Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.

Discovery of Asciminib (ABL001), an Allosteric Inhibitor of the Tyrosine Kinase Activity of BCR-ABL1.

Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) that target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding. We describe the discovery of asciminib (ABL001), the first allosteric BCR-ABL1 inhibitor to reach the clinic. Asciminib binds to the myristate pocket of BCR-ABL1 and maintains activity against TKI-resistant ATP-site mutations. Although resistance can emerge due to myristate-site mutations, these are sensitive to ATP-competitive inhibitors so that combinations of asciminib with ATP-competitive TKIs suppress the emergence of resistance. Fragment-based screening using NMR and X-ray yielded ligands for the myristate pocket. An NMR-based conformational assay guided the transformation of these inactive ligands into ABL1 inhibitors. Further structure-based optimization for potency, physicochemical, pharmacokinetic, and drug-like properties, culminated in asciminib, which is currently undergoing clinical studies in CML patients.

Inhibition of prenylated KRAS in a lipid environment.

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.

Characterization of the mechanism of action of the pan class I PI3K inhibitor NVP-BKM120 across a broad range of concentrations.

The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G(2)-M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-à-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-α-dependent model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 µmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor-bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K.

An in vitro assay to measure targeted drug delivery to bone mineral.

Targeted delivery of drugs to their site of action is a promising strategy to decrease adverse effects and enhance efficacy, but successful applications of this strategy have been scarce. Human bone is a tissue with unique properties due to its high hydroxyapatite mineral content. However, with the exception of bisphosphonates, bone mineral has not been targeted in a successful clinical application of drugs that act on bone, such as anti-resorptive or bone anabolic agents. Herein we present an NMR-based in vitro assay to measure binding affinities of small molecules to hydroxyapatite (HAP) or bone powder. Binding was shown to be specific and competitive, and the assay can be carried out in a direct binding format or in competition mode. A selection of clinically relevant bisphosphonates was ranked by their binding affinity for HAP. The binding affinity decreases in the order: pamidronate > alendronate > zoledronate > risedronate > ibandronate. The differences in binding affinities span a factor of 2.1 between pamidronate and ibandronate, consistent with previous studies. The rank order is very similar with bone powder, although the binding capacity of bone powder is smaller and binding kinetics are slower. A zoledronate derivative that lacks the central hydroxy group binds to HAP with 2.3-fold weaker affinity than zoledronate itself. Any small molecule can be analyzed for its binding to HAP or bone powder, and the binding of common bone-staining agents such as alizarin and its derivatives was confirmed in the new assay. This assay supports a strategy for targeted delivery of drugs to bone by attaching a bone-affinity tag to the active drug substance.