Identification and biophysical characterization of a novel domain-swapped camelid antibody specific for fentanyl.

Opioid use disorders (OUD) and overdoses are ever-evolving public health threats that continue to grow in incidence and prevalence in the United States and abroad. Current treatments consist of opioid receptor agonists and antagonists, which are safe and effective but still suffer from some limitations. Murine and humanized monoclonal antibodies (mAb) have emerged as an alternative and complementary strategy to reverse and prevent opioid-induced respiratory depression. To explore antibody applications beyond traditional heavy-light chain mAbs, we identified and biophysically characterized a novel single-domain antibody specific for fentanyl from a camelid variable-heavy-heavy (VHH) domain phage display library. Structural data suggested that VHH binding to fentanyl was facilitated by a unique domain-swapped dimerization mechanism, which accompanied a rearrangement of complementarity-determining region (CDR) loops leading to the formation of a fentanyl-binding pocket. Structure-guided mutagenesis further identified an amino acid substitution that improved the affinity and relaxed the requirement for dimerization of the VHH in fentanyl binding. Our studies demonstrate VHH engagement of an opioid and inform on how to further engineer a VHH for enhanced stability and efficacy, laying the groundwork for exploring the in vivo applications of VHH-based biologics against OUD and overdose.

Development of an Engineered Single-Domain Antibody for Targeting MET in Non-Small Cell Lung Cancer.

The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is upregulated or mutated in 5% of non-small-cell lung cancer (NSCLC) patients and overexpressed in multiple other cancers. We sought to develop a novel single-domain camelid antibody with high affinity for MET that could be used to deliver conjugated payloads to MET expressing cancers. From a naïve camelid variable-heavy-heavy (VHH) domain phage display library, we identified a VHH clone termed 1E7 that displayed high affinity for human MET and was cross-reactive with MET across multiple species. When expressed as a bivalent human Fc fusion protein, 1E7-Fc was found to selectively bind to EBC-1 (MET amplified) and UW-Lung 21 (MET exon 14 mutated) cell lines by flow cytometry and immunofluorescence imaging. Next, we investigated the ability of [89Zr]Zr-1E7-Fc to detect MET expression in vivo by PET/CT imaging. [89Zr]Zr-1E7-Fc demonstrated rapid localization and high tumor uptake in both xenografts with a %ID/g of 6.4 and 5.8 for EBC-1 and UW-Lung 21 at 24 h, respectively. At the 24 h time point, clearance from secondary and nontarget tissues was also observed. Altogether, our data suggest that 1E7-Fc represents a platform technology that can be employed to potentially both image and treat MET-altered NSCLC.

Childhood Adversity Among Adults With Chronic Pain: Prevalence and Association With Pain-related Outcomes.

OBJECTIVES: Adverse childhood experiences (ACEs) have been linked to the development and impact of chronic pain in adulthood. The goal of this study was to investigate the prevalence of ACEs in a treatment-seeking sample of adults with chronic pain and the relationship between number and type(s) of ACEs and pain-related outcomes. METHODS: Adults (N=1794) presenting for treatment at a multidisciplinary pain management center completed self-report measures of childhood adversity, pain, functioning, emotional distress, and adjustment to pain. RESULTS: Participants endorsing ≥4 ACEs had significantly worse pain-related outcomes and lower quality of life compared with individuals reporting fewer ACEs. Having ≥3 ACEs was associated with higher anxiety and depression levels. Experiences of childhood neglect negatively affected mental health-related outcomes independent of the number of ACEs. Significant sex differences were found in the number and type of ACEs reported but not in the relationship between ACEs and outcome variables. CONCLUSION: Findings suggest that the number and the type of self-reported ACE(s) are associated with pain-related variables and psychosocial functioning in adults with chronic pain. The results highlight the importance of assessment of ACEs and trauma-informed care with patients with chronic pain.

Mediators of change in depressed mood following pain rehabilitation among participants with mild, moderate, or severe depressive symptoms.

BACKGROUND: Prior research indicates that depression and chronic pain commonly co-exist and impact each other. Interdisciplinary pain rehabilitation programs (IPRPs) have been shown to lead to statistically and clinically significant improvements for patients who report both depressed mood and chronic pain, however there is a gap in the literature regarding the mechanisms by which these improvements occur. METHODS: This two-site, distinct sample study (Study 1: N = 303, 10-week, individual format, ACT-based program; Study 2: N = 406, 3-week, group format, CBT-based program) evaluated mediators of treatment improvement in depressive symptoms among adult IPRP participants who reported elevated depressive symptoms at program admission and examined treatment mechanisms for depressive symptoms. RESULTS: Self-reported pain self-efficacy and pain catastrophizing - particularly the helplessness domain - mediated the treatment-related change in depression among IPRP participants with elevated depressive symptoms across the two sites and samples. In one sample, full mediation was achieved while in the other sample, partial mediation was achieved. Participants in both samples showed improvement on all measures. LIMITATIONS: This study relied on self-report measures of depressive severity and not clinical diagnosis. Results may not generalize to other populations of patients with chronic pain. There was no control condition in either study. CONCLUSION: Increasing pain self-efficacy and decreasing a sense of helplessness are important treatment targets among IPRP participants who endorse symptoms of depression.

