Intact quantitation of cysteine-conjugated antibody-drug conjugates using native mass spectrometry.

RATIONALE: A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation. METHODS: We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach. RESULTS: A linear dynamic range of 5-100 µg/mL was achieved using 20 µL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement. CONCLUSIONS: We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo.

Broad Application of CYP3A4 Liquid Chromatography-Mass Spectrometry Protein Quantification in Hepatocyte Cytochrome P450 Induction Assays Identifies Nonuniformity in mRNA and Protein Induction Responses.

Screening for cytochrome P450 (CYP) induction potential is routine in drug development. Induction results in a net increase in CYP protein and is assessed typically by measuring indirect endpoints, i.e., enzyme activity and mRNA in vitro. Recent methodological advancements have made CYP protein quantification by liquid chromatography-mass spectrometry in vitro induction studies more accessible and amenable to routine testing. In this study, we evaluated CYP3A4 concentration dependence of induction response for 11 compounds (rifampin, rifabutin, carbamazepine, efavirenz, nitrendipine, flumazenil, pioglitazone, rosiglitazone, troglitazone, pazopanib, and ticagrelor) in plated hepatocytes from two or three donors incorporating in the assessment all three endpoints. In addition, the time-dependence of the induction was examined over 1, 2, or 3 days of treatment. For most compounds, mRNA, enzyme activity, and protein endpoints exhibited similarity in induction responses. Pazopanib and ticagrelor were notable exceptions as neither protein nor enzyme activity were induced despite mRNA induction of a magnitude similar to efavirenz, pioglitazone, or rosiglitazone, which clearly induced in all three endpoints. Static modeling of clinical induction responses supported a role for protein as a predictive endpoint. These data highlight the value of including CYP protein quantification as an induction assay endpoint to provide a more comprehensive assessment of induction liability. SIGNIFICANCE STATEMENT: Direct, liquid chromatography-mass spectrometry (LC-MS)-based quantification of cytochrome P450 (CYP) protein is a desirable induction assay endpoint; however such application has been limited due to inefficient workflows. Here, we incorporate recent advancements in protein quantitation methods to efficiently quantify CYP3A4 protein in in vitro induction assays with 11 compounds in up to 3 donors. The data indicate induction responses from mRNA do not always align with those of protein suggesting assessment of induction liability is more complex than thought previously.

Use of in-sample calibration curve approach for quantification of peptides with high-resolution mass spectrometry.

RATIONALE: The in-sample calibration curve (ISCC) approach of quantification utilizes the response of isotopologue ions from spiked-in stable isotope labeled internal standard (SIL-IS) to build a standard curve. The quantitative analysis of the study sample is achieved based on the response of selected monoisotopic analyte ion against the calibration curve. Although this methodology has been demonstrated to be feasible by unit and high-resolution mass spectrometers, quantitation on high-resolution mass spectrometer with product ions has not been tested. We tested the feasibility of this approach using product ions on an high-resolution mass spectrometer equipped with an Orbitrap detector. METHODS: Using a proteomics workflow for sample preparation, two surrogate peptides were quantified from a complex matrix of protein digest from human peripheral blood mononuclear cells (hPBMCs). SIL-IS was spiked in at different levels to construct calibration curves in a traditional manner. ISCCs were prepared using extracted ion chromatograms from isotopically resolved mass spectra and compared with traditional calibration curves. RESULTS: A linear response was observed with ISCC approach for at least two to three orders of magnitude in MS1 as well as targeted MS2 (tMS2). From protein digests, isobaric interferences were observed for endogenous peptides on the MS1 level; this was circumvented with product-ion-based quantitation where for one peptide, %CV for endogenous levels was more than 20% with ISCC but higher with the traditional calibration curve approach. For the second peptide, endogenous levels could not be determined in the traditional approach as calibrant levels did not bracket the lower end, and with the ISCC approach, isotopologues at abundances lower than the endogenous level allowed for quantitative assessments. CONCLUSIONS: ISCC demonstrated improved precision across surrogate peptides from endogenous protein digests. In samples where endogenous analyte concentrations were low in abundance, ISCC rescued what would have been a non-reportable result in a traditional bioanalytical assay as calibrant levels were not prepared at adequately low levels to bracket unknowns. ISCC using high-resolution mass spectrometer is feasible and ideal compared to unit resolution mass spectrometers. High-resolution mass spectrometer allows for isotopic resolution for analytes with > + 2 charge state and provides flexibility in quantification using multiple product ions. ISCC using high-resolution mass spectrometer allows for simultaneous assaying of low abundance isotopologues, the signal acquisition of which is not constrained by limits in data acquisition or calibrant preparation as with other approaches but rather limited by platform sensitivity. In contrast to unit resolution mass spectrometers, these features offered by high-resolution mass spectrometer could be especially useful for the drug discovery assay support where there is less lead time for assay development than for the assays to support the drug development studies.

