Extensive Metallosis in a Primary Knee Arthroplasty as a Result of Polyethylene Wear: Is It Avoidable?

Metallosis is known to occur in metal-on-metal arthroplasty and has been of concern to orthopaedic surgeons worldwide. It is a rare, late complication of total knee arthroplasty (TKA), in which metal-on-metal contact leads to metal debris deposition in the surrounding tissue. Reasons for metal-on-metal contact could range from wear of the polyethylene insert to abnormal joint biomechanics. Many components can affect the development of metallosis, with polyethylene wear being the most common cause of metallosis. This paper discusses the case of an 85-year-old man who developed metallosis, attributed to polyethylene wear, 24 years after undergoing TKA. It also highlights the different components of knee prostheses, evaluates the efficacy of different types of polyethylene, and explores whether ceramic coating can improve TKA outcomes and reduce complications such as metallosis.

Impact of surgical timing and type of operative procedure on outcomes in periprosthetic hip fractures: an observational study at an NHS trust centre in the UK.

PURPOSE: There is a global trend of increased periprosthetic fractures due to the growing number of arthroplasty procedures. The present study assessed the impact of factors such as time to surgery and type of surgery on the outcomes, which have been seldom evaluated for periprosthetic fractures. METHODS: An observational study was conducted on consecutive 87 patients within an NHS district hospital trust in the UK. Patients who underwent a complete hip replacement prior to the fracture, received fixation therapy, or underwent revision surgery within the specified time were screened. Patients were grouped in two ways: based on time to surgery and based on surgery type. Logistic regression models were performed to assess for statistically significant differences in post-operative complication, 30-day, and 1-year mortality rates between groups, whilst adjusting for age, gender, and ASA grade. RESULTS: Forty-one patients underwent open reduction and internal fixation (ORIF), 29 patients underwent revision arthroplasty, and 17 patients were subjected to both, ORIF and revision arthroplasty. Sixty of the 87 patients were operated on > 48 h of injury. The median hospital stay was significantly lower in the ORIF plus revision arthroplasty group, versus other surgical groups (p < 0.05) whilst it was significantly higher in the group of patients who underwent surgery after 48 h of injury (p < 0.05). Numerically higher mortality was noted in the revision arthroplasty group (31.03%, p > 0.05). The group that was operated after 48 h of injury showed greater mortality but was comparable to the other group (25% vs. 14.81%, p > 0.05). For post-operative complications, none of the variables were significantly predictive (p > 0.05). However, for 30-day mortality, ASA grade (p = 0.04) and intra-operative complications (p = 0.0001) were significantly predictive. Additionally, for 1-year mortality, ASA grade (p = 0.004) was noted to be significantly predictive. CONCLUSION: Revision and delayed periprosthetic fracture management (> 48 h after injury) group showed a numerically greater mortality risk; however, this finding was not statistically significant. ASA grading at baseline is predictive of mortality for periprosthetic fractures.

Use of a Trephine to Extract a Fractured Corail Femoral Stem During Revision Total Hip Arthroplasty: Tips From Our Case Report.

Despite the significant advancements in the field of total hip arthroplasty (THA) and prosthesis designs, fracture of the modular femoral stem after THA can occur rarely and needs attention. Orthopaedic surgeons face a daunting task when it comes to the removal of a broken stem. The use of a trephine reamer has been evaluated for extracting the distal femoral stem, and this case report tries to address some key tips for the same. A 67-year-old obese male, without any major medical comorbidities, presented to the Royal Lancaster Infirmary orthopaedic outpatient department with a complaint of acute-on-chronic right anterior thigh pain that worsened over a few weeks. He had a history of bilateral staged uncemented THA done around 12 years ago. The plain radiological images confirmed the presence of a fracture of the Corail femoral stem. A posterior approach was used to dislocate the hip and the distal broken part of the stem was removed using trephines. Reamers were used and care was taken to prevent thermal necrosis by using intermittent saline lavage. After the removal of the fractured femoral stem, a cemented femoral revision THA was performed, which was uneventful. The patient walked without any aid or thigh pain postoperatively during his last follow-up. Using trephines to remove broken femoral stems is an effective and safe method. Intraoperative measures can help in avoiding heat necrosis while using a trephine reamer for extracting the fractured femoral stem. Regular follow-up and counselling are important after THA, to avoid a delayed diagnosis of non-traumatic femoral stem fractures.

