X-ray Screening of an Electrophilic Fragment Library and Application toward the Development of a Novel ERK 1/2 Covalent Inhibitor.

Fragment-based drug discovery (FBDD) has become an established method for the identification of efficient starting points for drug discovery programs. In recent years, electrophilic fragment screening has garnered increased attention from both academia and industry to identify novel covalent hits for tool compound or drug development against challenging drug targets. Herein, we describe the design and characterization of an acrylamide-focused electrophilic fragment library and screening campaign against extracellular signal-regulated kinase 2 (ERK2) using high-throughput protein crystallography as the primary hit-finding technology. Several fragments were found to have covalently modified the adenosine triphosphate (ATP) binding pocket Cys166 residue. From these hits, 22, a covalent ATP-competitive inhibitor with improved potency (ERK2 IC50 = 7.8 µM), was developed.

Trauma Assessment Clinic: A virtual fracture clinic model that delivers on its PROMise!

BACKGROUND: The use of virtual fracture clinics across the United Kingdom and Ireland is growing and have been shown in an increasing number of studies to be safe, cost-effective and associated with good functional outcomes and patient satisfaction rates for certain fracture types. Initially pioneered at Glasgow Royal Infirmary, many centres have adopted similar templates, or variations of, and the overall aim of this study was to assess functional outcomes and injury recovery satisfaction rates of patients discharged directly following review in a specific virtual fracture clinic model known as the Trauma Assessment Clinic (TAC). METHODS: A prospective observational study was carried out of paediatric (aged <17 years) and adult (aged >17 years) patients, with the five most commonly observed fracture types, who were discharged directly following review at the TAC in a single hospital centre over a 12 month period from January to December 2018. Primary and secondary outcomes were assessed via telephone administered questionnaires and patient reported outcome measures (PROMs). RESULTS: A total of 198 patients were included in the study (n = 98 paediatric and n = 100 adult). Overall, 192 (97%) patients or parents/guardians of patients stated that they either strongly agreed (n = 148, 74.9%) or agreed (n = 44, 22.1%) that they were satisfied with their own or their child's recovery from their injury at a median follow-up of 9 months post direct discharge from the TAC. Adult patients had an EQ-5D-5L index median value of 1 (range 0-1), an EQ-VAS median of 87 (range 0-100), a QuickDASH median score of 0 (range 0-100) and a median LEFS of 80 (range 0-80). CONCLUSION: The virtual management of trauma patients via the TAC model is a safe and patient-centred approach to treating certain injuries and fracture patterns. This study reports excellent patient reported outcome measures and patient injury recovery satisfaction rates. The use of current available technology in tandem with up-to-date best clinical practice and guidelines play a central role in this novel care pathway.

Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

ASTX029, a Novel Dual-mechanism ERK Inhibitor, Modulates Both the Phosphorylation and Catalytic Activity of ERK.

The MAPK signaling pathway is commonly upregulated in human cancers. As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for the treatment of MAPK-activated cancers and for overcoming resistance to upstream inhibition. ASTX029 is a highly potent and selective dual-mechanism ERK inhibitor, discovered using fragment-based drug design. Because of its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK itself by MEK, despite not directly inhibiting MEK activity. This dual mechanism was demonstrated in cell-free systems, as well as cell lines and xenograft tumor tissue, where the phosphorylation of both ERK and its substrate, ribosomal S6 kinase (RSK), were modulated on treatment with ASTX029. Markers of sensitivity were highlighted in a large cell panel, where ASTX029 preferentially inhibited the proliferation of MAPK-activated cell lines, including those with BRAF or RAS mutations. In vivo, significant antitumor activity was observed in MAPK-activated tumor xenograft models following oral treatment. ASTX029 also demonstrated activity in both in vitro and in vivo models of acquired resistance to MAPK pathway inhibitors. Overall, these findings highlight the therapeutic potential of a dual-mechanism ERK inhibitor such as ASTX029 for the treatment of MAPK-activated cancers, including those which have acquired resistance to inhibitors of upstream components of the MAPK pathway. ASTX029 is currently being evaluated in a first in human phase I-II clinical trial in patients with advanced solid tumors (NCT03520075).

