Proteolysis-targeting chimeras with reduced off-targets.

Proteolysis-targeting chimeras (PROTACs) are molecules that induce proximity between target proteins and E3 ligases triggering target protein degradation. Pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other proteins, including zinc-finger (ZF) proteins, with vital roles in health and disease. This off-target degradation hampers the therapeutic applicability of pomalidomide-based PROTACs, requiring development of PROTAC design rules that minimize off-target degradation. Here we developed a high-throughput platform that interrogates off-target degradation and found that reported pomalidomide-based PROTACs induce degradation of several ZF proteins. We generated a library of pomalidomide analogues to understand how functionalizing different positions of the phthalimide ring, hydrogen bonding, and steric and hydrophobic effects impact ZF protein degradation. Modifications of appropriate size on the C5 position reduced off-target ZF degradation, which we validated through target engagement and proteomics studies. By applying these design principles, we developed anaplastic lymphoma kinase oncoprotein-targeting PROTACs with enhanced potency and minimal off-target degradation.

Development and Applications of Chimera Platforms for Tyrosine Phosphorylation.

Chimeric small molecules that induce post-translational modification (PTM) on a target protein by bringing it into proximity to a PTM-inducing enzyme are furnishing novel modalities to perturb protein function. Despite recent advances, such molecules are unavailable for a critical PTM, tyrosine phosphorylation. Furthermore, the contemporary design paradigm of chimeric molecules, formed by joining a noninhibitory binder of the PTM-inducing enzyme with the binder of the target protein, prohibits the recruitment of most PTM-inducing enzymes as their noninhibitory binders are unavailable. Here, we report two platforms to generate phosphorylation-inducing chimeric small molecules (PHICS) for tyrosine phosphorylation. We generate PHICS from both noninhibitory binders (scantily available, platform 1) and kinase inhibitors (abundantly available, platform 2) using cysteine-based group transfer chemistry. PHICS triggered phosphorylation on tyrosine residues in diverse sequence contexts and target proteins (e.g., membrane-associated, cytosolic) and displayed multiple bioactivities, including the initiation of a growth receptor signaling cascade and the death of drug-resistant cancer cells. These studies provide an approach to induce biologically relevant PTM and lay the foundation for pharmacologic PTM editing (i.e., induction or removal) of target proteins using abundantly available inhibitors of PTM-inducing or -erasing enzymes.

Endogenous endophthalmitis in post-COVID-19 patients: a case report.

Ocular involvement in coronavirus disease 2019 (COVID-19) can be due to direct viral invasion or indirectly due to an immunosuppressed state. Prolonged hospitalization also makes them susceptible to various secondary infections. The purpose of this case report is to report two rare cases of endogenous endophthalmitis (EE) in COVID-19 recovered patients. Case presentation: Two patients who were hospitalized and received treatment for COVID-19 pneumonia with remdesivir and systemic steroids presented with decreased vision. The first case had a severe anterior chamber reaction with a hypopyon and dense exudates in the vitreous. The second case had cells and flare in the anterior chamber and exudates in the vitreous. They were diagnosed with EE and underwent a diagnostic vitreous tap followed by pars plana vitrectomy and intravitreal antibiotic and steroid. The culture of vitreous fluid was negative for any bacteria and fungus in both cases. However, the first case demonstrated Escherichia coli in urine culture. The follow-up visual acuity was no perception of light and only perception of light in the first and second case, respectively. Clinical discussion: Severe COVID-19 patients who are hospitalized, receive systemic steroid and have associated comorbidities like diabetes mellitus are at high risk of EE. Conclusion: Delay in diagnosis and appropriate treatment in these patients leads to poor visual outcome.

Proximity-inducing modalities: the past, present, and future.

Living systems use proximity to regulate biochemical processes. Inspired by this phenomenon, bifunctional modalities that induce proximity have been developed to redirect cellular processes. An emerging example of this class is molecules that induce ubiquitin-dependent proteasomal degradation of a protein of interest, and their initial development sparked a flurry of discovery for other bifunctional modalities. Recent advances in this area include modalities that can change protein phosphorylation, glycosylation, and acetylation states, modulate gene expression, and recruit components of the immune system. In this review, we highlight bifunctional modalities that perform functions other than degradation and have great potential to revolutionize disease treatment, while also serving as important tools in basic research to explore new aspects of biology.