Quantitation of Plasma Membrane Drug Transporters in Kidney Tissue and Cell Lines Using a Novel Proteomic Approach Enabled a Prospective Prediction of Metformin Disposition.

The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na+/K+-ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na+/K+-ATPase and γ-glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (<2-fold). In vitro transporter kinetics of metformin were extrapolated to in vivo using the corresponding REFs in a PBPK model. The simulated metformin exposures were within 2-fold of clinical exposure. These results demonstrate that transporter REFs based on plasma membrane expression enable a prediction of transporter-mediated drug disposition. Such REFs may be estimated without the correction of plasma membrane recovery when the same procedure is applied between different matrices. SIGNIFICANCE STATEMENT: Transporter REFs based on plasma membrane expression enable in vitro-in vivo extrapolation of transporter kinetics. Plasma membrane recoveries as determined by the quantification of sodium-potassium adenosine triphosphatase were comparable between the in vitro and in vivo systems used in the present study, and therefore had minimal impact on the transporter REF values.

Assessing the Impact of Endogenously Derived Crystalline Drug on the in Vivo Performance of Amorphous Formulations.

Crystallization of drug from an amorphous formulation is expected to negatively impact its bioperformance following oral delivery. In evaluating this in vivo, neat crystalline drug is typically mixed with the amorphous formulation. However, this approach may not adequately mimic the effect of drug crystals that form within the amorphous matrix, because crystal properties are highly dependent on the crystallization environment. The aim of this study was to evaluate the in vivo impact of crystals formed in a generic tacrolimus amorphous formulation, relative to noncrystallized formulations and a reference suspension containing neat crystalline drug. Crystallization of tacrolimus was induced in the generic product by exposing it to moderate temperatures and high relative humidity. Controlled levels of crystallinity in the formulations were achieved by mixing maximally crystallized and fresh formulations at the desired ratios. These formulations were then characterized in vitro and used for oral dosing to beagle dogs. Analysis of blood concentrations versus time revealed that formulations containing 50 and 100% crystalline tacrolimus resulted in lower area under the curve (AUC) and maximum concentration (Cmax) values as compared to the fresh amorphous formulation. However, the AUC and the Cmax values for these formulations were significantly higher than those observed after dosing the pure crystalline tacrolimus suspension. The innovator formulation, Prograf, showed comparable pharmacokinetics before and after exposure to accelerated stability conditions, confirming the robustness of the innovator product to drug crystallization. This study provides insight into the impact of endogenously crystallized material on the oral absorption of a poorly water-soluble compound and highlights the importance of using representative crystalline material when undertaking risk assessment of amorphous formulations.

Mathematical and Experimental Validation of Flux Dialysis Method: An Improved Approach to Measure Unbound Fraction for Compounds with High Protein Binding and Other Challenging Properties.

