Targeted Library of Phosphonic-Type Inhibitors of Human Neutrophil Elastase.

Despite many years of research, human neutrophil elastase (HNE) still remains an area of interest for many researchers. This multifunctional representative of neutrophil serine proteases is one of the most destructive enzymes found in the human body which can degrade most of the extracellular matrix. Overexpression or dysregulation of HNE may lead to the development of several inflammatory diseases. Previously, we presented the HNE inhibitor with kinact/KI value over 2,000,000 [M-1s-1]. In order to optimize its structure, over 100 novel tripeptidyl derivatives of α-aminoalkylphosphonate diaryl esters were synthesized, and their activity toward HNE was checked. To confirm the selectivity of the resultant compounds, several of the most active were additionally checked against the two other neutrophil proteases: proteinase 3 and cathepsin G. The developed modifications allowed us to obtain a compound with significantly increased inhibitory activity against human neutrophil elastase with high selectivity toward cathepsin G, but none toward proteinase 3.

Diisothiocyanate-Derived Mercapturic Acids Are a Promising Partner for Combination Therapies in Glioblastoma.

Glioblastoma represents the most aggressive tumor of the central nervous system. Due to invasion of glioblastoma stem cells into the healthy tissue, chemoresistance, and recurrence of the tumor, it is difficult to successfully treat glioblastoma patients, which is demonstrated by the low life expectancy of patients after standard therapy treatment. Recently, we found that diisothiocyanate-derived mercapturic acids, which are isothiocyanate derivatives from plants of the Cruciferae family, provoked a decrease in glioblastoma cell viability. These findings were extended by combining diisothiocyanate-derived mercapturic acids with dinaciclib (a small-molecule inhibitor of cyclin-dependent kinases with anti-proliferative capacity) or temozolomide (TMZ, standard chemotherapeutic agent) to test whether the components have a cytotoxic effect on glioblastoma cells when the dosage is low. Here, we demonstrate that the combination of diisothiocyanate-derived mercapturic acids with dinaciclib or TMZ had an additive or even synergistic effect in the restriction of cell growth dependent on the combination of the components and the glioblastoma cell source. This strategy could be applied to inhibit glioblastoma cell growth as a therapeutic interference of glioblastoma.

The 45-second anterior knee pain provocation test: A quick test of knee pain and sporting function in 10-14-year-old adolescents with patellofemoral pain.

OBJECTIVE: To test 1) if the 45-second Anterior Knee Pain Provocation Test (AKPP-test) could differentiate between adolescents with patellofemoral pain (PFP) and pain-free controls and; 2) whether improvements in the AKPP-test over 12 weeks were associated with improvements in self-reported knee function and pain. DESIGN: Prospective cohort. PATIENTS: 151 with PFP and 50 pain-free controls (age 10-14 years). OUTCOMES: The AKPP-test was performed at baseline, 4- and 12-week follow-up. Pain and function were collected using Knee Injury and Osteoarthritis Outcome Score (KOOS). RESULTS: At baseline, the AKPP-test provoked pain to a median of 5 points (IQR: 3-7) on the 0-10 Numeric Pain Rating Scale in adolescents with PFP, compared to 0 (IQR 0-0) in controls. Higher pain during the AKPP-test was associated with worse KOOS-Sport/Rec (r = -0.33, P < 0.001), worse KOOS-Pain (r = -0.47, P < 0.001), and pain intensity (worst pain last 24 hours) (r = -0.39, P < 0.001) at baseline. Improvements in the AKPP-test over 12 weeks were associated with improvements in KOOS Pain (r = 0.48, P < 0.001) and KOOS Sport/Rec (r = 0.40, P < 0.001). CONCLUSIONS: Improvements in the AKPP-test were associated with improvements in self-report knee pain and limitations in sports, suggesting the AKPP-test may be a clinically responsive test of knee pain and sporting function in adolescents with PFP.

Is the Prognosis of Osgood-Schlatter Poorer Than Anticipated? A Prospective Cohort Study With 24-Month Follow-up.