Massively parallel base editing to map variant effects in human hematopoiesis.

Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.

Functional characterization of a hypothetical protein (TTHA1873) from Thermus thermophilus.

Thermus thermophilus is an extremely thermophilic organism that thrives at a temperature of 65°C. T. thermophilus genome has ~2218 genes, out of which 66% (1482 genes) have been annotated, and the remaining 34% (736 genes) are assigned as hypothetical proteins. In this work, biochemical and biophysical experiments were performed to characterize the hypothetical protein TTHA1873 from T. thermophilus. The hypothetical protein TTHA1873 acts as a nuclease, which indiscreetly cuts methylated and non-methylated DNA in divalent metal ions and relaxes the plasmid DNA in the presence of ATP. The chelation of metal ions with EDTA inhibits its activity. These results suggest that the hypothetical protein TTHA1873 would be a CRISPR-associated protein with non-specific DNase activity and ATP-dependent DNA-relaxing activity.

The Retinal Nerve Fiber Layer and Macular Thickness in Patients with Type 2 Diabetes Mellitus without Retinopathy Using Optical Coherence Tomography: A Comparative Study.

INTRODUCTION: Diabetes leads to an alteration in retinal nerve fiber layer (RNFL) thickness and macular thickness which can easily be detected with optical coherence tomography (OCT). OBJECTIVES: This study was done to compare the RNFL and macular thickness between diabetic patients without retinopathy and non-diabetic patients so that it would be useful in the early detection of retinal changes if present. The correlation between the RNFL and macular thickness with metabolic blood parameters of diabetic subjects was also studied. MATERIALS AND METHODS: This is an observational, cross-sectional, hospital-based study including 120 subjects who were further divided into two groups. Group A consisted of 60 diabetic patients without retinopathy and group B consisted of 60 non-diabetic patients. The blood parameters were recorded and the RNFL thickness and macular thickness were compared between the two groups after evaluation by OCT. RESULTS: The average central macular thickness was found to be more in group A but was statistically insignificant (p=0.29). Macular thickness in the superior quadrant was significantly higher among group A when compared with group B (p=0.01). Whereas RNFL thickness difference between the two groups was statistically insignificant (p=0.53). Blood urea showed significant positive correlation (r=0.269) with central macular thickness (p=0.03). CONCLUSION: Our study showed that diabetic patients without retinopathy could have increased macular thickness in the superior quadrant when compared with normal people whereas RNFL thickness may not alter. The blood urea levels of the diabetic patients can provide us clues regarding possible retinal changes.

Scleritis as the harbinger of Granulomatosis with polyangiitis.

Introduction: Ocular and orbital involvement in Granulomatosis with polyangiitis (GPA) is common. GPA can lead to life and sight threatening complications due to necrosis and tissue melting. Cases: We report four cases presenting with ocular pain and redness for varied durations. One had diminution of vision. All of them had deep sectoral/diffuse congestion with one having scleral thinning. All were diagnosed with anterior necrotizing/non-necrotizing scleritis. One had associated penetrating ulcerative keratitis. Topical steroids and systemic non-steroidal anti-inflammatory drugs were started in all cases and rheumatology consultation was taken. Pertinent investigations were sent, and GPA was diagnosed. Intravenous immunosuppressive regimens and oral steroid were started and significant improvements were seen, preventing untoward complications. Conclusion: Scleritis could be manifesting feature of GPA so cautious history taking and evaluation is important. Management often requires multidisciplinary care and ocular features could be the reference guidelines to adjust dose of systemic medications of GPA.

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules.

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.

Structure of the hypothetical protein TTHA1873 from Thermus thermophilus.

The crystal structure of an uncharacterized hypothetical protein, TTHA1873 from Thermus thermophilus, has been determined by X-ray crystallography to a resolution of 1.78 Šusing the single-wavelength anomalous dispersion method. The protein crystallized as a dimer in two space groups: P43212 and P6122. Structural analysis of the hypothetical protein revealed that the overall fold of TTHA1873 has a ß-sandwich jelly-roll topology with nine ß-strands. TTHA1873 is a dimeric metal-binding protein that binds to two Ca2+ ions per chain, with one on the surface and the other stabilizing the dimeric interface of the two chains. A structural homology search indicates that the protein has moderate structural similarity to one domain of cell-surface proteins or agglutinin receptor proteins. Red blood cells showed visible agglutination at high concentrations of the hypothetical protein.

