The cost of diagnostic uncertainty: a prospective economic analysis of febrile children attending an NHS emergency department.

BACKGROUND: Paediatric fever is a common cause of emergency department (ED) attendance. A lack of prompt and definitive diagnostics makes it difficult to distinguish viral from potentially life-threatening bacterial causes, necessitating a cautious approach. This may result in extended periods of observation, additional radiography, and the precautionary use of antibiotics (ABs) prior to evidence of bacterial foci. This study examines resource use, service costs, and health outcomes. METHODS: We studied an all-year prospective, comprehensive, and representative cohort of 6518 febrile children (aged < 16 years), attending Alder Hey Children's Hospital, an NHS-affiliated paediatric care provider in the North West of England, over a 1-year period. Performing a time-driven and activity-based micro-costing, we estimated the economic impact of managing paediatric febrile illness, with focus on nurse/clinician time, investigations, radiography, and inpatient stay. Using bootstrapped generalised linear modelling (GLM, gamma, log), we identified the patient and healthcare provider characteristics associated with increased resource use, applying retrospective case-note identification to determine rates of potentially avoidable AB prescribing. RESULTS: Infants aged less than 3 months incurred significantly higher resource use than any other age group, at £1000.28 [95% CI £82.39-£2993.37] per child, (p < 0.001), while lesser experienced doctors exhibited 3.2-fold [95% CI 2.0-5.1-fold] higher resource use than consultants (p < 0.001). Approximately 32.4% of febrile children received antibiotics, and 7.1% were diagnosed with bacterial infections. Children with viral illnesses for whom antibiotic prescription was potentially avoidable incurred 9.9-fold [95% CI 6.5-13.2-fold] cost increases compared to those not receiving antibiotics, equal to an additional £1352.10 per child, predominantly resulting from a 53.9-h increase in observation and inpatient stay (57.1 vs. 3.2 h). Bootstrapped GLM suggested that infants aged below 3 months and those prompting a respiratory rate 'red flag', treatment by lesser experienced doctors, and Manchester Triage System (MTS) yellow or higher were statistically significant predictors of higher resource use in 100% of bootstrap simulations. CONCLUSION: The economic impact of diagnostic uncertainty when managing paediatric febrile illness is significant, and the precautionary use of antibiotics is strongly associated with increased costs. The use of ED resources is highest among infants (aged less than 3 months) and those infants managed by lesser experienced doctors, independent of clinical severity. Diagnostic advances which could increase confidence to withhold antibiotics may yield considerable efficiency gains in these groups, where the perceived risks of failing to identify potentially life-threatening bacterial infections are greatest.

A covalent fragment-based strategy targeting a novel cysteine to inhibit activity of mutant EGFR kinase.

Several generations of ATP-competitive anti-cancer drugs that inhibit the activity of the intracellular kinase domain of the epidermal growth factor receptor (EGFR) have been developed over the past twenty years. The first-generation of drugs such as gefitinib bind reversibly and were followed by a second-generation such as dacomitinib that harbor an acrylamide moiety that forms a covalent bond with C797 in the ATP binding pocket. Resistance emerges through mutation of the T790 gatekeeper residue to methionine, which introduces steric hindrance to drug binding and increases the Km for ATP. A third generation of drugs, such as osimertinib were developed which were effective against T790M EGFR in which an acrylamide moiety forms a covalent bond with C797, although resistance has emerged by mutation to S797. A fragment-based screen to identify new starting points for an EGFR inhibitor serendipitously identified a fragment that reacted with C775, a previously unexploited residue in the ATP binding pocket for a covalent inhibitor to target. A number of acrylamide containing fragments were identified that selectively reacted with C775. One of these acrylamides was optimized to a highly selective inhibitor with sub-1 µM activity, that is active against T790M, C797S mutant EGFR independent of ATP concentration, providing a potential new strategy for pan-EGFR mutant inhibition.

PAC-FragmentDEL - photoactivated covalent capture of DNA-encoded fragments for hit discovery.

We describe a novel approach for screening fragments against a protein that combines the sensitivity of DNA-encoded library technology with the ability of fragments to explore what will bind. Each of the members of the library consists of a fragment which is linked to a photoactivatable diazirine moiety. Split and pool synthesis combines each fragment with a set of linkers with the version of the library reported here containing some 70k different compounds, each with an individual DNA code. Incubation of the library with a protein sample is followed by photoactivation, washing and subsequent PCR and sequencing which allows the individual fragment hits to be identified. We illustrate how the approach allows successful hit fragment identification using only microgram quantities of material for two targets. PAK4 is a kinase for which conventional fragment screening has generated many advance leads. The as yet undrugged target, 2-epimerase, presents a more challenging active site for identification of hit compounds. In both cases, PAC-FragmentDEL identified fragments validated as hits by ligand-observed NMR measurements and crystal structure determination of off-DNA sample binding to the proteins.