A flux dialysis method to measure unbound fraction (fu) of compounds with high protein binding and other challenging properties was tested and validated. This method is based on the principle that the initial flux rate of a compound through a size-excluding dialysis membrane is proportional to the product of the compound initial concentration, fu, and unbound dialysis membrane permeability (Pmem). Therefore, fu can be determined from the initial concentration and flux rate, assuming membrane Pmem is known. Compound initial flux rates for 14 compounds were determined by dialyzing human plasma containing compound (donor side) versus compound-free plasma (receiver side) and measuring the rate of compound appearance into the receiver side. Eleven compounds had known fu values obtained from conventional methods (ranging from 0.000013 to 0.22); three compounds (bedaquiline, lapatinib, and pibrentasvir) had previously qualified fu values (e.g., <0.001).Pmem estimated from flux rates and known fu values did not meaningfully differ among the compounds and were consistent with previously published values, indicating that Pmem is a constant for the dialysis membrane. This Pmem constant and the individual compound flux rates were used to calculate fu values. The flux dialysis fu values for the 11 compounds were in good agreement with their reported fu values (all within 2.5-fold; R2 = 0.980), confirming the validity of the method. Furthermore, the flux dialysis method allowed discrete fu to be estimated for the three compounds with previously qualified fu Theoretical and experimental advantages of the flux dialysis method over other dialysis-based protein binding methods are discussed.

Ubiquitination of neuronal nitric-oxide synthase in the calmodulin-binding site triggers proteasomal degradation of the protein.

Nitric-oxide synthase, a cytochrome P450-like hemoprotein enzyme, catalyzes the synthesis of nitric oxide, a critical signaling molecule in a variety of physiological processes. Our laboratory has discovered that certain drugs suicide-inactivate neuronal nitric-oxide synthase (nNOS) and lead to the preferential ubiquitination of the inactivated nNOS by an Hsp70- and CHIP (C terminus of Hsc70-interacting protein)-dependent process. To further understand the process by which altered nNOS is recognized, ubiquitinated, and proteasomally degraded, we examined the sites of ubiquitination on nNOS. We utilized an in vitro ubiquitination system containing purified E1, E2 (UbcH5a), Hsp70, and CHIP that recapitulates the ability of the cells to selectively recognize and ubiquitinate the altered forms of nNOS. LC-MS/MS analysis of the tryptic peptides obtained from the in vitro ubiquitinated nNOS identified 12 ubiquitination sites. Nine of the sites were within the oxygenase domain and two were in the calmodulin-binding site, which links the oxygenase and reductase domains, and one site was in the reductase domain. Mutational analysis of the lysines in the calmodulin-binding site revealed that Lys-739 is a major site for poly-ubiquitination of nNOS in vitro and regulates, in large part, the CHIP-dependent degradation of nNOS in HEK293 cells, as well as in in vitro studies with fraction II. Elucidating the exact site of ubiquitination is an important step in understanding how chaperones recognize and trigger degradation of nNOS.

Signature peptide selection workflow for biomarker quantification using LC-MS-based targeted proteomics.

In contrast to quantification of biotherapeutics, endogenous protein biomarker and target quantification using LC-MS based targeted proteomics can require a much more stringent and time-consuming tryptic signature peptide selection for each specific application. While some general criteria exist, there are no tools currently available in the public domain to predict the ionization efficiency for a given signature peptide candidate. Lack of knowledge of the ionization efficiencies forces investigators to choose peptides blindly, thus hindering method development for low abundant protein quantification. Here, the authors propose a tryptic signature peptide selection workflow to achieve a more efficient method development and to improve success rates in signature peptide selection for low abundant endogenous target and protein biomarker quantification.

A Simplified Method for Determining Blood-to-Plasma Ratios in vitro and ex vivo by Matrix Matching with Blank Blood or Plasma.

OBJECTIVE: This work describes a simplified, 96-well plate method for determining the blood-to-plasma concentration ratio (BP ratio) for small molecules. METHODS: The need for calibration curves was eliminated using a matrix-matching approach in which blood samples were mixed with blank plasma and plasma samples were mixed with blank blood. As a result, both blood- and plasma-origin samples shared an equivalent matrix ahead of bioanalysis. In the in vitro assay, identical sample matrices were achieved by using the same source of blank plasma and blood. RESULTS: In humans, a good correlation (R2 = 0.84) was observed between the data obtained in this matrix-matching method and literature values for 11 commercial compounds possessing a wide range of logD values across multiple chemical classes. In addition, this method showed good agreement with in vitro BP ratios for 10 proprietary compounds determined radiometrically (R2 = 0.72) in human and preclinical species. Finally, the in vitro matrix matching method compared favorably to BP ratios determined ex vivo for 13 proprietary and literature compounds (R2 = 0.87) in rat. CONCLUSION: This method, suitable for in vitro and ex vivo BP ratio determinations, is operationally efficient, robust, and a useful improvement upon previously published methods.