Managing a Case of a Congenital Cystic Eyeball: Case Report with Review of Literature.

A congenital cystic eyeball is an extremely rare condition, with only 52 cases reported in the literature to date. An orbital cyst replaces the eyeball which occurs due to the complete or partial failure in invagination of the primary optic vesicle during the fourth week of gestation. We discuss a case of a congenital cystic eyeball in a 14-year-old female who presented to us for a cosmetic blemish due to a large swelling in the right eyelid with the absence of a right eyeball since birth. She underwent removal of the cyst followed by an orbital implant and later prosthesis. Diagnosis of the congenital cystic eyeball was made based on the clinical and ultrasound B-scan features, intraoperative findings, and histopathology report. This article adds one more case to the existing literature on the congenital cystic eyeball. Orbital implant with prosthesis after excision of the cyst provided definitive diagnosis and a good cosmetic outcome in our case.

Synthetic Reprogramming of Kinases Expands Cellular Activities of Proteins.

Phosphorylation-inducing chimeric small molecules (PHICS) can enable a kinase to act at a new cellular location or phosphorylate non-native substrates (neo-substrates)/ sites (neo-phosphorylations).[1, 2] We report a modular design and high-yielding synthesis of such PHICS that endowed multiple new activities to protein kinase C (PKC). For example, while PKC is unable to downregulate the activity of a gain-of-function variant (S180A) of Bruton's tyrosine kinase that evokes B cell malignancy phenotype, PHICS enabled PKC to induce inhibitory neo-phosphorylations on this variant. Furthermore, while PKC typically phosphorylates its membrane-associated substrates, PKC with PHICS phosphorylated multiple cytosol-based neo-substrates (e.g., BCR-ABL). Finally, a PHICS for BCR-ABL induced death of chronic myeloid leukemia cell lines. These studies show the power of synthetic chemistry to expand the chemical and functional diversity of proteins in cells using bifunctional molecules.

Profile of Destructive Ocular Surgery and its Indications: A Twelve-Year Review from Eastern Nepal.

BACKGROUND: Destructive ocular surgeries are performed for many conditions ranging from trauma to tumours, where the eyes cannot be salvaged. The objective of our research was to study the profile of destructive ocular surgery and their indications. METHODS: This retrospective study reviewed all patients who underwent evisceration, enucleation, and exenteration at B.P. Koirala Institute of Health Sciences, a tertiary eye hospital in Eastern Nepal, between January 2008 and December 2019. Medical records on patient demographics, type of surgery performed, and an indication of surgery during the study period were reviewed. RESULTS: One hundred thirty-four patients underwent destructive ocular surgeries. The median age of patients undergoing surgery was 14.5 (3-50) years. Children aged ten years or less accounted for 46.3% of the total patients. Fifty-two percent were male. The left eye was affected in more than half of the cases (56.7%). Enucleation was the most performed destructive ocular surgery (76 cases, 56.7%). Intraocular and ocular adnexal malignancy was the most common overall indication (62 cases, 46.3%). Ocular infection (19 cases, 41.3%) and trauma (15 cases, 32.6%) were the most common indication of evisceration. Retinoblastoma accounted for most cases of enucleation (43 cases, 56.6%). Malignancy was the only indication of exenteration (12 cases, 100%). CONCLUSIONS: Enucleation was the most common destructive ocular surgery. Malignancy accounted for most of the cases of destructive eye surgery, followed by ocular infection. Ocular infection and trauma were the most common indication of evisceration, whereas retinoblastoma and eyelid malignancy were responsible for most of the cases of enucleation and exenteration, respectively.

Orbital Lymphoma Masquerading as Orbital Cellulitis.

