Serine/Threonine Protein Kinases as Attractive Targets for Anti-Cancer Drugs-An Innovative Approach to Ligand Tuning Using Combined Quantum Chemical Calculations, Molecular Docking, Molecular Dynamic Simulations, and Network-like Similarity Graphs.

Serine/threonine protein kinases (CK2, PIM-1, RIO1) are constitutively active, highly conserved, pleiotropic, and multifunctional kinases, which control several signaling pathways and regulate many cellular functions, such as cell activity, survival, proliferation, and apoptosis. Over the past decades, they have gained increasing attention as potential therapeutic targets, ranging from various cancers and neurological, inflammation, and autoimmune disorders to viral diseases, including COVID-19. Despite the accumulation of a vast amount of experimental data, there is still no "recipe" that would facilitate the search for new effective kinase inhibitors. The aim of our study was to develop an effective screening method that would be useful for this purpose. A combination of Density Functional Theory calculations and molecular docking, supplemented with newly developed quantitative methods for the comparison of the binding modes, provided deep insight into the set of desirable properties responsible for their inhibition. The mathematical metrics helped assess the distance between the binding modes, while heatmaps revealed the locations in the ligand that should be modified according to binding site requirements. The Structure-Binding Affinity Index and Structural-Binding Affinity Landscape proposed in this paper helped to measure the extent to which binding affinity is gained or lost in response to a relatively small change in the ligand's structure. The combination of the physico-chemical profile with the aforementioned factors enabled the identification of both "dead" and "promising" search directions. Tests carried out on experimental data have validated and demonstrated the high efficiency of the proposed innovative approach. Our method for quantifying differences between the ligands and their binding capabilities holds promise for guiding future research on new anti-cancer agents.

A review on structure-function mechanism and signaling pathway of serine/threonine protein PIM kinases as a therapeutic target.

The proviral integration for the Moloney murine leukemia virus (PIM) kinases, belonging to serine/threonine kinase family, have been found to be overexpressed in various types of cancers, such as prostate, breast, colon, endometrial, gastric, and pancreatic cancer. The three isoforms PIM kinases i.e., PIM1, PIM2, and PIM3 share a high degree of sequence and structural similarity and phosphorylate substrates controlling tumorigenic phenotypes like proliferation and cell survival. Targeting short-lived PIM kinases presents an intriguing strategy as in vivo knock-down studies result in non-lethal phenotypes, indicating that clinical inhibition of PIM might have fewer adverse effects. The ATP binding site (hinge region) possesses distinctive attributes, which led to the development of novel small molecule scaffolds that target either one or all three PIM isoforms. Machine learning and structure-based approaches have been at the forefront of developing novel and effective chemical therapeutics against PIM in preclinical and clinical settings, and none have yet received approval for cancer treatment. The stability of PIM isoforms is maintained by PIM kinase activity, which leads to resistance against PIM inhibitors and chemotherapy; thus, to overcome such effects, PIM proteolysis targeting chimeras (PROTACs) are now being developed that specifically degrade PIM proteins. In this review, we recapitulate an overview of the oncogenic functions of PIM kinases, their structure, function, and crucial signaling network in different types of cancer, and the potential of pharmacological small-molecule inhibitors. Further, our comprehensive review also provides valuable insights for developing novel antitumor drugs that specifically target PIM kinases in the future. In conclusion, we provide insights into the benefits of degrading PIM kinases as opposed to blocking their catalytic activity to address the oncogenic potential of PIM kinases.

Imparting aromaticity to 2-pyridone derivatives by O-alkylation resulted in new competitive and non-competitive PIM-1 kinase inhibitors with caspase-activated apoptosis.