Obsessive-Compulsive Disorder (OCD): A Comprehensive Review of Diagnosis, Comorbidities, and Treatment Approaches.

Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder widely recognized for its recurrent obsessions and compulsions, which may cause severe impairment worldwide. This review explores the difficulties in diagnosing OCD, its comorbidities, and its treatment approaches. Psychiatry and neuroscience face noteworthy obstacles in treating OCD, which is frequently misdiagnosed and inadequately addressed. This illness, which causes upsetting symptoms that interfere with day-to-day living, affects not only adults but also children and adolescents to a great extent. Despite the availability of multiple therapy methods, such as pharmacological and psychological approaches, many patients exhibit resistance, emphasizing the necessity for alternative therapies. OCD and other psychiatric conditions like bipolar disorder, schizophrenia, and attention deficit hyperactivity disorder substantially overlap, highlighting the complexity of mental health diagnoses. Furthermore, its comorbidity with these diseases further highlights OCD's intricacy. Several therapy considerations have been mentioned, such as using larger dosages of medications and combining different therapeutic approaches. Their association suggests possible common pathogenic pathways between OCD and other psychiatric illnesses. The review concludes that, given the significant number of people who still struggle with chronic symptoms, new treatment techniques and ongoing research are necessary, even in the face of improvements in the understanding and treatment of OCD.

Evaluation of Change in the Facial Profile and Aesthetics in Relation to Incisor Position in Both Maxillary and Mandibular Arches.

Background The orthodontist is often confronted with the need to predict soft tissue profile changes that may result from the orthodontic treatment. The problem arises because the contribution of many of the factors influencing the soft tissue profile still needs to be fully understood. The complexity of the problem is increased in growing patients in whom the post-treatment soft tissue profile is the result of both growth and orthodontic treatment. A primary motivation for seeking orthodontic treatment is a desire to improve dental and facial aesthetics. To achieve balance in the facial profile treated orthodontically, it is essential to identify the underlying skeletal hard tissue and soft tissue parameters. The present study evaluated the changes in facial profile and aesthetics in relation to incisor position. Materials and methods Samples for this study consisted of pre-treatment lateral cephalograms of the Indian population (n = 450) having different incisor relationships. Subjects aged between 18 and 30 years were included. Angular and linear measurements were taken to analyse the incisor relationship with soft tissue parameters. Results The majority (61.2%) of subjects belonged to the age group of 18-30 years. The overall female-to-male ratio in the study was 7:3. The parameter U1 to L1 was abnormal in 86.8% of subjects. Similarly, the parameters S-line upper lip (UL), S-line lower lip (LL), E-line UL, and E-line LL were found abnormal in 93.9%, 86.8%, 82.6%, and 70.1% of subjects, respectively. A significant agreement was found between U1 to L1 and E-line UL and U1 to L1 and E-line LL. Conclusions The present study concludes that facial aesthetics combines soft and hard tissue corrections, not just based on occlusal relationships but also considering facial harmony. Thus, the incisor relationship is an important asset and strongly correlates with other soft tissue and hard tissue parameters that improve facial aesthetics for the individual undergoing orthodontic treatment.

Investigation of efficient photoconduction and enhanced luminescence characteristics of Ce-doped ZnO nanophosphors for UV sensors.