BACKGROUND: Osgood-Schlatter disease (OSD), an apophyseal injury of the tibial tuberosity, affects up to 1 in 10 adolescents. This condition has previously been assumed to be innocuous and to self-resolve with limited intervention. PURPOSE: To investigate the 24-month prognosis of OSD and examine if ultrasound (US) classification is associated with outcomes. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: This study included a preregistered prospective cohort of 51 adolescents (aged 10-14 years) diagnosed with OSD who were evaluated for 24 months. The primary outcome at 24-month follow-up was whether participants continued to experience OSD-related knee pain. Baseline US scans were collected and characterized by OSD type (De Flaviis classification) as well as maturation of the tibial tuberosity. Secondary outcomes included sports participation, Knee injury and Osteoarthritis Outcome Score (KOOS) Sport/Recreation subscale, and health-related quality of life (European Quality of Life-5 Dimensions-Youth [EQ-5D-Y]). All participants were invited for re-examination by US at follow-up. RESULTS: A total of 51 patients preregistered for the study, with 90% (n = 46) available at follow-up. Of these 46 participants, 37% (n = 17) still reported knee pain due to OSD. In this subgroup, the median duration since symptom onset was 42 months (interquartile range, 38-51 months). More than 1 in 5 participants reported stopping sport due to knee pain, and those who continued to experience knee pain reported significantly worse KOOS Sport/Recreation scores at follow-up compared with patients with no knee pain (mean 74 [95% CI, 63-84] vs 91 [95% CI, 85-97]). Participants with continued OSD-related pain also had lower health-related quality of life (mean difference in EQ-5D-Y, 0.11 [95% CI, 0.06-0.13]). Higher De Flaviis classification at baseline was significantly associated with an increased risk of knee pain at 2 years. Diagnostic US at follow-up demonstrated primarily tendon changes (thickening, positive Doppler signal), as well as an ununited ossicle in 32% of participants who underwent US scanning at follow-up. CONCLUSION: Over one-third of the study participants had knee pain at 2-year follow-up, which was associated with lower sports related function and health related quality of life. This questions the assumption that all patients with OSD experience quick recovery. Participants without any changes on imaging at baseline were less likely to report pain at follow-up.

Phosphonic Analogs of Alanine as Acylpeptide Hydrolase Inhibitors.

Acylpeptide hydrolase is a serine protease, which, together with prolyl oligopeptidase, dipeptidyl peptidase IV and oligopeptidase B, belongs to the prolyl oligopeptidase family. Its primary function is associated with the removal of N-acetylated amino acid residues from proteins and peptides. Although the N-acylation occurs in 50-90 % of eukaryotic proteins, the precise functions of this modification remains unclear. Recent findings have indicated that acylpeptide hydrolase participates in various events including oxidized proteins degradation, amyloid ß-peptide cleavage, and response to DNA damage. Considering the protein degradation cycle cross-talk between acylpeptide hydrolase and proteasome, inhibition of the first enzyme resulted in down-regulation of the ubiquitin-proteasome system and induction of cancer cell apoptosis. Acylpeptide hydrolase has been proposed as an interesting target for the development of new potential anticancer agents. Here, we present the synthesis of simple derivatives of (1-aminoethyl)phosphonic acid diaryl esters, phosphonic analogs of alanine diversified at the N-terminus and ester rings, as inhibitors of acylpeptide hydrolase and discuss the ability of the title compounds to induce apoptosis of U937 and MV-4-11 tumor cell lines.

Concurrent musculoskeletal complaints in elbows, shoulders, and necks after common hand and forearm injuries or conditions: A cross-sectional study among 600 patients.