Sex differences in interdisciplinary pain rehabilitation outcomes: a systematic review.

OBJECTIVES: Interdisciplinary pain rehabilitation programs (IPRPs) are evidence-based treatments for chronic pain. Previous research has demonstrated that initial presentations of adult men and women admitted to IPRPs differ, but less is known about sex differences in IPRP treatment outcomes. To summarize and synthesize the current literature base on this topic, a systematic literature review was conducted that asked: are sex differences present in participant outcomes upon completion of interdisciplinary pain rehabilitation programs for cisgender patients? Four core domains of outcome measures were assessed: depression, pain catastrophizing, pain interference, and pain intensity/severity. METHODS: Relevant studies meeting inclusion criteria were identified using a computer-aided search of the following electronic bibliographic databases: PubMed (MEDLINE), EMBASE, PsycINFO, CENTRAL (via Wiley Online Library), and CINAHL (via EBSCOhost). The reference list of relevant studies identified in the electronic searches was also screened to identify further studies. RESULTS: This review concluded that most studies did not find any differences related to sex using the four outcome measures included in this review. This implies that specific considerations based on sex may not be needed when providing interdisciplinary pain rehabilitation. CONCLUSIONS: Future research directions include comparison of additional outcome measures and exploring sex and gender issues in IPRP treatment in other formats than as a simple dichotomous variable.

Patients with Clinically Elevated Depressive Symptoms Report Improvements in Mood, Pain, and Functioning Following Participation in Interdisciplinary Pain Rehabilitation.

OBJECTIVE: Chronic pain and depression frequently co-occur and exacerbate one another; therefore, it is important to treat both conditions to improve patient outcomes. The current study evaluates an interdisciplinary pain rehabilitation program (IPRP) with respect to the following questions: 1) How do clinically elevated depressive symptoms impact pain-related treatment outcomes? and 2) To what extent does IPRP participation yield reliable and clinically significant change in depressed mood? METHODS: Participants in this study included 425 adults who engaged in a 10-week IPRP and completed self-report measures of pain, mood, and functioning at intake and discharge. Participants were categorized into 4 groups based on self-reported depressive symptoms (PROMIS Depression): within normal limits (WNL; n = 121), Mild (n = 115), Moderate (n = 153), and Severe (n = 36). RESULTS: Participants reported significant improvement in pain, pain-related life interference, health-related quality of life, pain catastrophizing, and depressed mood regardless of initial symptom level. In addition, 43.4% of patients with Mild, Moderate, or Severe depressed mood reported reliable and clinically significant improvement in depressive symptoms and 30.3% were in remission at the end of treatment. CONCLUSIONS: These findings support the assertion that IPRPs represent an effective treatment for patients with comorbid chronic pain and depression and that participation is associated with improvement in both conditions.

Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography.

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

Adverse Childhood Experiences and Chronic Pain Rehabilitation Treatment Outcomes in Adults.

OBJECTIVES: Adverse childhood experiences (ACEs) are commonly reported by individuals with chronic pain. However, little is known about how ACE exposure influences treatment outcomes. The goal of the current study was to evaluate group and treatment-related differences among adults with varying levels of ACE exposure participating in a pain rehabilitation treatment program. METHODS: Adult participants (N=269) were categorized as 0 ACEs (n=65), 1 to 2 ACEs (n=87), or ≥3 ACEs (n=117). Participants completed self-report measures of pain, physical functioning, and psychosocial functioning at intake and discharge from a 10-week interdisciplinary pain rehabilitation program. RESULTS: ACE exposure was frequently endorsed in this sample, with the majority of participants (78.5%) reporting at least 1 form of childhood adversity. Adults in the ≥3 ACEs group reported a greater level of impairment in mental health symptoms and adjustment to chronic pain; however, all groups endorsed treatment improvements and there were no differences in response to treatment. There were also no differences between groups on measures of pain or physical functioning at intake or discharge. DISCUSSION: ACE exposure appears common among treatment-seeking adults with chronic pain and is associated with increased clinical complexity. However, adults with and without exposure to ACEs endorsed significant improvements in pain and functioning following participation in an interdisciplinary pain rehabilitation program. This model of treatment may be especially well situated to address the biopsychosocial contributions to pain among those with a history of adversity.