Shielded hydrogen bonds as structural determinants of binding kinetics: application in drug design.

Time scale control of molecular interactions is an essential part of biochemical systems, but very little is known about the structural factors governing the kinetics of molecular recognition. In drug design, the lifetime of drug-target complexes is a major determinant of pharmacological effects but the absence of structure-kinetic relationships precludes rational optimization of this property. Here we show that almost buried polar atoms--a common feature on protein binding sites--tend to form hydrogen bonds that are shielded from water. Formation and rupture of this type of hydrogen bonds involves an energetically penalized transition state because it occurs asynchronously with dehydration/rehydration. In consequence, water-shielded hydrogen bonds are exchanged at slower rates. Occurrence of this phenomenon can be anticipated from simple structural analysis, affording a novel tool to interpret and predict structure-kinetics relationships. The validity of this principle has been investigated on two pairs of Hsp90 inhibitors for which detailed thermodynamic and kinetic data has been experimentally determined. The agreement between macroscopic observables and molecular simulations confirms the role of water-shielded hydrogen bonds as kinetic traps and illustrates how our finding could be used as an aid in structure-based drug discovery.

The Effect of Core Replacement on S64315, a Selective MCL-1 Inhibitor, and Its Analogues.

Following the identification of thieno[2,3-d]pyrimidine-based selective and potent inhibitors of MCL-1, we explored the effect of core swapping at different levels of advancement. During hit-to-lead optimization, X-ray-guided S-N replacement in the core provided a new vector, whose exploration led to the opening of the so-called deep-S2 pocket of MCL-1. Unfortunately, the occupation of this region led to a plateau in affinity and had to be abandoned. As the project approached selection of a clinical candidate, a series of core swap analogues were also prepared. The affinity and cellular activity of these compounds showed a significant dependence on the core structure. In certain cases, we also observed an increased and accelerated epimerization of the atropoisomers. The most potent core replacement analogues showed considerable in vivo PD response. One compound was progressed into efficacy studies and inhibited tumor growth.

Fragment-Derived Selective Inhibitors of Dual-Specificity Kinases DYRK1A and DYRK1B.

The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down's syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit. We have used fragment and structure-based discovery methods to identify a highly selective, well-tolerated, brain-penetrant DYRK1A inhibitor which showed in vivo activity in a tumor model. The inhibitor provides a useful tool compound for further exploration of the effect of DYRK1A inhibition in models of disease.

Discovery of cell-active phenyl-imidazole Pin1 inhibitors by structure-guided fragment evolution.

Pin1 is an emerging oncology target strongly implicated in Ras and ErbB2-mediated tumourigenesis. Pin1 isomerizes bonds linking phospho-serine/threonine moieties to proline enabling it to play a key role in proline-directed kinase signalling. Here we report a novel series of Pin1 inhibitors based on a phenyl imidazole acid core that contains sub-µM inhibitors. Compounds have been identified that block prostate cancer cell growth under conditions where Pin1 is essential.

Structure-guided design of alpha-amino acid-derived Pin1 inhibitors.

The peptidyl prolyl cis/trans isomerase Pin1 is a promising molecular target for anti-cancer therapeutics. Here we report the structure-guided evolution of an indole 2-carboxylic acid fragment hit into a series of alpha-benzimidazolyl-substituted amino acids. Examples inhibited Pin1 activity with IC(50) <100nM, but were inactive on cells. Replacement of the benzimidazole ring with a naphthyl group resulted in a 10-50-fold loss in ligand potency, but these examples downregulated biomarkers of Pin1 activity and blocked proliferation of PC3 cells.

Rapid optimisation of fragments and hits to lead compounds from screening of crude reaction mixtures.

Fragment based methods are now widely used to identify starting points in drug discovery and generation of tools for chemical biology. A significant challenge is optimization of these weak binding fragments to hit and lead compounds. We have developed an approach where individual reaction mixtures of analogues of hits can be evaluated without purification of the product. Here, we describe experiments to optimise the processes and then assess such mixtures in the high throughput crystal structure determination facility, XChem. Diffraction data for crystals of the proteins Hsp90 and PDHK2 soaked individually with 83 crude reaction mixtures are analysed manually or with the automated XChem procedures. The results of structural analysis are compared with binding measurements from other biophysical techniques. This approach can transform early hit to lead optimisation and the lessons learnt from this study provide a protocol that can be used by the community.

Establishing Drug Discovery and Identification of Hit Series for the Anti-apoptotic Proteins, Bcl-2 and Mcl-1.