Bioanalytical strategy for the characterization and bioanalysis of biologics: a global, nonregulated bioanalytical lab perspective.

Over the past two decades, we have seen an increase in the complexity and diversity of biotherapeutic modalities pursued by biopharmaceutical companies. These biologics are multifaceted and susceptible to post-translational modifications and in vivo biotransformation that could impose challenges for bioanalysis. It is vital to characterize the functionality, stability and biotransformation products of these molecules to enable screening, identify potential liabilities at an early stage and devise a bioanalytical strategy. This article highlights our perspective on characterization and bioanalysis of biologics using hybrid LC-MS in our global nonregulated bioanalytical laboratories. AbbVie's suite of versatile, stage-appropriate characterization assays and quantitative bioanalytical approaches are discussed, along with guidance on their utility in answering project-specific questions to aid in decision-making.

Integrity and efficiency: AbbVie's journey of building an integrated nonregulated bioanalytical laboratory.

While bioanalytical outsourcing is widely adopted in the pharmaceutical industry, AbbVie is one of the few large biopharmaceutical companies having an internal bioanalytical unit to support nearly all its drug metabolism and pharmacokinetic studies. This article highlights our experience and perspective in building an integrated and centralized laboratory to provide early discovery and preclinical-stage bioanalytical support with high operational efficiency, cost-effectiveness and data integrity. The advantages of in-house nonregulated bioanalytical support include better control of data quality, faster turnaround times, real-time knowledge sharing and troubleshooting, and lower near- and long-term costs. The success of an in-house model depends upon a comprehensively optimized and streamlined workflow, fueled by continuous improvements and implementation of innovative technologies.

Identification of Plasma Biomarkers from Rheumatoid Arthritis Patients Using an Optimized Sequential Window Acquisition of All THeoretical Mass Spectra (SWATH) Proteomics Workflow.

Rheumatoid arthritis (RA) is a systemic autoimmune and inflammatory disease. Plasma biomarkers are critical for understanding disease mechanisms, treatment effects, and diagnosis. Mass spectrometry-based proteomics is a powerful tool for unbiased biomarker discovery. However, plasma proteomics is significantly hampered by signal interference from high-abundance proteins, low overall protein coverage, and high levels of missing data from data-dependent acquisition (DDA). To achieve quantitative proteomics analysis for plasma samples with a balance of throughput, performance, and cost, we developed a workflow incorporating plate-based high abundance protein depletion and sample preparation, comprehensive peptide spectral library building, and data-independent acquisition (DIA) SWATH mass spectrometry-based methodology. In this study, we analyzed plasma samples from both RA patients and healthy donors. The results showed that the new workflow performance exceeded that of the current state-of-the-art depletion-based plasma proteomic platforms in terms of both data quality and proteome coverage. Proteins from biological processes related to the activation of systemic inflammation, suppression of platelet function, and loss of muscle mass were enriched and differentially expressed in RA. Some plasma proteins, particularly acute-phase reactant proteins, showed great power to distinguish between RA patients and healthy donors. Moreover, protein isoforms in the plasma were also analyzed, providing even deeper proteome coverage. This workflow can serve as a basis for further application in discovering plasma biomarkers of other diseases.

Expanding the Repertoire for "Large Small Molecules": Prodrug ABBV-167 Efficiently Converts to Venetoclax with Reduced Food Effect in Healthy Volunteers.

