N-tert-Butoxycarbonyl-N-(2-(tritylthio)ethoxy)glycine as a Building Block for Peptide Ubiquitination.

N-Boc-N-(2-(tritylthio)ethoxy)glycine has been developed as a building block for peptide ubiquitination, which is fully compatible with solid-phase Fmoc chemistry and common peptide modifications including phosphorylation, methylation, acetylation, biotinylation, and fluorescence labeling. The optimal conditions for peptide cleavage and auxiliary removal were obtained. The utility of this building block in peptide ubiquitination was demonstrated by the synthesis of seven ubiquitinated histone and Tau peptides bearing various modifications. Cys residues were well tolerated and did not require orthogonal protection. The structural integrity and folding of the synthesized ubiquitinated peptides were confirmed by enzymatic deubiquitination of a fluorescently labeled ubiquitin conjugate. The synthetic strategy using this building block provides a practical approach for the preparation of ubiquitinated peptides with diverse modifications.

A Multifaceted Hit-Finding Approach Reveals Novel LC3 Family Ligands.

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.

Incorporating NOE-Derived Distances in Conformer Generation of Cyclic Peptides with Distance Geometry.

Nuclear magnetic resonance (NMR) data from NOESY (nuclear Overhauser enhancement spectroscopy) and ROESY (rotating frame Overhauser enhancement spectroscopy) experiments can easily be combined with distance geometry (DG) based conformer generators by modifying the molecular distance bounds matrix. In this work, we extend the modern DG based conformer generator ETKDG, which has been shown to reproduce experimental crystal structures from small molecules to large macrocycles well, to include NOE-derived interproton distances. In noeETKDG, the experimentally derived interproton distances are incorporated into the distance bounds matrix as loose upper (or lower) bounds to generate large conformer sets. Various subselection techniques can subsequently be applied to yield a conformer bundle that best reproduces the NOE data. The approach is benchmarked using a set of 24 (mostly) cyclic peptides for which NOE-derived distances as well as reference solution structures obtained by other software are available. With respect to other packages currently available, the advantages of noeETKDG are its speed and that no prior force-field parametrization is required, which is especially useful for peptides with unnatural amino acids. The resulting conformer bundles can be further processed with the use of structural refinement techniques to improve the modeling of the intramolecular nonbonded interactions. The noeETKDG code is released as a fully open-source software package available at www.github.com/rinikerlab/customETKDG.

A novel method to produce synthetic murine CXCL10 for efficient screening of functional variants.

Antitumor immune responses depend on the infiltration of solid tumors by effector T cells, a process guided by chemokines. In particular, the chemokine CXCL10 has been shown to play a critical role in mediating recruitment of CXCR3 + cytolytic T and NK cells in tumors, though its use as a therapeutic agent has not been widely explored. One of the limitations is due to the rapid inactivation of CXCL10 by dipeptidyl peptidase 4 (DPP4), a broadly expressed enzyme that is active in plasma and other bodily fluids. In the present study, we describe a novel method to produce synthetic CXCL10 that is resistant to DPP4 N-terminal truncation. Using a Fmoc solid-phase peptide synthesis approach, synthetic murine WT CXCL10 was produced, showing similar biochemical and biological properties to the recombinant protein. This synthesis method supported production of natural (amino acid substitution, insertion or deletion) and non-natural (chemical modifications) variants of CXCL10. In association with a functional screening cascade that assessed DPP4-mediated cleavage, CXCR3 signaling potency and chemotactic activity, we successfully generated 20 murine CXCL10 variants. Among those, two non-natural variants with N-methylated Leu3 (MeLeu3) and a reduced amide bond between Pro2 and Leu3 (rLeu3), respectively, showed resistance to DPP4 truncation but decreased CXCR3 signaling and chemotactic activity. Interestingly, MeLeu3 and rLeu3 CXCL10 behaved as DPP4 inhibitors, preventing the truncation of WT CXCL10. This study highlights the potential of using Fmoc solid-phase chemistry in association with biochemical and biological characterization to rapidly identify CXCL10 variants with desired properties. These novel methods unlock the opportunity to develop DPP4 resistant CXCL10 variants, as well as other chemokine substrates, while maintaining chemotactic properties.

Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation.

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.