The Association of Gastrocnemius Tightness, Genu Valgum and Hallux Valgus: A Prospective Case-Control Study.

There has been much debate regarding the aetiology and pathogenesis of hallux valgus and it appears to be multifactorial with contracture or tightness of the Achilles tendon and more specifically the gastrocnemius being implicated as an intrinsic factor. The purpose of this study was to look at the association of gastrocnemius tightness, genu valgum and hallux valgus. A prospective case-control study with 25 patients in each group was carried out over a 12-month period. The case group observed adult patients who were referred primarily because of symptomatic hallux valgus and were assessed for the following: hallux valgus stage; presence or absence of isolated gastrocnemius tightness; presence or absence of genu valgum. The control group excluded those with pre-existing hallux valgus, genu valgum and rheumatoid arthritis and were assessed for isolated gastrocnemius tightness. There was a statistically significant association between the presence of genu valgum and hallux valgus when comparing both groups with a p < .001. There was also a statistically significant association between the Silfverskiöld test and the presence of hallux valgus, as well as the Silfverskiöld test and the presence of genu valgum with a p < .001. This study is the first to describe the association of gastrocnemius tightness, genu valgum and hallux valgus. Further studies are required to assess this relationship but knowledge and awareness of it can be applied by clinicians when considering the most appropriate management options with patients.

Acute compartment syndrome of the foot due to a twisting injury while playing sports.

Acute compartment syndrome (ACS) of the foot is one of the most severe injuries of the foot and typically results from a fracture, crush or vascular injury. ACS, isolated to a single foot compartment, is a rare complication following a simple twisting injury of the ankle. In this article, the authors present the case report of a 25-year-old man who developed ACS, isolated to the lateral compartment of the foot, secondary to rupture of the lateral ligament complex and subsequent haematoma formation. An emergency fasciotomy was performed and the patient had complete resolution of his symptoms. ACS is usually associated with significant trauma, however, there are reported cases in the literature associated with a minor injury. In this case report, the authors describe how ACS developed following a simple ankle sprain playing sports in the absence of a high-energy insult or fracture.

Dual-Mechanism ERK1/2 Inhibitors Exploit a Distinct Binding Mode to Block Phosphorylation and Nuclear Accumulation of ERK1/2.

The RAS-regulated RAF-MEK1/2-ERK1/2 signaling pathway is frequently deregulated in cancer due to activating mutations of growth factor receptors, RAS or BRAF. Both RAF and MEK1/2 inhibitors are clinically approved and various ERK1/2 inhibitors (ERKi) are currently undergoing clinical trials. To date, ERKi display two distinct mechanisms of action (MoA): catalytic ERKi solely inhibit ERK1/2 catalytic activity, whereas dual mechanism ERKi additionally prevents the activating phosphorylation of ERK1/2 at its T-E-Y motif by MEK1/2. These differences may impart significant differences in biological activity because T-E-Y phosphorylation is the signal for nuclear entry of ERK1/2, allowing them to access many key transcription factor targets. Here, we characterized the MoA of five ERKi and examined their functional consequences in terms of ERK1/2 signaling, gene expression, and antiproliferative efficacy. We demonstrate that catalytic ERKi promote a striking nuclear accumulation of p-ERK1/2 in KRAS-mutant cell lines. In contrast, dual-mechanism ERKi exploits a distinct binding mode to block ERK1/2 phosphorylation by MEK1/2, exhibit superior potency, and prevent the nuclear accumulation of ERK1/2. Consequently, dual-mechanism ERKi exhibit more durable pathway inhibition and enhanced suppression of ERK1/2-dependent gene expression compared with catalytic ERKi, resulting in increased efficacy across BRAF- and RAS-mutant cell lines.

