At the Speed of Light: The Systematic Implementation of Photoredox Cross-Coupling Reactions for Medicinal Chemistry Research.

The adoption of new and emerging techniques in organic synthesis is essential to promote innovation in drug discovery. In this Perspective, we detail the strategy we used for the systematic deployment of photoredox-mediated, metal-catalyzed cross-coupling reactions in AbbVie's medicinal chemistry organization, focusing on topics such as assessment, evaluation, implementation, and accessibility. The comprehensive evaluation of photoredox reaction setups and published methods will be discussed, along with internal efforts to build expertise and photoredox high-throughput experimentation capabilities. We also highlight AbbVie's academic-industry collaborations in this field that have been leveraged to develop new synthetic strategies, along with discussing the internal adoption of photoredox cross-coupling reactions. The work described herein has culminated in robust photocatalysis and cross-coupling capabilities which are viewed as key platforms for medicinal chemistry research at AbbVie.

Ocular proteomic and transcriptomic changes with aging in a rabbit model of lensectomy with intraocular lens insertion.

Children that undergo intraocular surgery have an exaggerated postoperative response compared to adults that can result in significant postoperative challenges and reduced post-operative visual acuity. Rabbits were used as an animal model for investigating aging differences, treatment options, and surgical techniques for anterior chamber surgical interventions due to similarities in anterior chamber size and decreasing postoperative response with age. In our study, juvenile and adult rabbits underwent lensectomy with intraocular lens (IOL) insertion to determine how ocular RNA transcripts and proteins change with age. Rabbits underwent lensectomy with IOL insertion, and aqueous humor (AH) was collected immediately prior to surgery and at the peak of the postoperative response on post-operative day 3. Proteins related to coagulation and inflammation were assessed using targeted mass spectrometry. In addition, the cornea and iris/ciliary body tissues were dissected, and transcripts analyzed using RNA sequencing. While clinically, juvenile rabbits have greater fibrin formation following intraocular surgery compared to older rabbits, this change does not appear to be related to relative abundance levels of coagulation and inflammatory proteins in the AH. Gene transcript levels from a variety of immune response and inflammatory pathways reflected significant increases when comparing operated to unoperated ocular tissues, indicating the significant impact that surgery has on each ocular structure. This work further advances our understanding of how the rabbit eye proteomic and transcriptomic changes in response to surgery with aging, as we seek to ultimately identify the mechanisms for the exaggerated postoperative responses after pediatric intraocular surgery.

Tissue Plasminogen Activator Effects on Fibrin Volume and the Ocular Proteome in a Juvenile Rabbit Model of Lensectomy.

Purpose: To investigate the use of tissue plasminogen activator (tPA) and its effects on the ocular proteome as a therapeutic intervention for postoperative inflammation and fibrin formation following intraocular lens (IOL) insertion in a juvenile rabbit model. Methods: Twenty-six rabbits, 6 to 7 weeks old, underwent lensectomy with IOL insertion. Following examination on day 3, 100 µL of either 25 µg of recombinant rabbit tPA or balanced salt solution (control) was injected into the anterior chamber. On postoperative day 4, rabbits underwent examination, and eyes were harvested and fixed for 9.4-Tesla magnetic resonance imaging (MRI). Three masked observers quantified fibrin scar volume using Horos Project software. Aqueous humor (AH) was collected immediately prior to surgery and on postoperative days 3 and 4. Proteins related to coagulation and inflammation were assessed in AH samples using targeted mass spectrometry via parallel reaction monitoring. Results: tPA significantly reduced the volume of fibrin 24 hours following administration compared with control eyes (0.560 mm3 vs. 3.29 mm3; P < 0.0001). Despite the reduced fibrin scar, proteins related to the coagulation and complement cascade were not significantly different following tPA injection. Conclusions: tPA may be a safe candidate for reduction of postoperative fibrin scarring after intraocular surgery. MRI can provide a quantitative value for fibrin volume changes. Translational Relevance: tPA is a candidate to treat ocular fibrin scarring. MRI can quantify the efficacy of treatments in future dose-response studies. Targeted mass spectrometry can provide critical data necessary to help decipher the effect on the abundance of targeted proteins following pharmacological intervention.