Association Between the Implementation of a Population-Based Primary Care Payment System and Achievement on Quality Measures in Hawaii.

Importance: Hawaii Medical Service Association (HMSA), the Blue Cross Blue Shield of Hawaii, introduced Population-based Payments for Primary Care (3PC), a new capitation-based primary care payment system, in 2016. The effect of this system on quality measures has not been evaluated. Objective: To evaluate whether the 3PC system was associated with changes in quality, utilization, or spending in its first year. Design, Setting, and Participants: Observational study using HMSA claims and clinical registry data from January 1, 2012, to December 31, 2016, and a propensity-weighted difference-in-differences method to compare 77 225 HMSA members in Hawaii attributed to 107 primary care physicians (PCPs) and 4 physician organizations participating in the first wave of the 3PC and 222 233 members attributed to 312 PCPs and 14 physician organizations that continued in a fee-for-service model in 2016 but had 3PC start dates thereafter. Exposures: Participation in the 3PC system. Main Outcomes and Measures: The primary outcome was the change in a composite measure score reflecting the probability that a member achieved an eligible measure out of 13 pooled Healthcare Effectiveness Data and Information Set quality measures. Primary care visits and total cost of care were among 15 secondary outcomes. Results: In total, the study included 299 458 HMSA members (mean age, 42.1 years; 51.5% women) and 419 primary care physicians (mean age, 54.9 years; 34.8% women). The risk-standardized composite measure scores for 2012 to 2016 changed from 75.1% to 86.6% (+11.5 percentage points) in the 3PC group and 74.3% to 83.5% (+9.2 percentage points) in the non-3PC group (differential change, 2.3 percentage points [95% CI, 2.1 to 2.6 percentage points]; P < .001). Of 15 prespecified secondary end points for utilization and spending, 11 showed no significant difference. Compared with the non-3PC group, the 3PC system was associated with a significant reduction in the mean number of primary care visits (3.3 to 3.0 visits vs 3.3 to 3.1 visits; adjusted differential change, -3.9 percentage points [95% CI, -4.6 to -3.2 percentage points]; P < .001), but there was no significant difference in mean total cost of care ($3344 to $4087 vs $2977 to $3564; adjusted differential change, 1.0% [95% CI, -1.3% to 3.4%]; P = .39). Conclusions and Relevance: In its first year, the 3PC population-based primary care payment system in Hawaii was associated with small improvements in quality and a reduction in PCP visits but no significant difference in the total cost of care. Additional research is needed to assess longer-term outcomes as the program is more fully implemented and to determine whether results are generalizable to other health care markets.

An allosteric switch for pro-HGF/Met signaling using zymogen activator peptides.

Stimulation of hepatocyte growth factor (HGF) signaling through the Met receptor is an attractive approach for promoting tissue repair and preventing fibrosis. Using structure-guided peptide phage display combined with an activity-based sorting strategy, we engineered allosteric activators of zymogen-like pro-HGF to bypass proteolytic activation and reversibly stimulate pro-HGF signaling through Met. Biochemical, structural and biological data showed that zymogen activator peptides (ZAPtides) potently and selectively bind the activation pocket within the serine protease-like ß-chain of pro-HGF and display titratable activation of pro-HGF-dependent Met signaling, leading to cell survival and migration. To further demonstrate the versatility of our ZAPtide platform, we identified allosteric activators for pro-macrophage stimulating protein and a zymogen serine protease, Protein C, which also provides evidence for target selectivity. These studies reveal that ZAPtides use molecular mimicry of the trypsin-like N-terminal insertion mechanism and establish a new paradigm for selective pharmacological activation of plasminogen-related growth factors and zymogen serine proteases.

Improving Adherence to Cardiovascular Therapies: An Economic Evaluation of a Randomized Pragmatic Trial.