Crystallographic screening using ultra-low-molecular-weight ligands to guide drug design.

We present a novel crystallographic screening methodology (MiniFrags) that employs high-concentration aqueous soaks with a chemically diverse and ultra-low-molecular-weight library (heavy atom count 5-7) to identify ligand-binding hot and warm spots on proteins. We propose that MiniFrag screening represents a highly effective method for guiding optimisation of fragment-derived lead compounds or chemical tools and that the high screening hit rates reflect enhanced sampling of chemical space.

Electronic referrals for virtual fracture clinic service using the National Integrated Medical Imaging System (NIMIS).

INTRODUCTION: Virtual fracture clinics (VFC) are now prevalent across many orthopaedic services in the UK and Ireland. The management of a variety of musculoskeletal injuries using the VFC model has been demonstrated to be safe, cost-effective and associated with high levels of patient satisfaction. Referrals were made available through the National Integrated Medical Imaging System (NIMIS). NIMIS allows for electronic movement of patient images throughout the Irish health service. METHODS: A retrospective review of 157 orthopaedic fracture referrals from a regional hospital was performed. The referrals were received during a 6-week period between May 2016 and June 2016. Each of these referrals was sent electronically. These referrals were reviewed each day by a consultant-led multi-disciplinary team. RESULTS: Thirty (93%) patients agreed or strongly agreed that they received adequate information in relation to the VFC when they attended the emergency department (ED). All patients except for one either agreed or strongly agreed that they were satisfied with their recovery (97%). Fifteen parents advised us that they would have had to take time off to attend fracture clinic with their child. Two patients attended their general practitioner (GP) or ED to seek further pain relief following their injuries. Only one patient reported a poor clinical outcome. Nine (28%) patients reported that they would have preferred a face-to-face appointment rather than being treated by the VFC. CONCLUSION: Virtual review of orthopaedic trauma patients results in satisfactory patient outcomes. Clinical outcomes were acceptable with minimal additional medical attention required following injury. Electronic transfer of information allows for the virtual service to operate from sites long distances from the primary orthopaedic centre. The NIMIS is a safe and confidential means of collaborating with other institutions and has huge potential in the areas of trauma care delivery, clinical conferencing and other image-based disciplines.

The "National Integrated Medical Imaging System" [NIMIS]-friend, not nimesis!

INTRODUCTION: The "National Integrated Medical Imaging System" or NIMIS went live in 2011 and allows the movement of patient radiology imaging throughout the Irish health system. At the time of its launch, NIMIS was not only going to allow the filmless passage of patient radiology imaging but it was also envisaged that it would act as a medical image archive. The aim of this study was to assess the awareness and use of non-consultant hospital doctors and hospital consultants with regard to this medical image archive/referral function of NIMIS. METHODS: A survey was carried out on 50 doctors across all specialities and grades at Tullamore Hospital looking at different aspects of the use of NIMIS. RESULTS: Ninety-four percent of respondents use NIMIS on a daily basis and 6% use it on a weekly basis. The primary reason for using NIMIS was found to be "Viewing and Ordering Imaging" in 92% of those surveyed with 8% stating it was "Viewing imaging/reports". Ninety-eight percent surveyed said they had never used NIMIS to send a referral form or clinical photograph and 82% were not aware of this potential function. The majority of those surveyed stated that they either agreed or strongly agreed NIMIS is user-friendly. CONCLUSION: NIMIS allows the safe and confidential flow of patient images and clinical information in the Irish health system. It could provide definite potential in the areas of clinical conferencing, multidisciplinary meetings and remote patient assessment along with collaborative research and education.

General practitioner attitudes and experiences of orthopaedic services in the Irish midlands.

