Recommendations from people who use drugs in Philadelphia, PA about structuring point-of-care drug checking.

BACKGROUND: Adulterants, such as fentanyl and xylazine, among others, are present in a high percentage of the illicit drug supply, increasing the risk for overdose and other adverse health events among people who use drugs (PWUD). Point-of-care drug checking identifies components of a drug sample and delivers results consumers. To successfully meet the diverse needs of PWUD, more information is needed about the utility of drug checking, motivations for using services contextualized in broader comments on the drug supply, hypothesized actions to be taken after receiving drug checking results, and the ideal structure of a program. METHODS: In December 2021, semi-structured interviews were conducted with 40 PWUD who were accessing harm reduction services in Philadelphia, PA. Participants were asked about opinions and preferences for a future drug checking program. Interviews were audio recorded, transcribed and coded using content analysis to identify themes. RESULTS: Participants were primarily White (52.5%) and male (60%). Heroin/fentanyl was the most frequently reported drug used (72.5%, n = 29), followed by crack cocaine (60.0%, n = 24) and powder cocaine (47.5%, n = 19). Emerging themes from potential drug checking consumers included universal interest in using a drug checking program, intentions to change drug use actions based on drug checking results, deep concern about the unpredictability of the drug supply, engaging in multiple harm reduction practices, and concerns about privacy while accessing a service. CONCLUSIONS: We offer recommendations for sites considering point-of-care drug checking regarding staffing, safety, logistics, and cultural competency. Programs should leverage pre-existing relationships with organizations serving PWUD and hire people with lived experiences of drug use. They should work with local or state government to issue protections to people accessing drug checking programs and ensure the service is anonymous and that data collection is minimized to keep the program low-threshold. Programs will ideally operate in multiple locations and span "atmosphere" (e.g., from clinical to a drop-in culture), offer in-depth education to participants about results, engage with a community advisory board, and not partner with law enforcement.

Biophysical characterization of full-length human phenylalanine hydroxylase provides a deeper understanding of its quaternary structure equilibrium.

Dysfunction of human phenylalanine hydroxylase (hPAH, EC 1.14.16.1) is the primary cause of phenylketonuria, the most common inborn error of amino acid metabolism. The dynamic domain rearrangements of this multimeric protein have thwarted structural study of the full-length form for decades, until now. In this study, a tractable C29S variant of hPAH (C29S) yielded a 3.06 Å resolution crystal structure of the tetrameric resting-state conformation. We used size-exclusion chromatography in line with small-angle X-ray scattering (SEC-SAXS) to analyze the full-length hPAH solution structure both in the presence and absence of Phe, which serves as both substrate and allosteric activators. Allosteric Phe binding favors accumulation of an activated PAH tetramer conformation, which is biophysically distinct in solution. Protein characterization with enzyme kinetics and intrinsic fluorescence revealed that the C29S variant and hPAH are otherwise equivalent in their response to Phe, further supported by their behavior on various chromatography resins and by analytical ultracentrifugation. Modeling of resting-state and activated forms of C29S against SAXS data with available structural data created and evaluated several new models for the transition between the architecturally distinct conformations of PAH and highlighted unique intra- and inter-subunit interactions. Three best-fitting alternative models all placed the allosteric Phe-binding module 8-10 Å farther from the tetramer center than do all previous models. The structural insights into allosteric activation of hPAH reported here may help inform ongoing efforts to treat phenylketonuria with novel therapeutic approaches.

Simulations of the regulatory ACT domain of human phenylalanine hydroxylase (PAH) unveil its mechanism of phenylalanine binding.

Phenylalanine hydroxylase (PAH) regulates phenylalanine (Phe) levels in mammals to prevent neurotoxicity resulting from high Phe concentrations as observed in genetic disorders leading to hyperphenylalaninemia and phenylketonuria. PAH senses elevated Phe concentrations by transient allosteric Phe binding to a protein-protein interface between ACT domains of different subunits in a PAH tetramer. This interface is present in an activated PAH (A-PAH) tetramer and absent in a resting-state PAH (RS-PAH) tetramer. To investigate this allosteric sensing mechanism, here we used the GROMACS molecular dynamics simulation suite on the Folding@home computing platform to perform extensive molecular simulations and Markov state model (MSM) analysis of Phe binding to ACT domain dimers. These simulations strongly implicated a conformational selection mechanism for Phe association with ACT domain dimers and revealed protein motions that act as a gating mechanism for Phe binding. The MSMs also illuminate a highly mobile hairpin loop, consistent with experimental findings also presented here that the PAH variant L72W does not shift the PAH structural equilibrium toward the activated state. Finally, simulations of ACT domain monomers are presented, in which spontaneous transitions between resting-state and activated conformations are observed, also consistent with a mechanism of conformational selection. These mechanistic details provide detailed insight into the regulation of PAH activation and provide testable hypotheses for the development of new allosteric effectors to correct structural and functional defects in PAH.