRNA optimization and visualization that generates mRNA sequences with comparable if not superior characteristics to commercially optimized sequences. To encompass all aspects of mRNA design, we also interrogated the impact of uridine content, nucleoside analogs and UTRs on expression and immunogenicity.

Targeted protein degradation has emerged as a powerful technology – both as a biological tool and for broadening the therapeutic proteome. As tools to probe this approach on historically intractable targets, we have previously advanced ‘biodegraders’ — targeted degradation fusion constructs composed of mini-proteins/peptides linked to modified E3 ligase receptors. Herein, we gain deeper insights into the utility and potential of biodegraders, through a detailed study on Con1-SPOP, a biodegrader which rapidly degrades the potential cancer target, proliferating cell nuclear antigen (PCNA). In a variety of settings, the active biodegrader (Con1-SPOP) proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut). Specifically, in addition to more potent anti-proliferative effects in both 2D cell culture and 3D spheroids, PCNA degradation uniquely induced DNA damage, cell apoptosis and necrosis. Global proteomic profiling of a stable cell-line expressing Con1-SPOP under doxycycline (Dox) induction revealed that impaired mitotic division and mitochondria dysfunction is a direct consequence of PCNA degradation, effects not seen with the stoichiometric inhibitor protein. To evaluate the therapeutic potential of biodegraders, we showed that Dox-induced Con1-SPOP achieved complete tumor-growth inhibition in a xenograft model. To explore application of biodegraders as a novel therapeutic modality, modified mRNA encoding Con1-SPOP was synthesized and encapsulated into lipid nanoparticles (LNPs). The approach successfully delivered mRNA in vitro to deplete endogenous PCNA within hours of application and with nanomolar potency. Overall, our results demonstrate the utility of biodegraders as biological tools and highlight target-degradation as a more efficacious approach versus stoichiometric inhibition. Finally, once in vivo delivery and expression are optimized, biodegraders may be leveraged as an exciting therapeutic modality.

Peptide-based molecules hold great potential as targeted inhibitors of intracellular protein–protein interactions (PPIs). Indeed, the vast diversity of chemical space conferred through their primary, secondary and tertiary structures allows these molecules to be applied to targets that are typically deemed intractable via small molecules. However, the development of peptide therapeutics has been hindered by their limited conformational stability, proteolytic sensitivity and cell permeability. Several contemporary peptide design strategies are aimed at addressing these issues. Strategic macrocyclization through optimally placed chemical braces such as olefinic hydrocarbon crosslinks, commonly referred to as staples, may improve peptide properties by (i) restricting conformational freedom to improve target affinities, (ii) improving proteolytic resistance, and (iii) enhancing cell permeability. As a second strategy, molecules constructed entirely from D-amino acids are hyper-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. Since neither approach is a complete solution, we have combined these strategies to identify the first examples of all-D α-helical stapled and stitched peptides. As a template, we used a recently reported all D-linear peptide that is a potent inhibitor of the p53–Mdm2 interaction, but is devoid of cellular activity. To design both stapled and stitched all-D-peptide analogues, we used computational modelling to predict optimal staple placement. The resultant novel macrocyclic all D-peptide was determined to exhibit increased α-helicity, improved target binding, complete proteolytic stability and, most notably, cellular activity.