Since gaining approval for the treatment of chronic lymphocytic leukemia (CLL), the BCL-2 inhibitor venetoclax has transformed the treatment of this and other blood-related cancers. Reflecting the large and hydrophobic BH3-binding groove within BCL-2, venetoclax has significantly higher molecular weight and lipophilicity than most orally administered drugs, along with negligible water solubility. Although a technology-enabled formulation successfully achieves oral absorption in humans, venetoclax tablets have limited drug loading and therefore can present a substantial pill burden for patients in high-dose indications. We therefore generated a phosphate prodrug (3, ABBV-167) that confers significantly increased water solubility to venetoclax and, upon oral administration to healthy volunteers either as a solution or high drug-load immediate release tablet, extensively converts to the parent drug. Additionally, ABBV-167 demonstrated a lower food effect with respect to venetoclax tablets. These data indicate that beyond-rule-of-5 molecules can be successfully delivered to humans via a solubility-enhancing prodrug moiety to afford robust exposures of the parent drug following oral dosing.

Effects of microtubule-inhibiting small molecule and antibody-drug conjugate treatment on differentially-sized A431 squamous carcinoma spheroids.

Multicellular tumor spheroids have been increasingly used by researchers to produce more physiologically relevant experimental environments. However, tracking of spheroid growth and treatment-induced volume reduction has not been readily adopted. Here, squamous carcinoma cells were seeded at different starting cell numbers with growth and reduction kinetics monitored using live cell imaging. Following the initial growth phase, spheroids were treated with auristatin as small molecule (MMAE) or as antibody-drug conjugate containing non-cleavable auristatin drug payload (033-F). Compared to cells in monolayers, 033-F had notably weaker potency against spheroids despite potency levels of MMAE being similar against monolayers and spheroids. Accumulation of released payload from 033-F was reduced in higher volume spheroids, likely contributing to the potency differences. Despite lowered potency towards spheroids with 033-F, spheroid volume was still readily reduced by 033-F in a dose-dependent fashion, with >85% volume reductions at the highest concentrations for all spheroid sizes. Additionally, the core of the larger spheroids showed more resiliency towards microtubule inhibition. Overall, this work highlights how various in-vivo 'features' such as tumor penetration, cell interactions, and increased resistance to therapeutics can be integrated into a spheroid model and tracked over time by automated imaging technology.

Amorphous solid dispersions of enzalutamide and novel polysaccharide derivatives: investigation of relationships between polymer structure and performance.

Amorphous solid dispersion (ASD) is a widely employed formulation technique for drugs with poor aqueous solubility. Polymers are integral components of ASDs, but mechanisms by which polymers lead to the generation and maintenance of supersaturated solutions, which enhance oral absorption in vivo, are poorly understood. Herein, a diverse group of newly synthesized cellulose derivatives was evaluated for their ability to inhibit crystallization of enzalutamide, a poorly soluble compound used to treat prostate cancer. ASDs were prepared from selected polymers, specifically a somewhat hydrophobic polymer that was extremely effective at inhibiting drug crystallization, and a less effective, but more hydrophilic, crystallization inhibitor, that might afford better release. Drug membrane transport rate was evaluated in vitro and compared to in vivo performance, following oral dosing in rats. Good correlation was noted between the in vitro diffusion cell studies and the in vivo data. The ASD formulated with the less effective crystallization inhibitor outperformed the ASD prepared with the highly effective crystallization inhibitor in terms of the amount and rate of drug absorbed in vivo. This study provides valuable insight into key factors impacting oral absorption from enabling ASD formulations, and how best to evaluate such formulations using in vitro approaches.

Determination of IL-23 Pharmacokinetics by Highly Sensitive Accelerator Mass Spectrometry and Subsequent Modeling to Project IL-23 Suppression in Psoriasis Patients Treated with Anti-IL-23 Antibodies.