BACKGROUND: Orbital lymphomas are primarily non-Hodgkin type and can originate from the eyelids, extraocular muscles, soft tissue orbital adnexa, conjunctiva, or lacrimal glands. Orbital malignancies often represent a diagnostic dilemma for clinicians given their varying and atypical presentations. OBJECTIVE: To report a case of orbital lymphoma mimicking orbital cellulitis. CASE: A 66-year-old male patient presented with sudden onset of painful proptosis with visual impairment in the left eye for 15 days. On ocular examination, best-corrected visual acuity was 6/12 in the right eye and 2/60 in the left eye, abaxial proptosis with hypertropia, swollen and erythematous eyelids, restricted extraocular movement in all cardinal position of gaze, conjunctival congestion with chemosis and tortuous vessels, sluggish pupillary reaction, and chorioretinal folds in the inferior quadrants. The case was diagnosed as left eye orbital cellulitis, and the patient was treated with broad-spectrum intravenous antibiotics and oral steroids. No clinically discernible response was noted despite 7 days of antibiotics and steroids. Contrast-enhanced computed tomography (CECT) orbit showed features suggestive of orbital lymphoma involving the ipsilateral maxillary and ethmoid sinuses. ENT consultation with diagnostic nasal endoscopy and biopsy was done. Histopathological reports showed features of non-Hodgkin lymphoma. CONCLUSION: Orbital malignancies masquerading as orbital cellulitis can pose a diagnostic dilemma. A multidisciplinary approach involving ENT consultation, radiological investigation, and pathological sampling can help achieve a timely diagnosis and appropriate management.

Subconjunctival Mass as Rare Presentation of Even Rarer Intraocular Medulloepithelioma: A Case Report.

Medulloepithelioma is a rare childhood embryonal tumor arising from the non-pigmented ciliary epithelium of the pars plicata. We report a case of an 11-year-old male who presented with painless loss of vision of the right eye for the last three years and progressively increasing mass on the superior aspect of the globe for the last three months. On ocular examination, a firm, non-tender mass of 4cm x 3cm was noted in the superior aspect of the globe. CT-Scan of the orbit was suggestive of a foreign body with a haemorrhage or infection. The patient underwent enucleation with minimal manipulation. Histopathological examination of the enucleated globe revealed medulloepithelioma. The intraocular medulloepithelioma presentation is often late and masquerading, which may lead to extraocular extension and metastasis and ultimately unfavorable prognosis.

Managing Recurrent Orbital Lymphangioma in a Pubertal Female with Negative Pressure Aspiration and Intralesional Bleomycin Injection: A Case Report.

INTRODUCTION: Management of orbital lymphangioma is challenging. Complete surgical excision is often impossible due to its infiltrative nature. Sclerosing agents have been used in its management with variable outcomes. We report a case of recurrent orbital lymphangioma managed with intralesional bleomycin. CASE: A 14-year-old female presented with proptosis of the right eye for two weeks. She had a similar history at five years of age for which she underwent surgical excision. We performed negative pressure aspiration using a 20-gauge angiocatheter, injected bleomycin, and left the cannula in situ for repeat aspiration to maintain cyst collapse. OBSERVATION: The lymphangioma regressed, and there was no recurrence at six months of follow-up. CONCLUSION: This report highlights the use of negative pressure aspiration and intralesional bleomycin injection by minimal intervention using angiocatheter in the successful management of orbital lymphangioma.

Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes.

Prolonged Cas9 activity can hinder genome engineering as it causes off-target effects, genotoxicity, heterogeneous genome-editing outcomes, immunogenicity, and mosaicism in embryonic editing-issues which could be addressed by controlling the longevity of Cas9. Though some temporal controls of Cas9 activity have been developed, only cumbersome systems exist for modifying the lifetime. Here, we have developed a chemogenetic system that brings Cas9 in proximity to a ubiquitin ligase, enabling rapid ubiquitination and degradation of Cas9 by the proteasome. Despite the large size of Cas9, we were able to demonstrate efficient degradation in cells from multiple species. Furthermore, by controlling the Cas9 lifetime, we were able to bias the DNA repair pathways and the genotypic outcome for both templated and nontemplated genome editing. Finally, we were able to dosably control the Cas9 activity and specificity to ameliorate the off-target effects. The ability of this system to change the Cas9 lifetime and, therefore, bias repair pathways and specificity in the desired direction allows precision control of the genome editing outcome.