STUDY DESIGN: This is a cross-sectional study among 600 patients. INTRODUCTION: Isolated hand and forearm injuries or conditions are common in the emergency and orthopedic departments. So far, little is known about whether these patients suffer from concurrent musculoskeletal complaints (MSCs) besides their hand and forearm complaints. Neglecting concurrent MSCs in the upper limbs and necks could hamper rehabilitation and prolong the time taken to return to daily and work-related activities. PURPOSE OF THE STUDY: The purpose of this study was to investigate the prevalence of concurrent MSCs in the elbow, shoulder, and neck after common hand and/or forearm injuries or conditions. METHODS: This study included 600 patients with any type of diagnosis referred to rehabilitation after hand and/or forearm injuries or conditions. Basic characteristics, diagnoses, and location of patients' symptoms were collected and analyzed. RESULTS: The overall prevalence of concurrent MSCs was 40%. Twenty-eight percent of the whole sample developed concurrent MSCs after the hand and forearm injury or condition. The gender distribution was 68% women and 32% men. The most common location for complaints was the shoulder (62%), followed by the elbow (49%), and the neck (32%). DISCUSSION: The present results suggest that MSCs from the elbows, shoulders, or necks are very common in patients with hand and/or forearm injuries or conditions. CONCLUSION: Clinicians treating patients with isolated hand and forearm injuries or conditions should be aware of the high prevalence of concurrent MSCs. Future research should investigate if specific rehabilitation, focusing on concurrent MSCs, may influence the outcome in this population.

Activity Modification and Knee Strengthening for Osgood-Schlatter Disease: A Prospective Cohort Study.

BACKGROUND: Osgood-Schlatter disease (OSD) affects 1 in 10 adolescents. There is a lack of evidence-based interventions, and passive approaches (eg, rest and avoidance of painful activities) are often prescribed. PURPOSE: To investigate an intervention consisting of education on activity modification and knee-strengthening exercises designed for adolescents with OSD. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: This study included 51 adolescents (51% female; age range, 10-14 years) with OSD. The 12-week intervention consisted of an activity ladder designed to manage patellar tendon loading and pain, knee-strengthening exercises, and a gradual return to sport. The primary outcome was the global reporting of change at 12 weeks, evaluated with a 7-point Likert scale (successful outcome was considered "much improved" or "improved"). Additional endpoints were at 4, 8, 26, and 52 weeks. Secondary outcomes included the Knee injury and Osteoarthritis Outcome Score (KOOS), objective strength, and jump performance. RESULTS: Adolescents reported a mean pain duration of 21 months at enrollment. After 12 weeks, 80% reported a successful outcome, which increased to 90% at 12 months. At 12 weeks, 16% returned to playing sport, which increased to 69% at 12 months. The KOOS subscores of Pain, Activities of Daily Living, Sport and Recreation, and Quality of Life improved significantly (7-20 points), and there were improvements in knee extension strength (32%; P < .001), hip abduction strength (24%; P < .001), and jumping for distance (14%; P < .001) and height (19%; P < .001) at 12 weeks. CONCLUSION: An intervention consisting of activity modification, pain monitoring, progressive strengthening, and a return-to-sport paradigm was associated with improved self-reported outcomes, hip and knee muscle strength, and jumping performance. This approach may offer an alternative to passive approaches such as rest or wait-and-see, often prescribed for adolescents with OSD. REGISTRATION: NCT02799394 (ClinicalTrials.gov identifier).

Pain, Sports Participation, and Physical Function in Adolescents With Patellofemoral Pain and Osgood-Schlatter Disease: A Matched Cross-sectional Study.

OBJECTIVE: To compare pain, physical activity, quality of life, strength, and knee function in adolescents with patellofemoral pain (PFP) and Osgood-Schlatter disease (OSD) to those in pain-free controls. DESIGN: Cross-sectional study. METHODS: Self-report questionnaires were used to describe pain, physical activity, knee function, and quality of life in participants with PFP (n = 151) or OSD (n = 51) and in pain-free controls (n = 50) between 10 and 14 years of age. Hip and knee strength were measured by handheld dynamometry. Physical activity levels were measured using wearable accelerometers. RESULTS: Adolescents were highly active (accumulating greater than 120 minutes of vigorous physical activity per day), with no differences between the OSD, PFP, and control groups. Adolescents with PFP or OSD scored 22 to 56 points lower (P<.001) on the Knee injury and Osteoarthritis Outcome Score subscales compared with controls, with the lowest scores on the "sport and recreation" and "quality of life" subscales. Adolescents with OSD had lower knee extension strength compared to controls (P<.05; effect size, 1.25). Adolescents with PFP had lower hip extension strength compared to controls (P<.05; effect size, 0.73). CONCLUSION: Adolescents with PFP or OSD had high physical activity levels, despite reporting long-standing knee pain and impaired knee function that impacted on their sports participation and quality of life. Clinicians treating adolescents with PFP or OSD may use these findings to target treatment to the most common deficits to restore sports-related function and sports participation. J Orthop Sports Phys Ther 2020;50(3):149-157. Epub 6 Jan 2020. doi:10.2519/jospt.2020.8770.

