Discordance between preclinical and clinical testing of NaV1.7-selective inhibitors for pain.

ABSTRACT: The voltage-gated sodium channel NaV1.7 plays an important role in pain processing according to genetic data. Those data made NaV1.7 a popular drug target, especially since its relatively selective expression in nociceptors promised pain relief without the adverse effects associated with broader sodium channel blockade. Despite encouraging preclinical data in rodents, NaV1.7-selective inhibitors have not yet proven effective in clinical trials. Discrepancies between preclinical and clinical results should raise alarms. We reviewed preclinical and clinical reports on the analgesic efficacy of NaV1.7-selective inhibitors and found critical differences in several factors. Putting aside species differences, most preclinical studies tested young male rodents with limited genetic variability, inconsistent with the clinical population. Inflammatory pain was the most common preclinical chronic pain model whereas nearly all clinical trials focused on neuropathic pain despite some evidence suggesting NaV1.7 channels are not essential for neuropathic pain. Preclinical studies almost exclusively measured evoked pain whereas most clinical trials assessed average pain intensity without distinguishing between evoked and spontaneous pain. Nearly all preclinical studies gave a single dose of drug unlike the repeat dosing used clinically, thus precluding preclinical data from demonstrating whether tolerance or other slow processes occur. In summary, preclinical testing of NaV1.7-selective inhibitors aligned poorly with clinical testing. Beyond issues that have already garnered widespread attention in the pain literature, our results highlight the treatment regimen and choice of pain model as areas for improvement.

The RAF cysteine-rich domain: Structure, function, and role in disease.

RAF kinases, consisting of ARAF, BRAF and CRAF, are direct effectors of RAS GTPases and critical for signal transduction through the RAS-MAPK pathway. Driver mutations in BRAF are commonplace in human cancer, while germline mutations in BRAF and CRAF cause RASopathy development syndromes. However, there remains a lack of effective drugs that target RAF function, which is partially due to the complexity of the RAF activation cycle. Therefore, greater understanding of RAF regulation is required to identify new approaches that target its function in disease. A key piece of this puzzle is the RAF zinc finger, often referred to as the cysteine-rich domain (CRD). The CRD is a lipid and protein binding domain which plays complex and opposing roles in the RAF activation cycle. Firstly, it supports the RAS-RAF interaction during RAF activation by binding to phosphatidylserine (PS) in the plasma membrane and by making direct RAS contacts. Conversely, under quiescent conditions the CRD also plays a critical role in maintaining RAF in a closed, autoinhibited state. However, the interplay between these activities and their relative importance for RAF activation were not well understood. Recent structural and biochemical studies have contributed greatly to our understanding of these roles and identified functional differences between BRAF CRD and that of CRAF. This chapter provides an in-depth review of the CRDs roles in RAF regulation and how they may inform novel approaches to target RAF function.

Beyond Strain Release: Delocalization-Enabled Organic Reactivity.

The release of strain energy is a fundamental driving force for organic reactions. However, absolute strain energy alone is an insufficient predictor of reactivity, evidenced by the similar ring strain but disparate reactivity of cyclopropanes and cyclobutanes. In this work, we demonstrate that electronic delocalization is a key factor that operates alongside strain release to boost, or even dominate, reactivity. This delocalization principle extends across a wide range of molecules containing three-membered rings such as epoxides, aziridines, and propellanes and also applies to strain-driven cycloaddition reactions. Our findings lead to a "rule of thumb" for the accurate prediction of activation barriers in such systems, which can be easily applied to reactions involving many of the strained building blocks commonly encountered in organic synthesis, medicinal chemistry, polymer science, and bioconjugation. Given the significance of electronic delocalization in organic chemistry, for example in aromatic π-systems and hyperconjugation, we anticipate that this concept will serve as a versatile tool to understand and predict organic reactivity.

Regulation of RAF family kinases: new insights from recent structural and biochemical studies.

The RAF kinases are required for signal transduction through the RAS-RAF-MEK-ERK pathway, and their activity is frequently up-regulated in human cancer and the RASopathy developmental syndromes. Due to their complex activation process, developing drugs that effectively target RAF function has been a challenging endeavor, highlighting the need for a more detailed understanding of RAF regulation. This review will focus on recent structural and biochemical studies that have provided 'snapshots' into the RAF regulatory cycle, revealing structures of the autoinhibited BRAF monomer, active BRAF and CRAF homodimers, as well as HSP90/CDC37 chaperone complexes containing CRAF or BRAFV600E. In addition, we will describe the insights obtained regarding how BRAF transitions between its regulatory states and examine the roles that various BRAF domains and 14-3-3 dimers play in both maintaining BRAF as an autoinhibited monomer and in facilitating its transition to an active dimer. We will also address the function of the HSP90/CDC37 chaperone complex in stabilizing the protein levels of CRAF and certain oncogenic BRAF mutants, and in serving as a platform for RAF dephosphorylation mediated by the PP5 protein phosphatase. Finally, we will discuss the regulatory differences observed between BRAF and CRAF and how these differences impact the function of BRAF and CRAF as drivers of human disease.