This study involves the single-step, mass-scale productive synthesis, photoconduction, and luminescence characteristics of pure and cerium rare-earth-ion-doped ZnO (CZO) nanophosphors with different Ce concentrations (Ce: 0, 2, 4, 6, and 8 wt.%) synthesized using the solid-state reaction method. The synthesized nanophosphors were characterized for their structural, morphological, optical, and photoconductivity (PC) properties using X-ray diffraction (XRD), field-effect scanning electron microscopy (FE-SEM), energy dispersive spectroscopy, Fourier-transform infrared (FT-IR), photoluminescence (PL), and PC measurements. The sharp diffraction peaks of XRD results exhibit the formation of crystalline hexagonal wurtzite ZnO nanostructures. The decrease in diffraction peak intensities of CZO with an increase in Ce concentrations signifies the deterioration of the ZnO crystal. FE-SEM images exhibit the good crystalline quality of nanophosphors composed of spherical- and elongated-shaped nanoparticles that are distributed consistently on the surface. The energy dispersive X-ray pattern of the 4 wt.% Ce-doped ZnO (CZO4 ) sample confirms the doping of Ce in ZnO. The presence of chemical bonds and functional groups corresponds to transmittance peaks established using FT-IR spectroscopy. Deconvoluted PL spectra show two major emission peaks, one in the UV region, which is near-band-edge, and the other in the visible region ranging from ~456 to 561 nm. In PC studies, current-voltage (I-V) and current-time (I-T) characteristics, that is, rise/decayin current under dark as well as UV light conditions, are also investigated. Efficient photoconduction is observed in CZO samples. The obtained results indicate the suitability to luminescent and photosensor applications.

Cryo-EM structures of wild-type and E138K/M184I mutant HIV-1 RT/DNA complexed with inhibitors doravirine and rilpivirine.

Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in explaining the mechanisms of catalysis, inhibition, and drug resistance and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-electron microscopy (cryo-EM) has been challenging due to the enzyme's relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and differ from the typical conformation of nonnucleoside RT inhibitor (NNRTI)-bound RT/double-stranded DNA (dsDNA), RT/RNA-DNA, and RT/dsRNA complexes; the primer grip in RT/DNA/doravirine and the YMDD motif in RT/DNA/rilpivirine have large shifts. The DNA primer 3'-end in the doravirine-bound structure is positioned at the active site, but the complex is in a nonproductive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation opens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by an earlier kinetic study. These structural differences have implications for understanding molecular mechanisms of drug resistance and for drug design.

Insights into HIV-1 Reverse Transcriptase (RT) Inhibition and Drug Resistance from Thirty Years of Structural Studies.

The enzyme reverse transcriptase (RT) plays a central role in the life cycle of human immunodeficiency virus (HIV), and RT has been an important drug target. Elucidations of the RT structures trapping and detailing the enzyme at various functional and conformational states by X-ray crystallography have been instrumental for understanding RT activities, inhibition, and drug resistance. The structures have contributed to anti-HIV drug development. Currently, two classes of RT inhibitors are in clinical use. These are nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). However, the error-prone viral replication generates variants that frequently develop resistance to the available drugs, thus warranting a continued effort to seek more effective treatment options. RT also provides multiple additional potential druggable sites. Recently, the use of single-particle cryogenic electron microscopy (cryo-EM) enabled obtaining structures of NNRTI-inhibited HIV-1 RT/dsRNA initiation and RT/dsDNA elongation complexes that were unsuccessful by X-ray crystallography. The cryo-EM platform for the structural study of RT has been established to aid drug design. In this article, we review the roles of structural biology in understanding and targeting HIV RT in the past three decades and the recent structural insights of RT, using cryo-EM.

Batwing incision for a case of bilateral fungal zygomatic osteomyelitis.

Zygomatic osteomyelitis is a rare occurrence due to rich collateral blood supply of bone. A man in his 30s presented with complaints of pain over bilateral cheek and pus discharge below the eye on lateral aspect. He was a known case of COVID-19 associated mucormycosis postendoscopic debridement of sinuses 3 months back. Radiology revealed bilateral destruction of zygoma with discharging sinus. Microbiological analysis confirmed aseptate hyphae in pus, and a diagnosis of bilateral fungal zygomatic osteomyelitis made. Under general anaesthesia, sequestrectomy done using bilateral lateral rhinotomy with extended Dieffenbach's approach (batwing incision). Postsurgery 3000 mg of liposomal amphotericin was administered. There was no enophthalmos or restricted eye movements postoperatively. Follow-up MRI suggested minimal inflammatory enhancement in maxillary sinus. Patient was discharged on oral antifungals.