Activity Modification and Load Management of Adolescents With Patellofemoral Pain: A Prospective Intervention Study Including 151 Adolescents.

BACKGROUND: Patellofemoral pain (PFP) affects 7% of adolescents, especially those who are highly active. Exercise-focused treatments show limited effect and overlook activity modification and load management. As many adolescents continue at high levels of sports despite pain, a new strategy addressing this problem is warranted. PURPOSE: To investigate the effects of a treatment strategy for adolescents that focuses on activity modification and load management. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: Adolescents aged 10 to 14 years with PFP were included (N = 151). The 12-week intervention included 4 supervised sessions with a physical therapist, which adolescents and parents were required to attend. The intervention included activity modification (weeks 1-4) to reduce loading of the patellofemoral joint via an activity ladder and pain monitoring, home-based exercises (weeks 5-8), and return-to-sport guidance (weeks 9-12). Primary outcome was a 7-point global rating of change, ranging from "much improved" to "much worse." Adolescents were considered to have a successful outcome if they reported "much improved" or "improved." The primary endpoint was at 12 weeks, with additional follow-up at 4, 24, and 52 weeks. Secondary outcomes included the Knee injury and Osteoarthritis Outcome Score (KOOS), hip and knee torque, sports participation, satisfaction with treatment, and use of painkillers. RESULTS: At 12 weeks, 87% completed the full questionnaire, of which 86% reported a successful outcome, as compared with 77% (95% CI, 68%-83%) at 6 months and 81% (95% CI, 73%-88%) at 12 months. There were large clinically relevant improvements in 3 KOOS subscales: Pain, Sport/Recreation, and Quality of Life (13-24 points). Hip and knee torque increased by 20% to 33%. In total, 68% were back playing sport after 3 months, which increased to 79% at 6 months and 81% at 12 months. The majority were satisfied with the treatment (90%) and would recommend it to a friend (95%). No specific patient characteristics were associated with prognosis. CONCLUSION: A treatment strategy focusing on activity modification and load management was associated with high rates of successful outcome among adolescents with PFP at 12 and 52 weeks. These short- and longer-term outcomes were supported by improvements in symptoms and objective measures of hip and knee torque. REGISTRATION: NCT02402673 (ClinicalTrials.gov identifier).

Viability of glioblastoma stem cells is effectively reduced by diisothiocyanate-derived mercapturic acids.

Glioblastoma is the most aggressive tumor of the central nervous system and is manifested by diffuse invasion of glioblastoma stem cells into the healthy tissue, chemoresistance and recurrence. Despite aggressive therapy, consisting of maximal surgical resection, radiotherapy and chemotherapy with temozolomide (Temodal®), life expectancy of patients with glioblastoma is typically less than 15 months. In general, natural isothiocyanates isolated from plants of the Cruciferae family are selectively cytotoxic to tumor cells. It has been demonstrated previously that diisothiocyanate-derived mercapturic acids are highly cytotoxic to colon cancer cells. In the present study, the application of diisothiocyanate-derived mercapturic acids led to a decrease in the viability of an established glioblastoma cell line, primary patient-derived sphere-cultured stem cell-enriched cell populations (SCs), and cells differentiated from SCs. Consequently, targeting glioblastoma cells by diisothiocyanate-derived mercapturic acids is a promising approach to restrict tumor cell growth and may be a novel therapeutic intervention for the treatment of glioblastoma.

Phosphorus-containing isothiocyanate-derived mercapturic acids as a useful alternative for parental isothiocyanates in experimental oncology.