We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods. Fragment screening using ligand-observed NMR experiments identified several series of compounds for each protein. The series were assessed for their potential for subsequent optimization using 1H and 15N heteronuclear single-quantum correlation NMR, surface plasmon resonance, and isothermal titration calorimetry measurements to characterize and validate binding. Crystal structures could not be determined for the early hits, so NMR methods were developed to provide models of compound binding to guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine series was optimized to a K d of 40 µM before a thienopyrimidine hit series was identified which subsequently led to the lead series from which the clinical candidate S 64315 (MIK 665) was identified. For Bcl-2, the fragment-derived series were difficult to progress, and a compound derived from a published tetrahydroquinone compound was taken forward as the hit from which the clinical candidate (S 55746) was obtained. For both the proteins, the work to establish a portfolio of assays gave confidence for identification of compounds suitable for optimization.

Interactions of designer antibiotics and the bacterial ribosomal aminoacyl-tRNA site.

The X-ray crystal structures for the complexes of three designer antibiotics, compounds 1, 2, and 3, bound to two models for the ribosomal aminoacyl-tRNA site (A site) at 2.5-3.0 Angstroms resolution and that of neamine at 2.8 Angstroms resolution are described. Furthermore, the complex of antibiotic 1 bound to the A site in the entire 30S ribosomal subunit of Thermus thermophilus is reported at 3.8 Angstroms resolution. Molecular dynamics simulations revealed that the designer compounds provide additional stability to bases A1492 and A1493 in their extrahelical forms. Snapshots from the simulations were used for free energy calculations, which revealed that van der Waals and hydrophobic effects were the driving forces behind the binding of designer antibiotic 3 when compared to the parental neamine.

Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: role of the number of rings and positive charges in the specific binding leading to miscoding.

The crystal structures of six complexes between aminoglycoside antibiotics (neamine, gentamicin C1A, kanamycin A, ribostamycin, lividomycin A and neomycin B) and oligonucleotides containing the decoding A site of bacterial ribosomes are reported at resolutions between 2.2 and 3.0 A. Although the number of contacts between the RNA and the aminoglycosides varies between 20 and 31, up to eight direct hydrogen bonds between rings I and II of the neamine moiety are conserved in the observed complexes. The puckered sugar ring I is inserted into the A site helix by stacking against G1491 and forms a pseudo base pair with two H-bonds to the Watson-Crick sites of the universally conserved A1408. This central interaction helps to maintain A1492 and A1493 in a bulged-out conformation. All these structures of the minimal A site RNA complexed to various aminoglycosides display crystal packings with intermolecular contacts between the bulging A1492 and A1493 and the shallow/minor groove of Watson-Crick pairs in a neighbouring helix. In one crystal, one empty A site is observed. In two crystals, two aminoglycosides are bound to the same A site with one bound specifically and the other bound in various ways in the deep/major groove at the edge of the A sites.

Design of Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors Using a Crystallographic Surrogate Derived from Checkpoint Kinase 1 (CHK1).

Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson's disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LRRK2-pSer935 IC50 values for 22 in mouse brain and kidney being 1.3 and 5 nM, respectively.

Application of Off-Rate Screening in the Identification of Novel Pan-Isoform Inhibitors of Pyruvate Dehydrogenase Kinase.

Libraries of nonpurified resorcinol amide derivatives were screened by surface plasmon resonance (SPR) to determine the binding dissociation constant (off-rate, kd) for compounds binding to the pyruvate dehydrogenase kinase (PDHK) enzyme. Parallel off-rate measurements against HSP90 and application of structure-based drug design enabled rapid hit to lead progression in a program to identify pan-isoform ATP-competitive inhibitors of PDHK. Lead optimization identified selective sub-100-nM inhibitors of the enzyme which significantly reduced phosphorylation of the E1α subunit in the PC3 cancer cell line in vitro.

Crystal structure of the bacterial ribosomal decoding site complexed with amikacin containing the gamma-amino-alpha-hydroxybutyryl (haba) group.

Amikacin is the 4,6-linked aminoglycoside modified at position N1 of the 2-deoxystreptamine ring (ring II) by the L-haba group. In the present study, the crystal structure of a complex between oligonucleotide containing the bacterial ribosomal A site and amikacin has been solved at 2.7 A resolution. Amikacin specifically binds to the A site in practically the same way as its parent compound kanamycin. In addition, the L-haba group interacts with the upper side of the A site through two direct contacts, O2*...H-N4(C1496) and N4*-H...O6(G1497). The present crystal structure shows how the introduction of the L-haba group on ring II of aminoglycoside is an effective mutation for obtaining a higher affinity to the bacterial A site.

A helical twist-induced conformational switch activates cleavage in the hammerhead ribozyme.