OBJECTIVE: Preplanned economic analysis of a pragmatic trial using electronic-medical-record-linked interactive voice recognition (IVR) reminders for enhancing adherence to cardiovascular medications (i.e., statins, angiotensin-converting enzyme inhibitors [ACEIs], and angiotensin receptor blockers [ARBs]). METHODS: Three groups, usual care (UC), IVR, and IVR plus educational materials (IVR+), with 21,752 suboptimally adherent patients underwent follow-up for 9.6 months on average. Costs to implement and deliver the intervention (from a payer perspective) were tracked during the trial. Medical care costs and outcomes were ascertained using electronic medical records. RESULTS: Per-patient intervention costs ranged from $9 to $17 for IVR and from $36 to $47 for IVR+. For ACEI/ARB, the incremental cost-effectiveness ratio for each percent adherence increase was about 3 times higher with IVR+ than with IVR ($6 and $16 for IVR and IVR+, respectively). For statins, the incremental cost-effectiveness ratio for each percent adherence increase was about 7 times higher with IVR+ than with IVR ($6 and $43 for IVR and IVR+, respectively). Considering potential cost offsets from reduced cardiovascular events, the probability of breakeven was the highest for UC, but the IVR-based interventions had a higher probability of breakeven for subgroups with a baseline low-density lipoprotein (LDL) level of more than 100 mg/dl and those with two or more calls. CONCLUSIONS: We found that the use of an automated voice messaging system to promote adherence to ACEIs/ARBs and statins may be cost-effective, depending on a decision maker's willingness to pay for unit increase in adherence. When considering changes in LDL level and downstream medical care offsets, UC is the optimal strategy for the general population. However, IVR-based interventions may be the optimal choice for those with elevated LDL values at baseline.

Identification of a small peptide that inhibits PCSK9 protein binding to the low density lipoprotein receptor.

PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 µm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å(2) largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 µm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the ß-strand and a discontinuous short α-helix, and it engaged in the same ß-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9.

Development of orthogonally protected hypusine for solid-phase peptide synthesis.

An orthogonally protected hypusine reagent was developed for solid-phase synthesis of hypusinated peptides using the Fmoc/t-Bu protection strategy. The reagent was synthesized in an overall yield of 27% after seven steps from Cbz-Lys-OBzl and (R)-3-hydroxypyrrolidin-2-one. The side-chain protecting groups (Boc and t-Bu) are fully compatible with standard Fmoc chemistry and can be readily removed during the peptide cleavage step. The utility of the reagent was demonstrated by solid-phase synthesis of hypusinated peptides.

Personal health record use and association with immunizations and well-child care visits recommendations.

OBJECTIVE: To determine the association of parental use of integrated personal health records (PHRs) with children's adherence to immunization and well-child care (WCC) visit recommendations. STUDY DESIGN: For the immunization and WCC visit measures, we retrospectively analyzed, respectively, 766 and 639 matched pairs at Kaiser Permanente (KP) Hawaii and 2795 and 2448 pairs at KP Northwest who were ≤ 31 days old at enrollment and continuously enrolled for 2 years between January 2007 and July 2011. The independent variable (≥ 1 PHR feature used vs none) was matched using propensity scores on parental and children characteristics. The dependent variables were 2 measures from the 2010 Healthcare Effectiveness Data and Information Set: combination 2 immunization (all immunizations vs .05; KP Northwest OR 1.2, 95% CI 1.0-1.3, P < .05). PHR use was associated with better adherence to WCC visit recommendations for both KP Hawaii (OR 1.9, 95% CI 1.3-2.9, P < .001) and KP Northwest (OR 2.5, 95% CI 2.1-2.9, P < .001). CONCLUSIONS: Young children whose parents used a PHR were more likely to adhere to the recommended WCC visits in both regions but immunizations in only 1 region.

Geographic variation in cardiovascular procedure use among Medicare fee-for-service vs Medicare Advantage beneficiaries.