For over 10 years, laser plasma acceleration (LPA) has been a rapidly growing technologyused to create electron beams on length-scales much smaller than that of a conventionalRF-accelerator [1]. As electron beam properties improve, research for LPAs is expanding totake advantage of the creation and accessibility of high-quality electron beams from plasmatargets. Two applications which are currently being explored are a multi-stage plasmaaccelerator to reach energies greater than those a single-stage accelerator can achieve andexploring the possibility of an LPA based free-electron laser (FEL) light source. Researchsupporting both of these efforts has been performed on the 50 TW TREX laser system atthe BELLA Center at the Lawrence Berkeley National Lab, and the results of these effortsare described in this dissertation.Using chirped-pulsed amplification to produce high-quality laser pulses up to petawattlevels, experimental results have yielded laser driven electron beam energies up to 4.25GeV [2]. By tuning the density of the target, the accelerating gradients sustained by theplasma can grow beyond 100 GeV/m [3] (10^3 times larger than that of a conventional RFaccelerator). However, limiting factors such as dephasing of the electron beam from theplasma wake, defocusing of a laser pulse, and energy depletion of the laser into the plasmalimit the maximum sensible length of a plasma accelerator. Staging the LPA with two ormore accelerating modules could be the next step towards producing beams with energiesgreater than those possible with a single stage.One requirement for staged acceleration is that the laser pulse used to drive the firstaccelerating stage must be coupled out of the beamline, and a fresh laser pulse must becoupled in for the second stage to post accelerate the electrons. To do this while maintaininga short scale length between the two stages requires an optic to be placed near the final focus of the second laser pulse. Because damage will occur when the laser pulse interacts with a steering optic near focus, the coupling optic must be capable of replacing the surfacefollowing damage on each successive shot. This thesis comprises a detailed investigation of the physics of using a plasma mirror (PM) from a tape by reflecting ultrashort pulses froma laser-triggered surface plasma. The tapes used in the characterization of the PM are VHSand computer data storage tape. The tapes are 6.6 m (computer storage tape) and 15 m(VHS) thick. Each tape is 0.5 inches wide, and 10s of meters of tape are spooled using a tape drive; providing thousands of shots on a single reel of tape. The amount of reflected energy of the PM was studied for different input intensities. The fluence was varied by translating the focus of the laser upstream and downstream of the tape, which changed the spot size on the tape surface and hence changed the fluence. This study measured reflectances from both sides of the two tapes, and for input light of both s and p-polarizations. Lastly, an analytic model was developed to understand the reflectance as a function of fluence for each tape material and polarization.Another application that benefits from the advancements of LPA technology is an LPAbasedFEL. By sending a high quality electron bunch through an undulator (a periodicstructure of positive and negative magnetic poles), the electrons oscillate transversely to thepropagation axis and produce radiation. The 1.5 m THUNDER undulator [4] at the BELLACenter has been commissioned using electron beams of 400MeV beams with broad energyspread (35%) [5]. To produce a coherent LPA-based FEL, the beam quality would need toimprove to sub-percent level energy spread. A seed source could be used to help inducebunching of the electron beam within the undulator.This thesis described the experimental investigation of the physics of using solid-basedsurface high-harmonic generation (SHHG) from a thin tape as a possible seed source foran FEL. A thin tape placed within centimeters of the undulator's entrance could act asa harmonic generating source, while simultaneously transmitting an electron beam. Thisremoves the need for transport optics for the XUV photons and the need for additionaloptics to overlap the seed beam with the electron beam at the undulator entrance.By operating at sub-relativistic laser strengths, harmonics up to the 17th order of 800nm light are produced using an SHHG technique known as coherent wake emission (CWE).CWE pulse properties such as divergence, energy, conversion efficiency, and spectrum aremeasured for a wide range of tape materials and drive laser conditions. A clear correlationbetween surface roughness and harmonic beam divergence is found. The measured pulseproperties for the 15th harmonic from VHS tape (conversion efficiency 6.5x10^-?7 andan rms divergence of 12 mrad), the 100 mJ-level, 40-50 fs-class drive laser, produces peakpowers of several MW's of XUV pulses. The results of a 1D model indicate that these CWEpulses with MW level powers are sufficient for seed-induced FEL gain.

This study investigated relationships between academic success and aspects of health and illness in college students. College juniors and seniors completed a survey addressing ratings of health, measures of sleep, mental health, screen time, the frequency of illness-related absences and access of healthcare. Measures of academic performance (GPA and academic status) were gathered from university records. Significant relationships to academic performance were found for overall health, sleep, mental health, time accessing social media, and frequency of accessing healthcare and school due to illness. Collectively, health measures predicted 10% of the variance in GPA.

Mutations to RAS proteins (H-, N-, and K-RAS) are amongst the most common oncogenic drivers and tumors harboring these lesions are some of the most difficult to treat. Although the recently discovered covalent small molecules against the KRASG12C mutant have shown promising efficacy against lung cancers, traditional barriers remain for drugging the more prevalent KRASG12D and KRASG12V mutants. Targeted degradation has emerged as an attractive alternative approach but for KRAS, identification of the required high-affinity ligands continues to be a challenge. Another significant hurdle is the discovery of a hybrid molecule that appends an E3 ligase-recruiting moiety in a manner that satisfies the precise geometries required for productive polyubiquitin transfer while maintaining favorable drug-like properties. As a tool to gain insights into the advantages and feasibility of KRAS targeted-degradation, we applied the bioPROTAC approach. This workflow centers on the intracellular expression of a chimeric protein consisting of a high-affinity target-binding domain fused to an engineered E3 ligase adapter. We generated a series of anti-RAS bioPROTACs that span different RAS isoform/nucleotide-state specificities and leverage different E3 ligases. Overall, our results provide definitive evidence for the degradability of RAS proteins. We further elucidate the functional consequences of RAS degradation, the susceptibility and degradation kinetics of various mutant KRAS, and the prevalence of different nucleotide-states in WT and mutant KRAS. Finally, if delivery challenges can be addressed, anti-RAS bioPROTACs will be exciting candidates for clinical development.

Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)-engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.