We have captured the structure of a pre-catalytic conformational intermediate of the hammerhead ribozyme using a phosphodiester tether formed between I and Stem II. This phosphodiester tether appears to mimic interactions in the wild-type hammerhead RNA that enable switching between nuclease and ligase activities, both of which are required in the replicative cycles of the satellite RNA viruses from which the hammerhead ribozyme is derived. The structure of this conformational intermediate reveals how the attacking nucleophile is positioned prior to cleavage, and demonstrates how restricting the ability of Stem I to rotate about its helical axis, via interactions with Stem II, can inhibit cleavage. Analogous covalent crosslinking experiments have demonstrated that imposing such restrictions on interhelical movement can change the hammerhead ribozyme from a nuclease to a ligase. Taken together, these results permit us to suggest that switching between ligase and nuclease activity is determined by the helical orientation of Stem I relative to Stem II.

A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction.

We have investigated the chemical basis for a previously observed 7.8 A conformational change in the hammerhead ribozyme that positions the substrate for in-line attack. We have found that the conformational change can only be observed at or above pH 8.5 (in the presence of Co(2+)) and requires the presence of an ionizable 2'-OH at the cleavage site, and note that this observed apparent pK(a) of 8.5 for the conformational change is within experimental error (+/-0.5) of the previously reported apparent kinetic pK(a) of 8.5 for the hammerhead ribozyme in the presence of Co(2+). We have solved two crystal structures of hammerhead ribozymes having 2'-OCH(3) or 2'-F substitutions at the cleavage site and have found that these will not undergo a conformational change equivalent to that observed for the hammerhead ribozyme having an unmodified attacking nucleophile under otherwise identical conditions. We have also characterized the kinetics of cleavage in the crystal. In addition to verifying that the particular sequence of RNA that we crystallized cleaves faster in the crystal than in solution, we also find that the extent of cleavage in the crystal is complete, unlike in solution where this and most other hammerhead ribozyme substrates are cleaved only to about 70 % completion. The initial cleavage rate in the crystal obeys the expected log-linear relation between cleavage-rate and pH with a slope of 0.7, as has been observed for other hammerhead ribozyme sequences in solution, indicating that in both the crystal and in solution the pH-dependent step is rate-limiting. However, the cleavage rate in the crystal is biphasic, with the most dramatic distinction between initial (slower) and final (faster) phases appearing at pH 6.0. The initial phase corresponds to the pH-dependent cleavage rate observed in solution, but the second, faster phase is roughly pH-independent and closely parallels the cleavage rate observed at pH 8 (0.4/minute). This result is particularly remarkable because it entails that the rapidly cleaving phase at pH 6 is comparable to the cleavage rate for the fastest cleaving hammerhead ribozymes at pH 6. Based upon these observations, we conclude that the pH-dependent conformational change is the rate-determining step under standard conditions for the hammerhead ribozyme self-cleavage reaction, and that an ionizable 2'-proton at cleavage site is required for this conformational change. We further hypothesize that deprotonation of the cleavage-site 2'-oxygen drives this conformational change.

Off-rate screening (ORS) by surface plasmon resonance. An efficient method to kinetically sample hit to lead chemical space from unpurified reaction products.

The dissociation rate constant kd (off-rate) is the component of ligand-protein binding with the most significant potential to enhance compound potency. Here we provide theoretical and empirical data to show that this parameter can be determined accurately from unpurified reaction products containing designed test compounds. This screening protocol is amenable to parallel chemistry, provides efficiencies of time and materials, and complements existing methodologies for the hit-to-lead phase in fragment-based drug discovery.

Experiences in fragment-based lead discovery.

This chapter summarizes the experience at Vernalis over the past decade in developing and applying fragment-based discovery methods across a range of different targets. The emphasis will be on the practical aspects of the different biophysical techniques (surface plasmon resonance (SPR), differential scanning fluorimetry (DSF), isothermal titration calorimetry, nuclear magnetic resonance, and X-ray crystallography) that can be used to identify fragments that bind to targets and a discussion of the criteria and strategies for selecting and evolving fragments to lead compounds.

Adenosine-derived inhibitors of 78 kDa glucose regulated protein (Grp78) ATPase: insights into isoform selectivity.

78 kDa glucose-regulated protein (Grp78) is a heat shock protein (HSP) involved in protein folding that plays a role in cancer cell proliferation. Binding of adenosine-derived inhibitors to Grp78 was characterized by surface plasmon resonance and isothermal titration calorimetry. The most potent compounds were 13 (VER-155008) with K(D) = 80 nM and 14 with K(D) = 60 nM. X-ray crystal structures of Grp78 bound to ATP, ADPnP, and adenosine derivative 10 revealed differences in the binding site between Grp78 and homologous proteins.