Expanding the Medicinal Chemist Toolbox: Comparing Seven C(sp2)-C(sp3) Cross-Coupling Methods by Library Synthesis.

Despite recent advances in the field of C(sp2)-C(sp3) cross-couplings and the accompanying increase in publications, it can be hard to determine which method is appropriate for a given reaction when using the highly functionalized intermediates prevalent in medicinal chemistry. Thus a study was done comparing the ability of seven methods to directly install a diverse set of alkyl groups on "drug-like" aryl structures via parallel library synthesis. Each method showed substrates that it excelled at coupling compared with the other methods. When analyzing the reactions run across all of the methods, a reaction success rate of 50% was achieved. Whereas this is promising, there are still gaps in the scope of direct C(sp2)-C(sp3) coupling methods, like tertiary group installation. The results reported herein should be used to inform future syntheses, assess reaction scope, and encourage medicinal chemists to expand their synthetic toolbox.

Quantitative proteomic analysis of aqueous humor after rabbit lensectomy reveals differences in coagulation and immunomodulatory proteins.

Compared to adults, children experience increased postoperative scarring and inflammation following intraocular surgery. While the underlying causes of the exaggerated immune response in children are not understood, proteins play key roles in postoperative scarring and wound healing processes. To identify and quantify proteins associated with the robust postoperative immune response, this study applied quantitative proteomics approaches to a juvenile rabbit model of lensectomy with intraocular lens (IOL) insertion. Twenty-six 6-7 week-old New Zealand white rabbits underwent unilateral portions of lensectomy with IOL insertion including: anterior chamber paracentesis, corneal incision with wound suture, lensectomy only, and lensectomy with IOL insertion. Aqueous humor was collected immediately prior and three days after each procedure. Semi-quantitative protein discovery was achieved by label-free quantitation using data dependent and data independent acquisition modes. Based on the discovery results, targeted quantitation by parallel reaction monitoring of 3 proteins of interest, fibrinogen-beta chain, transforming growth factor beta-2, and retinol binding protein 3, was used to confirm the observed quantitative trends. Total protein concentration levels increased with each progressive surgical step of lensectomy with IOL insertion. Proteins related to the complement and coagulation cascades were found to increase in relative abundance, while proteins related to ocular immunosuppression decreased in abundance following surgery. These data provide insights into the postoperative response by providing the first surgical step-wise views of the AH proteome before and after surgery. Overall, this work provides the foundation for future investigations targeting specific proteins for therapeutic interventions aimed at minimizing postoperative complications after pediatric intraocular surgery.

Assessing Retinal Structure in Patients with Parkinson's Disease.

OBJECTIVE: The retina is an extension of the central nervous system (CNS), and ocular symptoms can precede manifestations of CNS disorders. Given that several neurodegenerative conditions that affect the brain exhibit ocular symptoms, the retina may be an accessible biomarker to monitor disease progression. Dopamine, the key neurotransmitter related to Parkinson's disease (PD), is contained in amacrine and interplexiform cells, which reside in specific retinal layers. Understanding how loss of dopaminergic cells affects retinal anatomy could be relevant for monitoring disease progression. Here, our objective is to evaluate retinal structure (foveal pit morphology and thickness) in patients with PD. METHODS: Thirty-three Caucasian subjects diagnosed with PD and 40 age-matched Caucasian control subjects underwent retinal imaging with spectral-domain optical coherence tomography (SD-OCT). Axial length measurements were used to correct the lateral scale of each macular volume scan. From these corrected volumes, foveal morphology was quantified with previously described algorithms, and Early Treatment Diabetic Retinopathy Study (ETDRS) grids of retinal thickness were generated and incorporated into a logistic regression model to predict PD. RESULTS: Interocular foveal morphology measurements were highly symmetrical in PD patients and control subjects. There were no significant differences in foveal pit morphology between PD patients and control subjects. Using a model incorporating sex and axial length corrected ETDRS regions, we generated a receiver operating characteristic curve with a C-statistic of 0.80. CONCLUSION: Our study, which to our knowledge is the first to properly scale OCT measurements when quantifying retinal thickness, demonstrates that PD patients retain foveal symmetry between eyes. When constructing a model to predict PD, sex, along with the center 1 mm and temporal outer ETDRS regions, were significant predictors of PD. In addition to proper scaling of OCT measures, gender and racial differences in retinal anatomy should be considered in building future predictive PD models when using OCT.

