Development of selective FLT3 inhibitors

FLT3-summarySummary

Vichem offers a compound family which selectively targets the FMS like tyrosine kinase 3 (FLT3). We are confident that this compound family can be developed into an effective drug for FLT3(ITD) positive acute myeloid leukemia (AML). Other cancer types identified with FLT3 driver mutations could also be treated with an FLT3 inhibitor.

Biological significance

AML causes high mortality comparing to other leukemia types. According to the National Cancer Institute, 45394 patients died of acute myeloid leukemia in the U.S. between 2007-2011.

It was observed that in the malignant tumors of the hematopoietic system, especially in AML (~70-100%), overexpression of FLT3 kinase is particularly common, moreover FLT3 it is the most important driver gene in AML.

FLT3 is a cytokine receptor and belongs to the family of the receptor tyrosine kinase class III. It plays an essential role in the the normal development of haematopoietic stem cells and progenitor cells. The activated FLT3 kinase uses several signal transduction pathways to transmit the information towards the nucleus.

The most common mutations of FLT3 are the internal tandem duplication (ITD) and the D835 point mutation.

ID FLT3(ITD)
inhibition (%)
at 10 µM
FLT3(ITD)
IC50 (µM)
MV4-11 cells
IC50 (µM)
VCC485702 86.1 0.76 0.32
VCC986749 92.3 0.55 0.25
VCC217510 99.8 0.20 0.15
VCC344049 95.4 0.65 ND
VCC282750 96.1 0.33 0.2
VCC376116 79.2 0.91 0.44
VCC904227 82.1 0.61 0.38
VCC515162 89.9 0.41 0.03
VCC788082 94.4 0.87 0.1
VCC997912 91 0.24 0.01
VCC319860 92.1 0.44 ND
VCC501244 100 0.58 ND
VCC145108 100 0.87 ND
VCC350485 92.4 2.01 2.34

The compound family

We developed a synthetic route and prepared 124 novel, potential inhibitors.

Biological results

Structure-activity relationships and enzyme inhibitions were determined using in-house IMAP biochemical assay.

Our lead compounds inhibit FLT3(ITD) with IC50 = 0.2 – 0.9 µM in biochemical enzymatic assays and show promising results on MV4-11 acute myeloid leukemia cell line. Notably, MV4-11 cell line contains the FLT3(ITD) mutation and increased expression is also manifested.

We checked the selectivity against wild type FLT3 as well. The compounds showed less activity on wild type FLT3 which is important to prevent the later side effects.

We used flow cytometry (FACS) to determine the cell death process. The results revealed that our inhibitors induce programmed cell death (apoptosis) instead of necrosis.

Solubility and permeability of the compounds

The kinetic solubility of the compounds was measured in two pH conditions using HPLC.

ID Solubility at pH=7.4 (µM) Solubility at pH=2.0 (µM)
VCC423492 110 88
VCC430361 119 94
VCC350485 96 96
VCC868837 113 103
VCC612359 66 104
VCC247875 114 110
VCC376116 56 113
VCC465259 114 96
VCC811647 119 >120
VCC485702 10 >120
VCC490798 107 >120
VCC975648 58 >120
VCC986749 116 119
VCC217510 108 >120
ID Solubility at pH=7.4 (µM) Solubility  at pH=2.0 (µM)
VCC344049 93 >120
VCC721842 74 >120
VCC904227 8 93
VCC282750 119 >120
VCC211167 23 102
VCC515162 12 110
VCC115373 18 104
VCC492193 107 113
VCC501244 5 118
VCC145108 36 >120
VCC788082 23 111
VCC151908 25 113
VCC997912 8 112
VCC319860 12 115

With the permeability assay (PAMPA) we determined that the compounds have a good penetration value (9-16×10-6 cm/s and can cross the cell membranes with passive diffusion.

Selectivity of compound VCC350485 at 5 µM

Kinase Group Family Displacement (%)
FLT3 TK PDGFR 81
FLT3(D835H) TK PDGFR 97
FLT3(D835Y) TK PDGFR 95
FLT3(ITD) TK PDGFR 96
FLT3(K663Q) TK PDGFR 80
FLT3(N841I) TK PDGFR 95
CSNK1A1 CK1 CK1 77
RIOK1 Atypical RIO 63
GAK Other NAK 65
VCC350485-selectivity

The selectivity illustrated on the kinase tree. Target proteins are shown on Figure A and B.

Objectives

Development of a new FLT3 selective drug for the treatment of acute myeloid leukemia. Our in-house developed molecules inhibit both the FLT3(ITD) and FLT3(D835) mutants and show promising selectivity against to other protein kinases.

Keys to success

Although the mostly selective FLT3 inhibitor quizartinib showed high effect in clinical trials, its application is limited due to the emerging resistance. An additional mutation (D835 point mutation) occurs after treatment and this new mutation prevent the binding of quizartinib to the protein. The other drug candidates under clinical trials cause too many side-effects due to their moderate selectivity not to mention their poor pharmacokinetic properties.

Moreover, the ITD mutation was determined as a driver mutation in AML which showed the worst prognosis considering drug resistance and survival. Therefore there is a high demand on the market for a selective FLT3 inhibitor.

Current project status and pipeline

FLT3-pipelineThe project time frame can be approximately 18-24 months.

Project activities

  • Design of further FLT3 specific chemical structures
  • In vitro biochemical enzyme activity assays on wild type FLT3, FLT3(ITD) and FLT3(D835) to determine the enzyme inhibitory effect
  • In vitro cellular viability assays for cancer cell lines in which FLT3 is overexpressed and/or mutated and/or amplified
  • Define clear Structure Activity Relationships
  • Conduct and manage early ADMET studies
  • Chemical synthesis of the structures that are already planned or arise during the course of the project based on SAR instructions
  • ADME/Toxicology studies of the most potential lead compounds
  • In vivo efficacy studies using human AML cancer model on mice

Vichem Chemie Ltd. contributes FLT3 kinase inhibitor compounds that have been synthesized using a quinazoline scaffold. Synthesis of further compounds can be planned, according to the activity and pharmacokinetic measurements. It is anticipated that future optimization cycles and detailed ADMET properties determinations will yield drug candidate compounds, which can be used for in vivo animal testing.