Bioluminescence Resonance Energy Transfer (BRET)-based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells.

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

A Flexible and Efficient Approach to Missing Transverse Momentum Reconstruction.

Missing transverse momentum is a crucial observable for physics at hadron colliders, being the only constraint on the kinematics of "invisible" objects such as neutrinos and hypothetical dark matter particles. Computing missing transverse momentum at the highest possible precision, particularly in experiments at the energy frontier, can be a challenging procedure due to ambiguities in the distribution of energy and momentum between many reconstructed particle candidates. This paper describes a novel solution for efficiently encoding information required for the computation of missing transverse momentum given arbitrary selection criteria for the constituent reconstructed objects. Pileup suppression using information from both the calorimeter and the inner detector is an integral component of the reconstruction procedure. Energy calibration and systematic variations are naturally supported. Following this strategy, the ATLAS Collaboration has been able to optimise the use of missing transverse momentum in diverse analyses throughout Runs 2 and 3 of the Large Hadron Collider and for future analyses.

Synthesis of Heterocycle-Substituted Bicyclo[3.1.1]heptanes and Aza-bicyclo[3.1.1]heptanes via Photocatalytic Minisci Reaction.

A route toward heterocycle-functionalized bicyclo[3.1.1]heptanes (BCHeps) and aza-bicyclo[3.1.1]heptanes (aza-BCHeps) has been developed, using mild, photocatalytic Minisci-like conditions to introduce various heterocycles at the bridgehead position from readily available N-hydroxyphthalimide esters of the corresponding carboxylic acids. This chemistry enables access to heterocycle-functionalized BCHep-containing structures that are highly relevant in medicinal chemistry research as potential bioisosteres of meta-substituted arenes and pyridines.

Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.

ACR Appropriateness Criteria® Staging and Post-Therapy Assessment of Head and Neck Cancer.

Imaging of head and neck cancer at initial staging and as part of post-treatment surveillance is a key component of patient care as it guides treatment strategy and aids determination of prognosis. Head and neck cancer includes a heterogenous group of malignancies encompassing several anatomic sites and histologies, with squamous cell carcinoma the most common. Together this comprises the seventh most common cancer worldwide. At initial staging comprehensive imaging delineating the anatomic extent of the primary site, while also assessing the nodal involvement of the neck is necessary. The treatment of head and neck cancer often includes a combination of surgery, radiation, and chemotherapy. Post-treatment imaging is tailored for the evaluation of treatment response and early detection of local, locoregional, and distant recurrent tumor. Cross-sectional imaging with CT or MRI is recommended for the detailed anatomic delineation of the primary site. PET/CT provides complementary metabolic information and can map systemic involvement. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment.

Protocol for measuring BRAF autoinhibition in live cells using a proximity-based NanoBRET assay.

BRAF is frequently activated via mutation in human cancer and the RASopathy syndromes; however, for BRAF activation to occur, autoinhibitory interactions between the regulatory and catalytic domains must be relieved. Here, we present a proximity-based NanoBRET (bioluminescence resonance energy transfer) assay for real-time measurement of BRAF autoinhibition in live cells. We describe steps for seeding, transfecting, and replating cells. We then detail procedures for reading the NanoBRET emissions and confirming protein expression. For complete details on the use and execution of this protocol, please refer to Spencer-Smith et al. (2022).1.

Mediating Effects of Thought Suppression in the Relationship Between Mindfulness and Substance Use Craving.

Background: Substance use disorder (SUD) is a significant issue in the United States, characterized by chronic relapse following periods of abstinence. One of the primary precursors to relapse is craving. Findings from several studies have shown a negative association between trait mindfulness and craving in clinical samples; however, further research is needed to understand mechanisms underlying this relationship. Purpose/Objectives: The current study assessed thought suppression as a partial mediator of the relationship between trait mindfulness and craving. Methods: The current study used data from a previous randomized controlled trial of adults (N = 244) enrolled in community-based treatment for substance use disorder (SUD). Results: Analyses showed a significant moderate positive association between thought suppression and craving, a significant moderate negative association between thought suppression and trait mindfulness, and a significant moderate negative association between trait mindfulness and craving. Subsequent analyses confirmed a partial mediating role of thought suppression in the relationship between trait mindfulness and craving, indicating the inverse relationship between trait mindfulness and craving was partially explained by thought suppression. Conclusions/importance: These findings may inform treatment for SUD. Specifically, targeting thought suppression through mindfulness-based treatment approaches may be a mechanism through which craving can be reduced.