Partner Abuse Among Treatment-Seeking Individuals with Chronic Pain: Prevalence, Characteristics, and Association with Pain-Related Outcomes.

OBJECTIVE: This study assessed the prevalence of abusive partner relationships among individuals presenting for chronic pain treatment. In addition, this study examined the association between partner abuse histories and pain-relevant outcome variables. DESIGN: Cross-sectional. SETTING: This study took place at a specialty pain rehabilitation treatment center in the Midwestern United States. SUBJECTS: Participants in this study (N = 108) included adults (Mage = 45.73 [15.95] years) presenting for chronic pain treatment who consented to participate in a research study on stress, relationships, and chronic pain. METHODS: Participants completed self-report measures about relationship and abuse histories, physical and mental health, and demographic information. Participants were categorized into the following groups: no intimate partner violence (IPV), past IPV (>12 months ago), or current/recent IPV (≤12 months ago). RESULTS: Results indicated that over half (56%) of the sample endorsed a history of partner abuse and around one-third (29%) of the sample had experienced abuse within the past year. Psychological/emotional abuse was the most common form of abuse reported. Those with current/recent abuse histories reported greater impairment in pain interference, post-traumatic stress symptoms, mental health functioning, and pain self-efficacy compared with those who had not experienced abuse in the past year. CONCLUSIONS: Partner abuse appears common among individuals with chronic pain and is associated with pain-relevant outcomes, warranting additional clinical attention and research in this area.

Treatment Outcomes and Mechanisms for an ACT-Based 10-Week Interdisciplinary Chronic Pain Rehabilitation Program.

BACKGROUND: Interdisciplinary pain rehabilitation programs are an evidence-based biopsychosocial treatment approach for chronic pain. The purpose of the current study is to assess outcomes for a 10-week interdisciplinary, acceptance and commitment therapy (ACT)-based, outpatient treatment model and to evaluate the relationship between psychological process variables (ie, pain catastrophizing, pain acceptance, pain self-efficacy) and treatment outcomes. METHODS: 137 adults with chronic pain completed an interdisciplinary pain rehabilitation program. Measures of pain, pain interference, health-related quality of life, anxiety, depressed mood, insomnia, pain catastrophizing, pain acceptance, and pain self-efficacy were completed at admission and discharge. Data were also collected on demographic and clinical variables, including opioid use. RESULTS: Results indicated significant changes in all measures at program discharge compared to admission. Opioid doses were also reduced. Results of within-subjects meditational analyses indicated that pain catastrophizing accounted for a significant portion of the treatment effect for pain severity, pain interference, and depressed mood. Pain acceptance was a mediator for change in depressed mood, whereas pain self-efficacy was a mediator for pain interference outcomes. CONCLUSIONS: This study supports a 10-week, ACT-based treatment model for interdisciplinary chronic pain rehabilitation. In addition, pain catastrophizing, pain acceptance, and pain self-efficacy were each found to be mechanisms by which individuals achieve successful treatment outcomes. This research provides further support for interdisciplinary rehabilitation approaches for chronic pain.

Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states.

Protein kinases undergo large-scale structural changes that tightly regulate function and control recognition by small-molecule inhibitors. Methods for quantifying the conformational effects of inhibitors and linking them to an understanding of selectivity patterns have long been elusive. We have developed an ultrafast time-resolved fluorescence methodology that tracks structural movements of the kinase activation loop in solution with angstrom-level precision, and can resolve multiple structural states and quantify conformational shifts between states. Profiling a panel of clinically relevant Aurora kinase inhibitors against the mitotic kinase Aurora A revealed a wide range of conformational preferences, with all inhibitors promoting either the active DFG-in state or the inactive DFG-out state, but to widely differing extents. Remarkably, these conformational preferences explain broad patterns of inhibitor selectivity across different activation states of Aurora A, with DFG-out inhibitors preferentially binding Aurora A activated by phosphorylation on the activation loop, which dynamically samples the DFG-out state, and DFG-in inhibitors binding preferentially to Aurora A constrained in the DFG-in state by its allosteric activator Tpx2. The results suggest that many inhibitors currently in clinical development may be capable of differentiating between Aurora A signaling pathways implicated in normal mitotic control and in melanoma, neuroblastoma, and prostate cancer. The technology is applicable to a wide range of clinically important kinases and could provide a wealth of valuable structure-activity information for the development of inhibitors that exploit differences in conformational dynamics to achieve enhanced selectivity.

A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation.

Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100 fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift from the DFG-Out to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation triggers a switch within the DFG-In subpopulation from an autoinhibited DFG-In substate to an active DFG-In substate, leading to catalytic activation. This mechanism raises new questions about the functional role of the DFG-Out state in protein kinases.