Synthesis of anti-1,3 Amino Alcohol Motifs via Pd(II)/SOX Catalysis with the Capacity for Stereodivergence.

We report the development of a Pd(II)/(±)-MeO-SOX/2,5-dimethylbenzoquinone system that enables unprecedented access to anti-1,3 amino alcohol motifs in good yields (33 substrates, avg. 66% isolated yield, >20:1 dr) and high selectivities (avg. 10:1 dr). Switching ligands to (±)-CF3-SOX with the use of a less bulky quinone oxidant, the kinetic syn-1,3 amino alcohol motif can be accessed in comparable yields and selectivities. Advantages of the stereodivergent nature of this reaction are seen in the synthesis of anti- and syn-1,3 amino alcohol vitamin D3 analogue intermediates in half the steps and higher overall yield relative to previous routes. Additionally, all eight possible stereoisomers of a chiral diamino alcohol core are generated from two amino acids. Mechanistic studies reveal that the anti-isomer is furnished through concurrent Pd(II)(SOX) catalyzed C-H amination and Pd(0)(SOX) catalyzed isomerization cycles.

Use of a Juvenile Rabbit Animal Model to Evaluate Therapeutic Interventions for Postoperative Inflammation and Fibrin Formation After Lensectomy.

PURPOSE: We used the juvenile rabbit as a model for investigating therapeutic interventions for postoperative inflammation and fibrin formation following intraocular lens (IOL) insertion for management of pediatric cataracts. METHODS: Twelve 6- to 7-week-old, 600 to 900 g rabbits underwent bilateral clear-cornea lensectomy via irrigation and aspiration with IOL insertion. Following wound closure, enoxaparin 8 mg (n = 6 eyes), preservative-free triamcinolone 0.5 mg (n = 6), 8 mg enoxaparin plus 0.5 mg triamcinolone (n = 6), or balanced salt solution (n = 6) was injected into the anterior chamber. Slit-lamp examinations and optical coherence tomography (OCT) scans were performed postoperatively on days 3 through 7, and 14 to characterize levels of inflammation and fibrin. Using 17 additional rabbits, enzyme-linked immunosorbent assays (ELISAs) with 100 µL of aqueous humor were performed to quantify the amount of fibrinogen and fibrin preoperatively and on postoperative day 3. Immunohistochemistry was performed to confirm the presence of fibrin. RESULTS: Enoxaparin alone and combined with triamcinolone reduced the amount of fibrin present in the anterior chamber compared to untreated eyes, which corresponded to an increase in OCT signal strength. Despite the clear visual axis shown in clinical images, the combination treatment group had the highest levels of soluble fibrin when assessed by ELISA. Immunohistochemistry confirmed the presence of insoluble fibrin seen clinically. CONCLUSIONS: A combination of enoxaparin and triamcinolone appears to provide the most therapeutic benefit by reducing fibrin formation and postoperative inflammation. TRANSLATIONAL RELEVANCE: The juvenile rabbit is an excellent model to investigate inflammation and fibrin formation following lensectomy with IOL insertion and possibly any intraocular surgery in children.

Assessing photoreceptor structure in patients with traumatic head injury.