IMPORTANCE: Little is known about how different financial incentives between Medicare Advantage and Medicare fee-for-service (FFS) reimbursement structures influence use of cardiovascular procedures. OBJECTIVE: To compare regional cardiovascular procedure rates between Medicare Advantage and Medicare FFS beneficiaries. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study of Medicare beneficiaries older than 65 years between 2003-2007 comparing rates of coronary angiography, percutaneous coronary intervention (PCI), and coronary artery bypass graft (CABG) surgery across 32 hospital referral regions in 12 states. MAIN OUTCOMES AND MEASURES: Rates of coronary angiography, PCI, and CABG surgery. RESULTS: We evaluated a total of 878,339 Medicare Advantage patients and 5,013,650 Medicare FFS patients. Compared with Medicare FFS patients, Medicare Advantage patients had lower age-, sex-, race-, and income-adjusted procedure rates per 1000 person-years for angiography (16.5 [95% CI, 14.8-18.2] vs 25.9 [95% CI, 24.0-27.9]; P < .001) and PCI (6.8 [95% CI, 6.0-7.6] vs 9.8 [95% CI, 9.0-10.6]; P < .001) but similar rates for CABG surgery (3.1 [95% CI, 2.8-3.5] vs 3.4 [95% CI, 3.1-3.7]; P = .33). There were no significant differences between Medicare Advantage and Medicare FFS patients in the rates per 1000 person-years of urgent angiography (3.9 [95% CI, 3.6-4.2] vs 4.3 [95% CI, 4.0-4.6]; P = .24) or PCI (2.4 [95% CI, 2.2-2.7] vs 2.7 [95% CI, 2.5-2.9]; P = .16). Procedure rates varied widely across hospital referral regions among Medicare Advantage and Medicare FFS patients. For angiography, the rates per 1000 person-years ranged from 9.8 to 40.6 for Medicare Advantage beneficiaries and from 15.7 to 44.3 for Medicare FFS beneficiaries. For PCI, the rates ranged from 3.5 to 16.8 for Medicare Advantage and from 4.7 to 16.1 for Medicare FFS. The rates for CABG surgery ranged from 1.5 to 6.1 for Medicare Advantage and from 2.5 to 6.0 for Medicare FFS. Across regions, we found no statistically significant correlation between Medicare Advantage and Medicare FFS beneficiary utilization for angiography (Spearman r = 0.19, P = .29) and modest correlations for PCI (Spearman r = 0.33, P = .06) and CABG surgery (Spearman r = 0.35, P = .05). Among Medicare Advantage beneficiaries, adjustment for additional cardiac risk factors had little influence on procedure rates. CONCLUSIONS AND RELEVANCE: Although Medicare beneficiaries enrolled in capitated Medicare Advantage programs had lower angiography and PCI procedure rates than those enrolled in Medicare FFS, the degree of geographic variation in procedure rates was substantial among Medicare Advantage beneficiaries and was similar in magnitude to that observed among Medicare FFS beneficiaries.

Evaluating Translational Efficiency of Noncanonical Amino Acids to Inform the Design of Druglike Peptide Libraries.

Advances in genetic code reprogramming have allowed the site-specific incorporation of noncanonical functionalities into polypeptides and proteins, providing access to wide swaths of chemical space via in vitro translation techniques like mRNA display. Prior efforts have established that the translation machinery can tolerate amino acids with modifications to both the peptide backbone and side chains, greatly broadening the chemical space that can be interrogated in ligand discovery efforts. However, existing methods for confirming the translation yield of new amino acid building blocks for these technologies necessitate multistep workups and, more importantly, are not relevant for measuring translation within the context of a combinatorial library consisting of multiple noncanonical amino acids. In this study, we developed a luminescence-based assay to rapidly assess the relative translation yield of any noncanonical amino acid in real time. Among the 59 amino acids tested here, we found that many translate with high efficiency, but translational yield is not necessarily correlated to whether the amino acid is proteinogenic or has high tRNA acylation efficiency. Interestingly, we found that single-template translation data can inform the library-scale translation yield and that shorter peptide libraries are more tolerant of lower-efficiency amino acid monomers. Together our data show that the luminescence-based assay described herein is an essential tool in evaluating new building blocks and codon table designs within mRNA display toward the goal of developing druglike peptide-based libraries for drug discovery campaigns.

Trifluoroacetyl Lysine as a Bromodomain Binding Mimic of Lysine Acetylation.

Genetic code expansion has proven invaluable to the elucidation of functions of defined protein modifications through the site-specific incorporation of noncanonical amino acids. The use of nonhydrolyzable derivatives of post-translational modifications can greatly increase site stoichiometry and half-life. Investigating acetyllysine reader domain (bromodomain) interactions with acetylated nonhistone proteins is challenging due to the limited tools available and dynamic nature of this post-translational modification. Here, we demonstrate that bromodomains bind acetyllysine peptides and those substituted with an acetyllysine derivative, trifluoroacetyllysine, with similar affinity and selectivity. Importantly, both trifluoroacetyllysine and acetyllysine can be site-specifically incorporated into proteins expressed in bacterial and mammalian cells, and the strong electron-withdrawing trifluoro substituent makes the latter resistant to deacetylation by sirtuins (SIRTs). The controlled expression of SIRT-resistant, site-specifically acetylated transcription factors expands the set of available tools for determining the function of acetylation, and it serves as a template for investigating bromodomain interactions with acetylated transcription factors.

