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.

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.

Computerized Tailored Interventions to Enhance Prevention and Screening for Hepatitis C Virus Among People Who Inject Drugs: Protocol for a Randomized Pilot Study.

BACKGROUND: Hepatitis C virus (HCV) infection is a growing problem among people who inject drugs. Strategies to reduce disease transmission (eg, syringe exchange programs) and facilitate HCV screening and linkage are available but are under-utilized in many communities affected by injection drug use. Novel approaches to increasing the use of these strategies are needed. OBJECTIVE: The goals of this project are to (1) develop and pilot test a computerized tailored intervention for increasing HCV screening and decreasing risky drug use behavior among people who inject drugs and (2) determine the feasibility of disseminating such an intervention using peer-based referrals in the setting of a community-based syringe exchange program. METHODS: This 2-arm, randomized pilot study is being conducted in a large-volume, multisite syringe exchange program in southern Wisconsin. A social network-based strategy was used to recruit a total of 235 adults who reported past-month injection of opioids, cocaine, or methamphetamine. Network recruiters were identified among clients requesting services from the syringe exchange program and were enlisted to refer eligible peers to the study. All participants completed a computer-adapted questionnaire eliciting information about risk behaviors and their knowledge, attitudes, and prior experiences related to HCV screening. Subjects were then randomly assigned to receive usual care, consisting of standard counseling by syringe exchange staff, or the Hep-Net intervention, which provides algorithm-based, real-time tailored feedback and recommendations for behavior change in the style of motivational interviewing. Changes in drug use behaviors and attitudes will be assessed during a second session between 90 and 180 days after the baseline visit. Frequency of repeat HCV testing and HCV incidence will be assessed through a database search 1 year after study completion. RESULTS: Recruitment for this study was completed in April 2015. Follow-up of enrolled participants is expected to continue until March 2016. Network recruiters were enrolled who referred a total of 195 eligible peers (overall N=235). At baseline, the median age was 34 years; 41.3% (97/235) were non-white; and 86.4% (203/235) reported predominantly injecting heroin. Most participants (161/234, 68.8%) reported sharing injection equipment in the past and of these, 30.4% (49/161) had never been tested for HCV. CONCLUSIONS: This study will provide preliminary evidence to determine whether incorporating computerized behavioral interventions into existing prevention services at syringe exchange programs can lead to adoption of healthier behaviors. TRIAL REGISTRATION: ClinicalTrials.gov NCT02474043; https://clinicaltrials.gov/ct2/show/NCT02474043 (Archived by WebCite at http://www.webcitation.org/6dbjUQG7J).

A practical approach to glare assessment for train cabs.

The assessment of glare is a key consideration in the design of a railway driver's cab. However, unlike assessment of other factors, such as forward visibility, there are no standardised approaches for performing assessments of glare. This paper describes an approach for assessing the impact of glare in a full size mock-up of a railway cab. While it is unrealistic to evaluate every possible lighting condition that may potentially occur in the vehicle cab in service, a pragmatic and practical approach is taken to provide a good level of indicative information about the cab design's likely glare performance. This involves assessing internal light sources, such as internal lights and illuminated controls, and simulating external light sources (e.g. the sun, other trains' headlights) by illuminating the cab mock up windscreen, side and door windows with a single light source manually located in a sequence of discrete positions and orientations and assessing the resulting glare impacts. The paper describes a structured process for assessing and recording the impact of glare and recommending mitigations.