A series of phosphonates, phosphinates and phosphine oxides isothiocyanate-derived mercapturic acids were synthesized. A temperature dependence dynamic proton decoupled 31P NMR studies indicated that in most cases the compounds were obtained as a mixture of rotamers. Moreover, biologically relevant reversibility of mercapturic acids synthesis from the parental isothiocyanates was confirmed. All compounds were evaluated ashighly active antiproliferative agents in vitro in human colon cancer cell lines (LoVo and its doxorubicin-resistant subline LoVo/DX). The cell cycle progression and caspase-3 activity analyses revealed compounds moderate activity as apoptosis inducers and their poor influence on cell cycle progression in the LoVo cells. Our results confirm that isothiocyanate-derived mercapturic acids present a reasonable alternative for the parental compounds, and can replace them in the future studies on isothiocyanates potential as anticancer agents.

Selection of Effective HTRA3 Activators Using Combinatorial Chemistry.

Herein, we report selection, synthesis, and enzymatic evaluation of a peptidomimetic library able to increase proteolytic activity of HtrA3 (high temperature requirement A) protease. Iterative deconvolution in solution of synthesized modified pentapeptides yielded two potent HtrA3 activators acting in the micromolar range (HCOO-CH2O-C6H4-OCH2-CO-Tyr-Asn-Phe-His-Asn-OH and HCOO-CH2O-C6H4-OCH2-CO-Tyr-Asn-Phe-His-Glu-OH). Both compounds increased proteolysis of an artificial HtrA3 substrate over 40-fold in a selective manner. On the basis of molecular modeling, the selected compounds bind strongly to the PDZ domain.

Synthesis and biological activity of diisothiocyanate-derived mercapturic acids.

This Letter deals with new non-natural diisothiocyanates, their mercapturic acid derivatives-conjugated with N-acetylcysteine as well as their antiproliferative activity towards human colon cancer cell lines and their inhibitory potency towards histone deacetylase activity. The activity of analysed isothiocyanates is not significantly different than their N-acetylcysteine conjugates. In comparison to simple mono-isothiocyanate analogues, aliphatic diisothiocyanates and their conjugates are much more active than the simple presence of two isothiocyanate functionalities could indicate.

Synthesis of novel phosphonic-type activity-based probes for neutrophil serine proteases and their application in spleen lysates of different organisms.

Neutrophils are a type of granulocyte important in the "first line of defense" of the innate immune system. Upon activation, they facilitate the destruction of invading microorganisms by the production of superoxide radicals, as well as the release of the enzymatic contents of their lysozymes. These enzymes include specific serine proteases: cathepsin G, neutrophil elastase, proteinase 3, as well as the recently discovered neutrophil serine protease 4 (NSP4). Under normal conditions, the proteolytic activity of neutrophil proteases is tightly regulated by endogenous serpins; however, this mechanism can be subverted during tissue stress, thereby resulting in the uncontrolled activity of serine proteases, which induce chronic inflammation and subsequent pathology. Herein, we describe the development of low-molecular-weight activity-based probes that specifically target the active sites of neutrophil proteases.

Human neutrophil elastase phosphonic inhibitors with improved potency of action.

Herein, we present the synthesis and the measurement of the inhibitory activity of novel peptidyl derivatives of α-aminoalkylphosphonate diaryl esters as human neutrophil elastase inhibitors. Their selectivity against other serine proteases, including porcine pancreatic elastase, chymotrypsin, and trypsin, was also demonstrated. We also describe the preparation of single peptide diastereomers. The most active and selective compound developed possessed a k(inact)/K(I) of 2353000 M(-1) s(-1), which is the most potent irreversible peptidyl inhibitor of human neutrophil elastase reported to date. The peptidyl inhibitors were demonstrated to be stable in PBS buffer and human plasma, as were their complexes with HNE.

Simple phosphonic inhibitors of human neutrophil elastase.

Herein, we describe the synthesis and resulting activity of a complex series of α-aminophosphonate diaryl esters as irreversible human neutrophil elastase inhibitors and their selectivity preference for human neutrophil elastase over several other serine proteases such as porcine pancreatic elastase, trypsin, and chymotrypsin. We synthesized and examined the inhibitory potency of several new simple Cbz-protected α-aminoalkylphosphonate diaryl esters that yielded several new HNE inhibitors, where one of the obtained compounds Cbz-Val(P)(OC(6)H(4)-4-COOMe)(2) displayed an apparent second-order inhibition value at 33,015 M(-1) s(-1).