Role of Etophylline and Theophylline Prolonged Release Tablet in COVID-19 Associated Sinus Node Dysfunction.

During COVID-19 pandemic, one of the most common arrythmia reported with this illness is sinus bradycardia. Treatment for COVID-19 and associated cardiac dysfunction is still evolving. Temporary pacemaker insertion is difficult due to pandemic and risk of spread of infection to the additional staff involved. Orciprenaline stimulates the sino-atrial and atrioventricular nodes and accelerates atrioventricular conduction. Theophylline improves sinus node function in subjects with sinus bradycardia and enhances atrioventricular nodal conduction We report a case series of 10 patients admitted in dedicated COVID-19 ICUs and developed sinus node dysfunction. All of these patients were started on etophylline and theophylline prolonged release tablet (150mg) once a day. On subsequent follow up after 72 hours, all patients reported heart rate well within normal range. COVID-19 virus directly involves the myocardium by entering the cardiac myocytes resulting in inflammation and injury. As the sinus bradycardia due to COVID-19 is usually transient and respond well this drug, short course of this drug could be added to treat this arrythmia in future.

Seven phenotypes of varus osteoarthritic knees can be identified in the coronal plane.

PURPOSE: Recommendations for resecting distal femur and proximal tibia in mechanical and anatomical alignment techniques are standardized. Kinematic alignment propagates individualizing resection planes. Whether significant variation exists, to warrant departure from standardized resection planes, has not been shown thus far in a large cohort of knees and with a wide range of varus deformity. The null hypothesis of this study was that there was no phenotypic variation in varus osteoarthritic knees. The aim of this paper was to determine whether distinct phenotypes could be identified, based on variations in coronal femoral and tibial morphology, which could aid in surgical planning and categorizing varus knees for future studies. METHODS: 2129 full-leg weightbearing radiographs were analyzed (1704 preoperative; 425 of contralateral arthritic knee). Measurements made were of HKA (hip-knee-ankle angle), VCA (valgus correction angle), mLDFA (lateral mechanical distal femoral angle), aLDFA (lateral anatomical distal femoral angle), MPTA (medial proximal tibial angle), MNSA (medial neck shaft angle), TAMA (angle between tibial mechanical and anatomical axes), and TPDR (percentage length of tibia proximal to extra-articular deformity). RESULTS: Seven distinct types were identified covering 2021 knees, reducible to 4 broad phenotypes: 11% were Type 1 'Neutral' knees showing values close to reported normal knees (mean VCA 5.5°, mLDFA 87°, aLDFA 81°). 38% were Type 2 'Intra-articular varus' with medial intra-articular bone loss (mean mLDFA 90.9°, MPTA 85.4°, VCA of 5.7°). 41% were Type 3 'Extra-articular varus' with extra-articular deformity (EAD). Type 3a had proximal tibial EAD; Type 3b had tibial diaphyseal EAD; Type 3c had femoral EAD (mean VCA 8.7°, HKA 166°), and severe medial bone loss (mean mLDFA 92°, MPTA 83°). 9% were Type 4 'Valgoid type' with features of valgus knees: Type 4a had medial femoral bowing (mean VCA 2.9°); Type 4b had significant distal femoral valgus (mean mLDFA 85.3°, aLDFA 78.6°). CONCLUSIONS: The null hypothesis that there was no phenotypic variation in varus osteoarthritic knees was rejected as considerable variation was found in coronal morphology of femur and tibia. Four broad phenotypic groups could be identified. Plane of the knee joint articular surface was quite variable. This has relevance to planning and performance of corrective osteotomies, unicompartmental and total knee arthroplasty. LEVEL OF EVIDENCE: III, retrospective cohort study.

Valgus arthritic knees can be classified into nine phenotypes.

