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.

Background: Altiratinib is a Type II switch pocket inhibitor of MET kinase as well as TIE2 and VEGFR2 kinases. This profile provides an agent that exhibits anti-tumor activity in cancers driven by MET overexpression or genomic mutation, and also blocks angiogenic and metastatic processes mediated by the tumor microenvironment. Material and Methods: Altiratinib was evaluated in MET, TIE2, and VEGFR2 biochemical studies, including evaluation in a number of MET activation mutants. Cellular activity was evaluated in tumor cell lines exhibiting MET amplification (MKN-45 gastric, EBC-1 NSCLC) or overexpression (B16/F10 melanoma; U87 glioblastoma). In vivo pharmacokinetic/pharmacodynamic studies were performed in an MKN45 xenograft model. Efficacy was demonstrated in a battery of xenograft/ allograft models including: gastric (MKN-45), melanoma (B16/F10, A375), ovarian (SKOV-3), colorectal (COLO-205), lung (EBC-1), breast (PyMT), and glioblastoma (U87). Results: Altiratinib afforded balanced inhibition of MET, TIE2, and VEGFR2 kinases in the low nM range (IC50s 2−9 nM) and blocked HGF-, ANG-, or VEGFA-induced HUVEC activation and capillary tube formation. Altiratinib retained potency versus activation loop mutant forms of MET (D1228X, Y1230X, M1250X), inhibiting all forms with IC50 24 hr after a single 10mg/kg oral dose in an MKN-45 xenograft pharmacodynamic model. Altiratinib exhibited anti-tumor activity in melanoma (B16/F10, A375), gastric (MKN-45), lung (EBC-1), colorectal (COLO-205), breast (PyMT), ovarian (SKOV-3) and GBM (U87) xenograft or allograft models. In these in vivo studies, altiratinib was shown to inhibit tumor growth, angiogenesis, invasion and/or metastasis. Altiratinib also blocked recruitment of TIE2-expressing monocytes in the PyMT breast cancer model. In some models, altiratinib was shown to increase overall survival. In particular, in the i.c.v. orthotopically implanted U87 glioblastoma model, altiratinib extended survival by 1.7-fold vs vehicle (112 days vs 66 days), while the combination of altiratinib + bevacizumab extended survival by 2.5 fold vs vehicle and by 1.9-fold vs bevacizumab single agent (166 days vs. 88 days). Conclusions: Altiratinib is a balanced inhibitor of MET, TIE2, and VEGFR2 kinases. This profile provides robust inhibition of tumors driven by MET amplification or overexpression, and also provides the potential for altiratinib to block tumor microenvironment angiogenic resistance mechanisms and pro-tumoral effects of TIE2-expressing macrophages in the clinical setting. Altiratinib is currently in Phase 1 clinical trials in patients with solid tumors.

Abstract Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFR) signalling as being key to the molecular pathology of cancer. A fragment screening campaign was conducted against the tyrosine kinase domain of FGFR1 to detect low molecular weight compounds that bound to the hinge region of the kinase. The screening produced several fragment inhibitors (molecular weight <250 Da) in the micromolar range and their binding modes were confirmed by X-ray crystallography. We selected an imidazo[1,2-a]pyridine fragment that was 120 uM versus FGFR3 in the kinase inhibition bioassay. Subsequently, in the fragments-to-leads stage a detailed structural understanding of the binding interactions between the fragment and its protein kinase target, using X-ray crystallography, led to the identification of a 0.003 uM inhibitor of FGFR3 in the kinase bioassay, with significant selectivity versus VEGFR2 and FLT3. The poster will focus on the description of previously undescribed compounds bearing an imidazo[1,2-a]pyridine core scaffold where selectivity versus other protein kinases, for example FLT3, is obtained using the X-ray crystal structure and structure-based design. In summary we will illustrate how X-ray crystallography and fragment-based drug design (FBDD) can be used to discover compounds with activity in an FGFR driven xenograft model when dosed by the oral route. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1361. doi:10.1158/1538-7445.AM2011-1361

Abstract Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFr) signalling as being key to the molecular pathology of cancer. This poster will describe fragment based drug discovery using biophysical screening to identify initial fragments. Subsequently, in the fragments-to-leads stage a detailed structural understanding of the binding interactions between the fragment and its target protein utilised X-ray crystallography and NMR. Starting with different fragments allows several lead series to be identified, often by synthesizing only small numbers of compounds. A fragment screening campaign was conducted against the FGFr-1 to detect very low molecular weight compounds that bound to the hinge region of the kinase. The screening produced several fragment molecules (Molecular Weight <250 Da) which were in the micromolar range and confirmed binding mode in X-ray crystallography. One X-ray hit series that was 120 uM verse FGFr-3 will be described. Several iterations of structure-guided medicinal chemistry led to the identification of a lead compound with 3 nM affinity for FGFr-3, good cell activity and 30-fold selectivity verse VEGFr-2 with good oral activity. The lead was optimised to afford a compound that showed good PK/PD and efficacy. This poster represents first disclosure of the structure of the lead series and illustrates how a fragment-based drug discovery approach can be efficiently used to discover compounds advanced nanomolar compounds with oral bioavailability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5778.

The combined use of a rapid virtual screen of a small fragment library together with a single point enzyme assay has been used for the discovery of novel PNP inhibitors. The availability of readily soakable crystals of bovine PNP has allowed the approach to be experimentally validated by determining the crystal structure of one of the inhibitor-PNP complexes. Comparison of the experimentally determined binding mode with that predicted by the virtual screening shows them to be similar. This represents a starting point for the growth of the ligand into a higher affinity inhibitor.

Fragment screening offers an alternative to traditional screening for discovering new leads in drug discovery programs. This paper describes a fragment screening methodology based on high throughput X-ray crystallography. The method is illustrated against five proteins (p38 MAP kinase, CDK2, thrombin, ribonuclease A, and PTP1B). The fragments identified have weak potency (>100 microM) but are efficient binders relative to their size and may therefore represent suitable starting points for evolution to good quality lead compounds. The examples illustrate that a range of molecular interactions (i.e., lipophilic, charge-charge, neutral hydrogen bonds) can drive fragment binding and also that fragments can induce protein movement. We believe that the method has great potential for the discovery of novel lead compounds against a range of targets, and the companion paper illustrates how lead compounds have been identified for p38 MAP kinase starting from fragments such as those described in this paper.