Designing a commercial bundle for cardiac procedures: The Percutaneous Coronary Intervention Episode Payment Model.

BACKGROUND: Cardiac interventions account for a significant share of overall healthcare spending and have been the focus of several large-scale interventions to develop effective bundled payments. To date, however, none have proven successful in commercially insured populations. In 2018, we worked with Hawaii Medical Service Association (HMSA), the Blue Cross Blue Shield of Hawaii, to design a novel commercial bundled payment for percutaneous coronary interventions, the Percutaneous Coronary Intervention Episode Payment Model (PCI EPM). METHODS: Descriptive analysis of HMSA's PCI EPM, including its inclusion criteria, contents of the bundle, target prices, shared savings model, and incentivized quality metrics. We also compare HMSA's PCI EPM to Medicare's Bundled Payment for Care Improvement programs and the cancelled Cardiac Care Model. RESULTS: HMSA's PCI EPM was designed through an iterative process with cardiologists and is the first commercial bundle to specifically target a cardiac procedure. PCI EPM incorporates site neutrality and incentivizes providers to shift care to the outpatient setting when medically permissible. Compared to existing non-commercial models, PCI EPM incorporate first-dollar shared savings and incentivized fewer quality metrics. CONCLUSIONS: Reviewing features of the Percutaneous Coronary Intervention Episode Payment Model in comparison to existing Medicare programs is intended to help guide health plan and health policymakers when designing programs and policies related to cardiac interventions. IMPLICATIONS: Bundled commercial payments for interventional cardiology procedures are promising and should continue to be further explored. LEVEL OF EVIDENCE: VI.

Heterobifunctional Molecules Induce Dephosphorylation of Kinases-A Proof of Concept Study.

Heterobifunctional molecules have proven powerful tools to induce ligase-dependent ubiquitination of target proteins. We describe here a chemical strategy for controlling a different post-translational modification (PTM): phosphorylation. Heterobifunctional molecules were designed to promote the proximity of a protein phosphatase (PP1) to protein targets. The synthesized molecules induced the PP1-dependent dephosphorylation of AKT and EGFR. To our knowledge, this work represents the first examples of small molecules recruiting non-native partners to induce removal of a PTM.

Small Molecule Dysregulation of TEAD Lipidation Induces a Dominant-Negative Inhibition of Hippo Pathway Signaling.

The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.

Changes in Outpatient Imaging Utilization and Spending Under a New Population-Based Primary Care Payment Model.

OBJECTIVE: To evaluate whether the implementation of a new population-based primary care payment system, Population-Based Payments for Primary Care (3PC), initiated by Hawaii Medical Service Association (HMSA; the Blue Cross Blue Shield of Hawaii), was associated with changes in spending and utilization for outpatient imaging in its first year. METHODS: In this observational study, we used claims data from January 1, 2012, to December 31, 2016. We used a propensity-weighted difference-in-differences design to compare 70,284 HMSA patients in Hawaii attributed to 107 primary care physicians (PCPs) and 4 physician organizations participating in 3PC in its first year of implementation (2016) and 195,902 patients attributed to 312 PCPs and 14 physician organizations that used a fee-for-service model during the study period. The primary outcome was total spending on outpatient imaging tests, and secondary outcomes included spending and utilization by modality. RESULTS: The study included 266,186 HMSA patients (mean age of 43.3 years; 51.7% women) and 419 PCPs (mean age of 54.9 years; 34.8% women). The 3PC system was not significantly associated with changes in total spending for outpatient imaging. Of 12 secondary outcomes, only 3 were statistically significant, including changes in nuclear medicine spending (adjusted differential change = -20.1% [95% confidence interval = -27.5% to -12.1%]; P < .001) and utilization (adjusted differential change = -18.1% [95% confidence interval = -23.8 to -11.9%]; P < .001). DISCUSSION: The HMSA 3PC system was not associated with significant changes in total spending for outpatient imaging, though spending and utilization on nuclear medicine tests decreased.