The pro-inflammatory cytokine interleukin (IL)-23 is a key modulator of the immune response, making it an attractive target for the treatment of autoimmune disease. Correspondingly, several monoclonal antibodies against IL-23 are either in development or approved for autoimmune indications such as psoriasis. Despite being a clinical validated target, IL-23 pharmacokinetics (e.g., IL-23 synthesis and elimination rates) and the degree of target suppression (i.e., decrease in free "active" IL-23) associated with clinical efficacy are not well understood, primarily due to its ultra-low circulating levels and the lack of sensitive and accurate measurement methods. In the current work, this issue was overcome by using accelerator mass spectrometry (AMS) to measure the concentration and pharmacokinetics of human recombinant [14C]-IL-23 following an intravenous trace-dose in cynomolgus monkeys. IL-23 pharmacokinetic parameters along with clinical drug exposure and IL-23 binding affinities from four different anti-IL-23 antibodies (ustekinumab, tildrakizumab, guselkumab, and risankizumab) were used to build a pharmacokinetics/pharmacodynamics (PK/PD) model to assess the time course of free IL-23 over one year in psoriasis patients following different dosing regimens. The predicted rank order of reduction of free IL-23 was consistent with their reported rank order of Psoriasis Area and Severity Index (PASI) 100 scores in clinical efficacy trials (ustekinumab < tildrakizumab < guselkumab < risankizumab), thus demonstrating the utility of highly sensitive AMS for determining target pharmacokinetics to inform PK/PD modeling and assessing target suppression associated with clinical efficacy.

High-Throughput, Multispecies, Parallelized Plasma Stability Assay for the Determination and Characterization of Antibody-Drug Conjugate Aggregation and Drug Release.

The stability of antibody-drug conjugates (ADCs) in circulation is critical for maximum efficacy and minimal toxicity. An ADC reaching the intended target intact can deliver the highest possible drug load to the tumor and reduce off-target toxicity from free drug in the blood. As such, assessment of ADC stability is a vital piece of data during development. However, traditional ADC stability assays can be manually intensive, low-throughput, and require large quantities of ADC material. Here, we introduce an automated, high-throughput plasma stability assay for screening drug release and aggregation over 144 h for up to 40 ADCs across five matrices simultaneously. The amount of ADC material during early drug development is often limited, so this assay was implemented in 384-well format to minimize material requirements to <100 µg of each ADC and 100 µL of plasma per species type. Drug release and aggregation output were modeled using nonlinear regression equations to calculate formation rates for each data type. A set of 15 ADCs with different antibodies and identical valine-citrulline-p-aminobenzylcarbamate-monomethylauristatin E linker-drug payloads was tested and formation rates were compared across ADCs and between species, revealing several noteworthy trends. In particular, a wide range in aggregation was found when altering only the antibody, suggesting a key role for plasma stability screening early in the development process to find and remove antibody candidates with the potential to create unstable ADCs. The assay presented here can be leveraged to provide stability data on new chemistry and antibody screening initiatives, select the best candidate for in vivo studies, and provide results that highlight stability issues inherent to particular ADC designs throughout all stages of ADC development.

Tetrahydrobiopterin depletion and ubiquitylation of neuronal nitric oxide synthase.

Tetrahydrobiopterin is a necessary cofactor for the synthesis of nitric oxide by the hemeprotein enzyme, NO-synthase (NOS). It is widely thought that inadequate levels of tetrahydrobiopterin lead to tissue injury and organ dysfunction due, in part, to formation of superoxide from pterin-deficient NOS. In the course of studies on the ubiquitylation of neuronal NOS (nNOS), we have found that certain substrate analogs, such as N(G)-nitro-L-arginine, stabilize the dimeric form of nNOS and protect the enzyme from ubiquitylation. Since tetrahydrobiopterin is known to bind near heme and confers stability to the active dimeric structure of nNOS, we wondered if the loss of tetrahydrobiopterin could be an endogenous signal for nNOS ubiquitylation and degradation. We show here in HEK293 cells stably transfected with nNOS that depletion of tetrahydrobiopterin by treatment with 2,4-diamino-6-hydroxypyrimidine leads to destabilization of the dimeric form and enhances ubiquitylation of nNOS. Sepiapterin, a precursor to tetrahydrobiopterin in the salvage pathway, completely reverses the effect of 2,4-diamino-6-hydroxypyrimidine on nNOS ubiquitylation. Consistent with that found in cells, the in vitro ubiquitylation of nNOS by reticulocyte proteins decreases when tetrahydrobiopterin is present. Thus, inadequate amounts of tetrahydrobiopterin may lead to a sustained decrease in the steady state level of nNOS that is not readily reversed.