INTRODUCTION: Disorders of the musculoskeletal system are the main cause of disability and lost working days worldwide, and osteoarthritis affects almost half a million people in Ireland. Appropriate access and resourcing of general practice and orthopaedics is a necessary measure for the provision of a safe and efficient health service. One area that remains particularly challenging in Ireland is that of outpatient waiting lists, and the purpose of this study was to assess the attitudes and experiences of general practitioners in the Irish midlands with regard to orthopaedic services and to evaluate these in the context of national strategies and international best practice. METHODS: A survey was sent to general practitioners in the midlands looking at five main areas: elective services, trauma services, allied health services, patient access and practice demographics. RESULTS: 98.7% of general practitioners surveyed stated they either agree or strongly agree that there is a significant difference in terms of access between public and private services. The average waiting time for an elective orthopaedic outpatient clinic is more than 1 year as per 92.3% of GPs surveyed with 89.7% of GPs stating that the average waiting time for an elective private outpatient appointment being between 0 and 3 months. Over three quarters of GPs surveyed either disagree or strongly disagree that there is adequate access to physiotherapy and occupational therapy services in the community with nearly 80% and 93.6% stating they have no physiotherapist or occupational therapist respectively attached to their practice. CONCLUSION: MSK disorders are a significant burden on the Irish health service and inadequate investment in general practice, allied health practitioner-led facilities and orthopaedic services remains a serious challenge that requires considerable attention to insure adequate patient care, safety and best practice.

Quantitation of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe.

Target engagement is a key concept in drug discovery and its direct measurement can provide a quantitative understanding of drug efficacy and/or toxicity. Failure to demonstrate target occupancy in relevant cells and tissues has been recognised as a contributing factor to the low success rate of clinical drug development. Several techniques are emerging to quantify target engagement in cells; however, in situ measurements remain challenging, mainly due to technical limitations. Here, we report the development of a non-covalent clickable probe, based on SCH772984, a slow off-rate ERK1/2 inhibitor, which enabled efficient pull down of ERK1/2 protein via click reaction with tetrazine tagged agarose beads. This was used in a competition setting to measure relative target occupancy by selected ERK1/2 inhibitors. As a reference we used the cellular thermal shift assay, a label-free biophysical assay relying solely on ligand-induced thermodynamic stabilization of proteins. To validate the EC50 values measured by both methods, the results were compared with IC50 data for the phosphorylation of RSK, a downstream substrate of ERK1/2 used as a functional biomarker of ERK1/2 inhibition. We showed that a slow off-rate reversible probe can be used to efficiently pull down cellular proteins, significantly extending the potential of the approach beyond the need for covalent or photoaffinity warheads.

Educational impact of joint replacement school for patients undergoing total hip and knee arthroplasty: a prospective cohort study.

INTRODUCTION: Enhanced recovery programmes for hip and knee arthroplasty surgery have been shown to decrease length of stay and improve patient outcomes in the elective setting. There are limited studies looking at pre-operative patient education alone and its role, however, more recent studies have demonstrated that it can help reduce length of stay and health care costs. The elective orthopaedic unit at Tullamore Hospital was the first unit in the Irish public health system to have a joint school, pre-operative, educational session, and the aim of this study was to assess our patient understanding of this session to ensure a sustained, high level of patient care, quality assurance and educational standards. METHODS: A sample size of n = 51 was calculated to adequately power the study. Final sample size was 57. Data was collected across four joint school sessions. Patients were asked to complete a questionnaire before and after the session. RESULTS: Twenty-seven male (47.3%) and 30 female (52.7%) patients completed the surveys. Mean age overall was 64.5 years. All survey questions except those related to anaesthesia and physiotherapy showed a statistically significant improvement after the joint school session. A total knowledge score was calculated and revealed a statistically significant difference between pre- and post-joint school survey answering (P value < 0.001). CONCLUSION: Our joint school, pre-operative educational session remains a very effective way of delivering content to patients regarding their surgery. Continuous auditing is paramount to its success and there is potential scope for web-based learning as an adjunct to this validated session.