Porous silicon rugate photonic crystals are an attractive optical sensor material due to their high surface area, naked eye response, and controllable optical, morphological, and chemical characteristics. This thesis presents new ways to improve the selectivity, reversibility, and stability against interferents of remotely interrogated porous silicon sensors. After a brief introduction to rugate porous silicon, Chapters 2-5 present methods of directly sensing the interaction of organic vapors with the porous layer by chemical and physical sensor modulation. A 0.3 mm² fiber optic-coupled porous silicon sensor is constructed in Chapter 2 and implanted in a bed of activated carbon, demonstrating carbon bed end-of-service-life sensing. Chapter 3 furthers this concept by incorporating chemically modified sensor surfaces, with selectivity between water vapor, isopropanol, and heptane vapors demonstrated using acetylated and oxidized sensor chemistries. Chapter 4 introduces physical modulation of the porous silicon sensor, with thermal modulation of the photonic crystal to 160°C employed to rapidly and repeatedly desorb methyl salicylate and octanol vapors that foul the sensor response. Thermal modulation is applied to discrimination of pure heptane, cyclohexane, and isopropanol vapors in Chapter 5 by rapidly cycling a rugate sensor between 25- 80°C while exposed to partial pressures of organic vapors up to 7.5 Torr. Sensor responses to the thermally modulated sorption equilibrium allow discrimination of these pure analyte vapors. The final three chapters describe using porous silicon as tailored interference filters that increase the specificity of standoff optical detection. In Chapter 6, the stop bands of rugate filters are tuned to match mid-infrared molecular absorbance bands, including the 1250 cm⁻¹ P=O bond stretch. Standoff gas sensing is demonstrated with filters matched to the 2350cm⁻¹ stretch of CO₂. In Chapter 7, selective 2-D imaging of target compounds is demonstrated by matching filters to visible emission peaks of photoluminescent dyes that bind to dipicolinic acid, found in anthrax spores. Finally, wavelength separated, ratiometric referencing is demonstrated in Chapter 8, where a pH-responsive NH₃(g) sensor based on a dye-infused rugate filter with two stop bands encoded into the porous layer is shown to compensate for large fluctuations in probe light intensity and increase signal to noise

Targeted degradation approaches have recently generated much excitement as a paradigm shift to address human disease in unprecedented ways. Amongst these, small molecule based approaches such as Proteolysis targeting chimeras (PROTACs) have attracted the lion’s share of attention due to their potential to tackle historically intractable targets and achieve greater potency, efficacy, and specificity over traditional small molecule inhibitors. Despite their promise, the identification of high-affinity ligands that can serve as starting points for PROTAC strategies remains challenging. As a complementary approach, we describe herein a class of intracellular biologics termed bioPROTACs. The substrate binding component of these fusion proteins consists of a peptide or an antibody-mimetic which allows for an unprecedented diversity of protein targets that can be addressed. The high-affinity binder is linked directly to an E3 ubiquitin ligase to harness the power of targeted degradation. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. Indeed, 9 out of 16 binder-E3 combinations tested resulted in greater than 70% target clearance. Through a systematic approach, we then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA), a sliding DNA clamp with critical roles in DNA replication and repair. The bioPROTAC, termed Con1-SPOP, elicited rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Compared to RNAi-based approaches which typically take days to manifest, PCNA knockdown using Con1-SPOP was evident within 4 h. The advantage of degradation versus stoichiometric inhibition was also clearly demonstrated with bioPROTAC strategies. Combining superior pharmacological inhibition and relative ease of development, bioPROTACs are powerful tools for interrogating the degradability of a substrate, for guiding the identification of the fittest E3 ligase, for studying the functional consequences associated with target protein down-regulation, and potentially for making therapeutic impacts.

Following Hurricane Katrina, the uniformed US Public Health Service created an updated system through which its officers participated in emergency responses. The Rapid Deployment Force (RDF) concept, begun in 2006, involved five teams of officers with diverse clinical and public health skill sets organized into an incident command system led by a team commander. Each team can deploy within 12 hours, according to a defined but flexible schedule.The core RDF mission is to set up and provide care for up to 250 patients, primarily persons with chronic diseases or disabilities, in a temporary federal medical station. Between 2006 and 2016, the RDF 3 team deployed multiple times in response to natural disasters and public health emergencies. Notable responses included Hurricane Sandy in 2012, the unaccompanied children mission in 2014, and the Louisiana floods in 2016.Lessons learned from the RDF 3 experience include the need for both clinical and public health capacity, the value of having special mental health resources, the benefits of collaboration with other federal medical responders, and recognition of the large burden of chronic disease management issues following natural disasters.

Acute chest syndrome (ACS) is a leading cause of death for those with sickle cell disease (SCD). ACS is defined by the development of a new pulmonary infiltrate on chest X-ray, with fever and respiratory symptoms. Efforts have been made to apply various technologies in the hospital setting to provide earlier detection of ACS than X-ray, but they are expensive, increase radiation exposure to the patient, and are not technologies that are easily transferrable for home use to help with early diagnosis. We present preliminary studies on patients suggesting that acoustical measurements recorded quantitatively with contact sensors (electronic stethoscopes) and analyzed using advanced computational analysis methods may provide an earlier diagnostic indicator of the onset of ACS than is possible with current clinical practice. Novel in silico models of respiratory acoustics utilizing image-based and algorithmically developed lungs with full conducting airway trees support and help explain measured acoustic trends and provide guidance on the next steps in developing and translating a diagnostic approach. More broadly, the experimental and computational techniques introduced herein, while focused on monitoring and predicting the onset of ACS, could catalyze further advances in mobile health (mhealth)-enabled, computer-based auscultative diagnoses for a wide range of cardiopulmonary pathologies.