Phosphorylation-Inducing Chimeric Small Molecules.

Small molecules have been classically developed to inhibit enzyme activity; however, new classes of small molecules that endow new functions to enzymes via proximity-mediated effect are emerging. Phosphorylation (native or neo) of any given protein-of-interest can alter its structure and function, and we hypothesized that such modifications can be accomplished by small molecules that bring a kinase in proximity to the protein-of-interest. Herein, we describe phosphorylation-inducing chimeric small molecules (PHICS), which enable two example kinases-AMPK and PKC-to phosphorylate target proteins that are not otherwise substrates for these kinases. PHICS are formed by linking small-molecule binders of the kinase and the target protein, and exhibit several features of a bifunctional molecule, including the hook-effect, turnover, isoform specificity, dose and temporal control of phosphorylation, and activity dependent on proximity (i.e., linker length). Using PHICS, we were able to induce native and neo-phosphorylations of BRD4 by AMPK or PKC. Furthermore, PHICS induced a signaling-relevant phosphorylation of the target protein Bruton's tyrosine kinase in cells. We envision that PHICS-mediated native or neo-phosphorylations will find utility in basic research and medicine.

Structural and functional characterization of oligomeric states of proteins in RecFOR pathway.

RecFOR pathway is the principal repair pathway for double strand break and single strand gap repair in Thermus thermophilus. RecF and RecR exist as monomer and dimer in solution, interestingly; they undergo condition-dependent dimerization and tetramerization, respectively during the DNA break repair. However, their importance in protein-protein and protein-DNA interactions remains elusive. In this study, the three-dimensional crystal structures of the wild type RecF and RecR proteins are determined. Thereafter, the structural information is used to mutate the interface residues to cysteine to stabilize the dimeric and tetrameric states of the RecF and RecR proteins, respectively. A comparative study for their interactions with other cognate proteins and ssDNA in native and SSB (single strand binding protein) bound states was performed. RecF or RecFR complex displays a negligible affinity towards ssDNA. Conversely, the RecF mutants and its complexes with wild type RecR showed affinity towards ssDNA, suggesting, distinct modes of interaction of RecF and RecFR complex for ssDNA binding. In the presence of RecO, the stabilized tetrameric RecR showed a lower binding affinity for ssDNA as compared to the SSB bound ssDNA, indicating the importance of tetrameric RecR in stabilizing the RecOR complex on the SSB coated ssDNA. This provides an insight into the reduction of the binding affinity of SSB proteins with the ssDNA, which in turn enhances the recruitment of RecA for strand exchange.

Insights into product release dynamics through structural analyses of thymidylate kinase.

Several studies on enzyme catalysis have pointed out that the product release event could be a rate limiting step. In this study, we have compared the release event of two products, Adenosine di-phosphate (ADP) and Thymidine di-phosphate (TDP) from the active-site of human and Thermus thermophilus thymidine mono-phosphate kinase (TMPK), referred to as hTMPK and ttTMPK, respectively. TMPK catalyses the conversion of Thymidine mono-phosphate (TMP) to TDP using ATP as phosphoryl donor in the presence of Mg2+ ion. Most of the earlier studies on this enzyme have focused on understanding substrate binding and catalysis, but the critical product release event remains elusive. Competitive binding experiments of the substrates and the products using ttTMPK apo crystals have indicated that the substrate (TMP) can replace the bound product (TDP), even in the presence of an ADP molecule. Further, the existing random accelerated molecular dynamics (RAMD) simulation program was modified to study the release of both the products simultaneously from the active site. The RAMD simulations on product-bound structures of both ttTMPK and hTMPK, revealed that while several exit patterns of the products are permissible, the sequential exit mode is the most preferred pattern for both ttTMPK and hTMPK enzymes. Additionally, the product release from the hTMPK was found to be faster and more directional as compared to ttTMPK. Structural investigation revealed that the critical changes in the residue composition in the LID-region of ttTMPK and hTMPK have an effect on the product release and can be attributed to the observed differences during product release event. Understanding of these dissimilarities is of considerable utility in designing potent inhibitors or prodrugs that can distinguish between eukaryotic and prokaryotic homologues of thymidylate kinase.