OBJECTIVE: Previous work using adaptive optics scanning light ophthalmoscopy (AOSLO) imaging has shown photoreceptor disruption to be a common finding in head and ocular trauma patients. Here an expanded trauma population was examined using a novel imaging technique, split-detector AOSLO, to assess remnant cone structure in areas with significant disruption on confocal AOSLO imaging and to follow photoreceptor changes longitudinally. METHODS AND ANALYSIS: Eight eyes from seven subjects with head and/or ocular trauma underwent imaging with spectral domain optical coherence tomography, confocal AOSLO and split-detector AOSLO to assess foveal and parafoveal photoreceptor structure. RESULTS: Confocal AOSLO imaging revealed hyporeflective foveal regions in two of eight eyes. Split-detector imaging within the hyporeflective confocal areas showed both remnant and absent inner-segment structure. Both of these eyes were imaged longitudinally and showed variation of the photoreceptor mosaic over time. Four other eyes demonstrated subclinical regions of abnormal waveguiding photoreceptors on multimodal AOSLO imagery but were otherwise normal. Two eyes demonstrated normal foveal cone packing without disruption. CONCLUSION: Multimodal imaging can detect subtle photoreceptor abnormalities not necessarily detected by conventional clinical imaging. The addition of split-detector AOSLO revealed the variable condition of inner segments within confocal photoreceptor disruption, confirming the usefulness of dual-modality AOSLO imaging in assessing photoreceptor structure and integrity. Longitudinal imaging demonstrated the dynamic nature of the photoreceptor mosaic after trauma. Multimodal imaging with dual-modality AOSLO improves understanding of visual symptoms and photoreceptor structure changes in patients with head and ocular trauma.

Findings in Persistent Retinopathy of Prematurity.

BACKGROUND AND OBJECTIVE: To determine whether retinopathy of prematurity (ROP) that persists beyond a postmenstrual age (PMA) of 45 weeks has abnormalities that can be documented by fundus photography or fluorescein angiography (FA). PATIENTS AND METHODS: Fundus photographs and FAs were reviewed for all premature infants who underwent FA for persistent ROP after 45 weeks PMA. RESULTS: Of the 487 infants who were screened for ROP, 16 (3.3%) demonstrated ROP beyond 45 weeks. Seven (43.8%) infants received prior treatment with intravitreal bevacizumab (IVB) for Type 1 ROP. FAs were obtained in eight cases; four subjects were previously treated with IVB. Leakage at the vascular-avascular border was demonstrated in seven subjects (87.5%). Shunt vessels, posterior retinal nonperfusion, and absence of the foveal avascular zone was limited to the IVB group. CONCLUSIONS: There are persistent vascular abnormalities among infants with ROP beyond 45 weeks. Findings that may be missed by RetCam fundus photographs were highlighted with FA. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:497-503.].

Cone structure in subjects with known genetic relative risk for AMD.

PURPOSE: Utilize high-resolution imaging to examine retinal anatomy in patients with known genetic relative risk (RR) for developing age-related macular degeneration (AMD). METHODS: Forty asymptomatic subjects were recruited (9 men, 31 women; age range, 51 to 69 years; mean age, 61.4 years). Comprehensive eye examination, fundus photography, and high-resolution retinal imaging using spectral domain optical coherence tomography and adaptive optics were performed on each patient. Genetic RR scores were developed using an age-independent algorithm. Adaptive optics scanning light ophthalmoscope images were acquired in the macula extending to 10 degrees temporal and superior from fixation and were used to calculate cone density in up to 35 locations for each subject. RESULTS: Relative risk was not significantly predictive of fundus grade (p = 0.98). Only patients with a high RR displayed drusen on Cirrus or Bioptigen OCT. Compared to an eye with a grade of 0, an eye with a fundus grade equal to or greater than 1 had a 12% decrease in density (p < 0.0001) and a 5% increase in spacing (p = 0.0014). No association between genetic RR and either cone density (p = 0.435) or spacing (p = 0.538) was found. Three distinct adaptive optics scanning light ophthalmoscope phenotypical variations of photoreceptor appearance were noted in patients with grade 1 to 3 fundi. These included variable reflectivity of photoreceptors, decreased waveguiding, and altered photoreceptor mosaic overlying drusen. CONCLUSIONS: Our data demonstrate the potential of multimodal assessment in the understanding of early anatomical changes associated with AMD. Adaptive optics scanning light ophthalmoscope imaging reveals a decrease in photoreceptor density and increased spacing in patients with grade 1 to 3 fundi, as well as a spectrum of photoreceptor changes, ranging from variability in reflectivity to decreased density. Future longitudinal studies are needed in genetically characterized subjects to assess the significance of these findings with respect to the development and progression of AMD.

Structural basis for selective small molecule kinase inhibition of activated c-Met.

The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix αC and the G loop to generate a viable active site. Helix αC adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.