Investigation of the pharmacokinetics and metabolic fate of Fasiglifam (TAK-875) in male and female rats following oral and intravenous administration.

The metabolism and pharmacokinetics of fasiglifam (TAK-875, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl]methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid), a selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist, were studied following intravenous (5 mg/kg) and oral administration (10 and 50 mg/kg) to male and female Sprague Dawley rats.Following intravenous dosing at 5 mg/kg, peak observed plasma concentrations of 8.8/9.2 µg/ml were seen in male and female rats respectively.Following oral dosing, peak plasma concentrations at 1 h of ca. 12.4/12.9 µg/ml for 10 mg/kg and 76.2/83.7 µg/ml for 50 mg/kg doses were obtained for male and female rats respectively. Drug concentrations then declined in the plasma of both sexes with t1/2's of 12.4 (male) and 11.2 h (female). Oral bioavailability was estimated to be 85-120% in males and females at both dose levels.Urinary excretion was low, but in a significant sex-related difference, female rats eliminated ca. 10-fold more drug-related material by this route.Fasiglifam was the principal drug-related compound in plasma, with 15 metabolites, including the acyl glucuronide, also detected. In addition to previously identified metabolites, a novel biotransformation, that produced a side-chain shortened metabolite via elimination of CH2 from the acetyl side chain was noted with implications for drug toxicity.

Rapid and Scalable Halosulfonylation of Strain-Release Reagents.

Sulfonylated aromatics are commonplace motifs in drugs and agrochemicals. However, methods for the direct synthesis of sulfonylated non-classical arene bioisosteres, which could improve the physicochemical properties of drug and agrochemical candidates, are limited. Here we report a solution to this challenge: a one-pot halosulfonylation of [1.1.1]propellane, [3.1.1]propellane and bicyclo[1.1.0]butanes that proceeds under practical, scalable and mild conditions. The sulfonyl halides used in this chemistry feature aryl, heteroaryl and alkyl substituents, and are conveniently generated in situ from readily available sulfinate salts and halogen atom sources. This methodology enables the synthesis of an array of pharmaceutically and agrochemically relevant halogen/sulfonyl-substituted bioisosteres and cyclobutanes, on up to multidecagram scale.

De-Novo Depression, Prophylactic Antidepressant, and Survival in Patients With Head and Neck Cancer.

OBJECTIVE: To study the association between the development of moderate or greater depression during curative-intent therapy and overall survival (OS) in patients with stages II-IV head and neck cancer (HNC). METHODS: In this secondary analysis of a randomized double-blind placebo-controlled trial, of 148 eligible participants diagnosed with stages II-IV HNC but without baseline depression, 125 were evaluable and were randomly allocated to prophylactic escitalopram oxalate (n = 60) or placebo (n = 65). Participants were followed for development of moderate or greater depression, using Quick Inventory of Depressive Symptomatology-Self Report (QIDS-SR, range 0-27, score ≥11 indicated moderate or greater depression), and were stratified by demographics; cancer site and stage; and primary treatment modality (surgery with or without radiotherapy vs. radiotherapy with or without chemotherapy). Single variable and multivariable Cox proportional-hazard models were used to evaluate differences in OS. RESULTS: Clinically significant depression developed in 22 of 125 patients (17.6%) during HNC treatment. The mean follow-up was 5.0 years (SD 2.4). OS was similar for patient groups, when stratified by development of moderate or greater depression (HR 0.54 [CI, 0.21-1.43]) or use of prophylactic antidepressant (HR 0.64 [CI, 0.34-1.21]). CONCLUSION: There was no significant association between OS and development of moderate or greater depression in patients being treated for stages II-IV HNC, or between OS and use of prophylactic antidepressant escitalopram. Prophylactic antidepressant may be considered in patients with HNC for prevention of clinically significant depression and may offer improved quality of life outcomes. LEVEL OF EVIDENCE: 2 Laryngoscope, 133:856-862, 2023.

RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation.

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

Synthesis of meta-substituted arene bioisosteres from [3.1.1]propellane.