PURPOSE: It is not shown whether anatomical variations exist in valgus arthritic limbs as to support individualized component and limb alignment. The null hypothesis was that there was no phenotypic variation of coronal femoro-tibial morphology in valgus knees. The aim was to determine whether distinct phenotypes of valgus knees could be identified to help surgical planning and classifying valgus knees for outcome studies. METHODS: Full-leg weight-bearing radiographs of 233 knees (182 preoperative; 51 of contralateral arthritic knee) were measured for HKA (hip-knee-ankle angle), VCA (valgus correction angle), mLDFA (lateral mechanical distal femoral angle), aLDFA (lateral anatomical distal femoral angle), MPTA (medial proximal tibial angle), MNSA (medial neck shaft angle), TAMA (angle between tibial mechanical and anatomical axes), and TBA (tibial bowing angle). RESULTS: Nine phenotypes were identified encompassing all 233 knees which could be clubbed into 4 broad types. Type 1 Neutral knees (12.5%) had almost normal values (mean VCA 5.3°, mLDFA 86.9°, aLDFA 81.1°). Type 2 'Intra-articular valgus' (22.7%) showed lateral compartment bone loss (mean mLDFA 83.9°; MPTA 90.2°). Type 3 'Extra-articular valgus' (35.2%) had extra-articular deformity: 3a showed valgus femoral bowing (mean VCA 2.7°); 3b valgus tibial bowing; 3c showed valgus tibial bowing with lateral femoral condyle wear (mean mLDFA 84.3°). Type 4 'Varus' type (29.6%) had features of varus knees: 4a had varus femoral bowing (VCA 8.3°); distal femur in 4b was akin to varus knees (mean mLDFA 89.3°) with lateral tibial bone loss (mean MPTA 91.2°). 4c had varus tibial bowing and deficient lateral femoral condyle (mLDFA 83.7°). 4d had varus tibial bowing and lateral tibial bone loss (mean MPTA 89.8°). CONCLUSIONS: The study identified four broad groups of valgus arthritic knees with nine phenotypes based on coronal plane variations in femoro-tibial morphology. This study may be of value in planning and performing corrective osteotomies, and planning the optimal position of femoral and tibial components in unicompartmental and total knee arthroplasty. The classification presented in this study may aid in categorizing valgus knees for outcome studies. LEVEL OF EVIDENCE: IV.

Arthritic knees with more than 10° valgus can have soft-tissue imbalance in flexion.

PURPOSE: Valgus knees have inferior outcomes compared to varus knees. There is little data regarding soft-tissue balance in flexion which may influence outcome in valgus knees undergoing TKA. The purpose of this study was to evaluate whether there is imbalance between medial and lateral flexion gaps in valgus deformity. A secondary aim was to compare soft-tissue balance in knees with valgus deformity less than 10° with those exceeding 10°. The null hypothesis was that there was no soft-tissue imbalance in 90° of flexion irrespective of magnitude of deformity. METHODS: 64 valgus knees (52 female and 12 male) with deformity from 0.5 to 27.5° (mean 188.77, SD 6.21) were studied in 54 patients (mean age 67.81 y, SD 8.69) undergoing navigated TKA. Medial and lateral gaps in extension and at 90° of flexion were compared (using Independent-samples t test) between knees with valgus < 10° with those > 10° using a validated dynamic method after resection of cruciates, menisci and osteophytes, and then after final trialling. RESULTS: Mean initial medial-lateral (ML) gap difference in extension was 2.63 mm (SD 2.63) and 2.09 mm (SD 3.78) in flexion, being tighter laterally. Initial ML gap differences in extension and flexion correlated with valgus deformity (R = - 0.514; p = 0.00001; R = - 0.325; p = 0.01, respectively). Initial ML gap differences in extension correlated with those in flexion (R = 0.42; p = 0.0005). Mean ML flexion and extension gap differences were 1.30 mm (SD 3.67) and 1.26 mm (SD 1.92) in knees with < 10° valgus, and 3.17 mm (SD 3.71) and 4.29 mm (SD 2.45) in those > 10° valgus; p values were 0.026 and < 0.001 respectively. CONCLUSION: The lateral flexion gap in valgus knees may be narrower than the medial flexion gap, especially in knees with > 10° deformity. This contrasts with native and varus knees, in which it exceeds the medial gap. This novel study indicates the need to identify valgus knees with lateral flexion gap tightness by distracting the posterior femoral condyles from the proximal tibia by dynamic stressing of the soft-tissues after resection of cruciates, menisci, and osteophytes, with the knee flexed to 90°. These findings, highlighting the need for restoring flexion gap balance, may improve the inferior outcomes in valgus knees. LEVEL OF EVIDENCE: IV.

Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - targeting P-pocket by fragment screening.

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3'-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized, two of which show noticeable RT inhibition. An engineered RT/DNA aptamer complex could trap the transient P-pocket in solution, and structures of the RT/DNA complex were determined in the presence of an inhibitory fragment. A synthesized analog bound at P-pocket is further analyzed by single-particle cryo-EM. Identification of the P-pocket within HIV RT and the developed structure-based platform provide an opportunity for the design new types of polymerase inhibitors.

Exploring the dNTP -binding site of HIV-1 reverse transcriptase for inhibitor design.

HIV-1 reverse transcriptase (RT) plays a central role in the viral life cycle, and roughly half of the FDA-approved anti-HIV drugs are targeting RT. Nucleoside analogs (NRTIs) require cellular phosphorylation for binding to RT, and to bypass this rate-limiting path, we designed a new series of acyclic nucleoside phosphonate analogs as nucleoside triphosphate mimics, aiming at the chelation of the catalytic Mg2+ ions via a phosphonate and/or a carboxylic acid group. Novel synthetic procedures were developed to access these nucleoside phosphonate analogs. X-ray structures in complex with HIV-1 RT/dsDNA demonstrated that their binding modes are distinct from that of our previously reported compound series. The impact of chain length, chirality and linker atom have been discussed. The detailed structural understanding of these new compounds provides opportunities for designing new class of HIV-1 RT inhibitors.

Talin rod domain-containing protein 1 (TLNRD1) is a novel actin-bundling protein which promotes filopodia formation.

Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain-containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif-containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly.

Development of a Novel SPR Assay to Study CXCR4-Ligand Interactions.

G protein-coupled receptors (GPCRs) are involved in a plethora of different diseases. Consequently, these proteins are considered as an important class of drug targets. Measuring detailed kinetic information on these types of proteins has been challenging. Surface plasmon resonance (SPR) can provide this information, however, the use of SPR on GPCRs remains a complex issue. Here, we report an SPR assay to investigate the interactions between the full-length chemokine receptor CXCR4 and nanobody-Fc (Nb-Fc) ligands. Nb-Fcs consist of two monovalent VHH domains fused with an Fc domain of a human IgG molecule. The CXCR4 protein used in this assay was produced with a C-terminal 10x-histidine tag and was immobilized on a nitrilotriacetic acid chip. In order to verify the sensitivity and effectiveness of this assay, the results were compared to data obtained from cellular assays as well as from another SPR assay using CXCR4 virus-like particles (VLPs). CXCR4 remained intact and stable for at least 12 h, and the kinetic results correlated well with both the cellular assays and the VLP SPR assay results. Apart from determining the binding kinetics of Nb-Fc with CXCR4, our results contributed to understanding CXCR4 interaction dynamics. In conclusion, this assay provides a viable experimental platform that has high potential to be expanded for studying other molecules as well as other histidine-tagged GPCRs.

Lead Optimization of Phthalazinone Phosphodiesterase Inhibitors as Novel Antitrypanosomal Compounds.

Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of Africa. In this manuscript we describe the optimization of a family of phtalazinone derivatives. Phosphodiesterases have emerged as attractive molecular targets for a novel treatment for a variety of neglected parasitic diseases. Compound 1 resulted in being a potent TbrPDEB1 inhibitor with interesting activity against T. brucei in a phenotypic screen. Derivative 1 was studied in an acute in vivo mouse disease model but unfortunately showed no efficacy due to low metabolic stability. We report structural modifications to achieve compounds with an improved metabolic stability while maintaining high potency against TbrPDEB1 and T. brucei. Compound 14 presented a good microsomal stability in mouse and human microsomes and provides a good starting point for future efforts.

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors (Part 2).

Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action.

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors.

Several 3',5'-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.