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Engineering and purification of a thermostable, high-yield, variant of PfCRT, the Plasmodium falciparum chloroquine resistance transporter.

Historically chloroquine was used to treat the most deadly form of malaria, caused by the parasite Plasmodium falciparum. The selective pressure of chloroquine therapy led to the rapid emergence of chloroquine resistant parasites. Resistance has been attributed to the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT), an integral membrane protein of unknown structure. A PfCRT structure would provide new insights into how the protein confers chloroquine resistance and thereby also yield novel opportunities for developing anti-malarial therapies. Although PfCRT is an attractive target for characterisation and structure determination, very little work has been published on its expression and purification. Here we present a medium throughput protocol, employing Sf9 insect cells, for testing the expression, stability and purification yield of rationally designed PfCRT mutant constructs and constructs of a PfCRT orthologue from Neospora caninum (NcCRT). We have identified a conserved cysteine residue in PfCRT that results in elevated protein stability when mutated. Combining this mutation with the insertion of T4-lysozyme into a specific surface loop further augments PfCRT protein yield and thermostability. Screening also identified an NcCRT construct with an elevated purification yield. Furthermore it was possible to purify both PfCRT and NcCRT constructs at milligram-scales, with high purities and with size exclusion chromatography profiles that were consistent with monodispersed, homogeneous protein.

In-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor.

In-gel activity-based protein profiling (ABPP) offers rapid assessment of the proteome-wide selectivity and target engagement of a chemical tool. Here we demonstrate the use of the inverse electron demand Diels Alder (IEDDA) click reaction for in-gel ABPP by evaluating the selectivity profile and target engagement of a covalent ERK1/2 probe tagged with a trans-cyclooctene group. The chemical probe was shown to bind covalently to Cys166 of ERK2 using protein MS and X-ray crystallography, and displayed submicromolar GI50s in A375 and HCT116 cells. In both cell lines, the probe demonstrated target engagement and a good selectivity profile at low concentrations, which was lost at higher concentrations. The IEDDA cycloaddition enabled fast and quantitative fluorescent tagging for readout with a high background-to-noise ratio and thereby provides a promising alternative to the commonly used copper catalysed alkyne-azide cycloaddition.

Fragment-based approaches to the discovery of kinase inhibitors.

Protein kinases are one of the most important families of drug targets, and aberrant kinase activity has been linked to a large number of disease areas. Although eminently targetable using small molecules, kinases present a number of challenges as drug targets, not least obtaining selectivity across such a large and relatively closely related target family. Fragment-based drug discovery involves screening simple, low-molecular weight compounds to generate initial hits against a target. These hits are then optimized to more potent compounds via medicinal chemistry, usually facilitated by structural biology. Here, we will present a number of recent examples of fragment-based approaches to the discovery of kinase inhibitors, detailing the construction of fragment-screening libraries, the identification and validation of fragment hits, and their optimization into potent and selective lead compounds. The advantages of fragment-based methodologies will be discussed, along with some of the challenges associated with using this route. Finally, we will present a number of key lessons derived both from our own experience running fragment screens against kinases and from a large number of published studies.

Pharmacologic activation of PKM2 slows lung tumor xenograft growth.

Inactivation of the M2 form of pyruvate kinase (PKM2) in cancer cells is associated with increased tumorigenicity. To test the hypothesis that tumor growth may be inhibited through the PKM2 pathway, we generated a series of small-molecule PKM2 activators. The compounds exhibited low nanomolar activity in both biochemical and cell-based PKM2 activity assays. These compounds did not affect the growth of cancer cell lines under normal conditions in vitro, but strongly inhibited the proliferation of multiple lung cancer cell lines when serine was absent from the cell culture media. In addition, PKM2 activators inhibited the growth of an aggressive lung adenocarcinoma xenograft. These findings show that PKM2 activation by small molecules influences the growth of cancer cells in vitro and in vivo, and suggest that such compounds may augment cancer therapies.