Small-ring cage hydrocarbons are popular bioisosteres (molecular replacements) for commonly found para-substituted benzene rings in drug design1. The utility of these cage structures derives from their superior pharmacokinetic properties compared with their parent aromatics, including improved solubility and reduced susceptibility to metabolism2,3. A prime example is the bicyclo[1.1.1]pentane motif, which is mainly synthesized by ring-opening of the interbridgehead bond of the strained hydrocarbon [1.1.1]propellane with radicals or anions4. By contrast, scaffolds mimicking meta-substituted arenes are lacking because of the challenge of synthesizing saturated isosteres that accurately reproduce substituent vectors5. Here we show that bicyclo[3.1.1]heptanes (BCHeps), which are hydrocarbons for which the bridgehead substituents map precisely onto the geometry of meta-substituted benzenes, can be conveniently accessed from [3.1.1]propellane. We found that [3.1.1]propellane can be synthesized on a multigram scale, and readily undergoes a range of radical-based transformations to generate medicinally relevant carbon- and heteroatom-substituted BCHeps, including pharmaceutical analogues. Comparison of the absorption, distribution, metabolism and excretion (ADME) properties of these analogues reveals enhanced metabolic stability relative to their parent arene-containing drugs, validating the potential of this meta-arene analogue as an sp3-rich motif in drug design. Collectively, our results show that BCHeps can be prepared on useful scales using a variety of methods, offering a new surrogate for meta-substituted benzene rings for implementation in drug discovery programmes.

Identification of the nucleotide-free state as a therapeutic vulnerability for inhibition of selected oncogenic RAS mutants.

RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

Potent Anti-Inflammatory, Arylpyrazole-Based Glucocorticoid Receptor Agonists That Do Not Impair Insulin Secretion.

Glucocorticoids (GCs) are widely used in medicine for their role in the treatment of autoimmune-mediated conditions, certain cancers, and organ transplantation. The transcriptional activities GCs elicit include transrepression, postulated to be responsible for the anti-inflammatory activity, and transactivation, proposed to underlie the undesirable side effects associated with long-term use. A GC analogue that could elicit only transrepression and beneficial transactivation properties would be of great medicinal value and is highly sought after. In this study, a series of 1-(4-substituted phenyl)pyrazole-based GC analogues were synthesized, biologically screened, and evaluated for SARs leading to the desired activity. Activity observed in compounds bearing an electron deficient arylpyrazole moiety showed promise toward a dissociated steroid, displaying transrepression while having limited transactivation activity. In addition, compounds 11aa and 11ab were found to have anti-inflammatory efficacy comparable to that of dexamethasone at 10 nM, with minimal transactivation activity and no reduction of insulin secretion in cultured rat 832/13 beta cells.

Predictors of success following extracorporeal shock-wave lithotripsy in a contemporary cohort.

OBJECTIVES: The objectives of this study are to determine the predictors of success following extracorporeal shock-wave lithotripsy (ESWL) in a contemporary cohort at a high-volume stone center. METHODS: We conducted a retrospective review all patients who underwent an elective ESWL within our institution over a 24-month period (January 2014 to December 2015). Data on patient demographics, stone variables, and inpatient treatment outcomes were evaluated.The presence of residual stone fragments larger than 4 mm on follow-up imaging was considered to be treatment failure. Using this threshold, clinically relevant variables between the treatment success and failure groups were identified. Multivariable logistic regression analyses (MVA) of clinically relevant variables were used to determine the independent factors predicting ESWL success. RESULTS: Of 446 study eligible patients, 421 patients had complete follow-up data and were included in the analysis. Treatment was successful in 72.2% of patients in this study. Stone size, number of shocks delivered, and maximum treatment intensity were statistically different in the two groups. In a MVA where stone size, location, density, presence of ureteric stent, skin-stone distance (SSD), number of shocks, and maximum shock intensity were included, only stone size of <10 mm (odds ratio [OR] 3.4 [95% confidence interval [CI]: 1.98-5.84]) and SSD <15 cm (OR: 0.133, [95% CI: 0.027-0.65]) were the independent predictor of ESWL success. CONCLUSION: We have demonstrated "real world" outcomes with high-volume use of ESWL. In our experience that with diligent patient selection, ESWL remains an effective tool for the management of upper tract calculi.

Twofold Radical-Based Synthesis of N,C-Difunctionalized Bicyclo[1.1.1]pentanes.

Bicyclo[1.1.1]pentylamines (BCPAs) are of growing importance to the pharmaceutical industry as sp3-rich bioisosteres of anilines and N-tert-butyl groups. Here we report a facile synthesis of 1,3-disubstituted BCPAs using a twofold radical functionalization strategy. Sulfonamidyl radicals, generated through fragmentation of α-iodoaziridines, undergo initial addition to [1.1.1]propellane to afford iodo-BCPAs; the newly formed C-I bond in these products is then functionalized via a silyl-mediated Giese reaction. This chemistry also translates smoothly to 1,3-disubstituted iodo-BCPs. A wide variety of radical acceptors and iodo-BCPAs are accommodated, providing straightforward access to an array of valuable aniline-like isosteres.