New aromatic O-alkyl pyridine derivatives were designed and synthesised as Proviral Integration Moloney (PIM)-1 kinase inhibitors. 4c and 4f showed potent in vitro anticancer activity against NFS-60, HepG-2, PC-3, and Caco-2 cell lines and low toxicity against normal human lung fibroblast Wi-38 cell line. Moreover, 4c and 4f induced apoptosis in the four tested cancer cell lines with high percentage. In addition, 4c and 4f significantly induced caspase 3/7 activation in HepG-2 cell line. Furthermore, 4c and 4f showed potent PIM-1 kinase inhibitory activity with IC50 = 0.110, 0.095 µM, respectively. Kinetic studies indicated that 4c and 4f were both competitive and non-competitive inhibitors for PIM-1 kinase enzyme. In addition, in silico prediction of physiochemical properties, pharmacokinetic profile, ligand efficiency, ligand lipophilic efficiency, and induced fit docking studies were consistent with the biological and kinetic studies, and predicted that 4c and 4f could act as PIM-1 kinase competitive non-adenosine triphosphate (ATP) mimetics with drug like properties.

Stereoselective synthesis of 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-one derivatives as PIM kinase inhibitors inspired from marine alkaloids.

We previously demonstrated that natural product-inspired 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-ones derivatives delivered potent and selective PIM kinases inhibitors however with non-optimal ADME/PK properties and modest oral bioavailability. Herein, we describe a structure-based scaffold decoration and a stereoselective approach to this chemical class. The synthesis, structure-activity relationship studies, chiral analysis, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Compound 20c demonstrated excellent potency on PIM1 and PIM2 with exquisite kinases selectivity and PK properties that efficiently and dose-dependently promoted c-Myc degradation and appear to be promising lead compounds for further development.

Identification of Pim-1 Kinase Inhibitors by Pharmacophore Model, Molecular Docking-based Virtual Screening, and Biological Evaluation.

AIM: This study aimed at screening and development of Pim-1 inhibitors as anticancer agent. BACKGROUND: Pim-1, a member of the Ser/Thr kinase family, plays a crucial role in cell proliferation and is being regarded as a promising target for cancer therapeutics. OBJECTIVE: The present work focused on screening more potent Pim-1 inhibitors by in-silico method and biological evaluation. MATERIALS AND METHODS: To identify more potent Pim-1 inhibitors, a GALAHAD pharmacophore model was constructed based on nine known Pim-1 inhibitors and followed by in silico screening including pharmacophore and molecular docking-based virtual screening. The hit compounds were further assessed the Pim-1, 2, and 3 kinase activities and the anticancer inhibition property against human myeloma RPMI-8226 and U266 cells using cytotoxicity studies. RESULTS: Based on Qfit value (from pharmacophore), docking score and clustering analysis, six compounds including C445_0268, C470_0769, 4456_0744, 0806_0325, G395_1510 and V023_3227 were hit. Binding mode analysis showed that hydrogen bond, hydrophobic and π-π stacking interactions dominated the bindings of these compounds to Pim-1. The further biological evaluation indicated that compounds C445_0268 and C470_0769 possessed excellent pan-Pim kinase activities and inhibited the growths of RPMI-8226 and U266 cell lines with IC50 values lower than 3.75 µM. CONCLUSION: We reported a series of Pim-1 small molecule inhibitors that could serve as the lead compounds to develop new targeted anticancer therapeutics.

Topical advances in PIM kinases and their inhibitors: Medicinal chemistry perspectives.

Cytosolic PIM kinases are the members of serine/ threonine family play a crucial role in the cancer progression and development. Overexpression of PIM kinases is observed in various types of cancers including prostate, hematological, pancreatic, breast carcinoma and likewise. PIM kinases have now been considered as limelight target for the discovery of new molecules as novel anticancer agents as no drug is in the market targeting PIM kinases. In the last two decades, numerous PIM kinase inhibitors have been developed and few of them were in clinical trial phases but could not pass the pipeline of the clinical trials. The present comprehensive review intends to cover biological and the structural aspects of PIM kinases and also medicinal chemistry of PIM inhibitors developed in recent years.

Cryo-EM structure of hexameric yeast Lon protease (PIM1) highlights the importance of conserved structural elements.

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphological perturbations. Although structures of the bacterial and human Lon protease reveal a hexameric assembly, yeast PIM1 was speculated to form a heptameric assembly and is uniquely characterized by a ∼50-residue insertion between the ATPase and protease domains. To further understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-electron microscopy structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrates to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ∼15-residue C-terminal extension. These additional C-terminal residues form an α-helix located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1's enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

A systematic review on active sites and functions of PIM-1 protein.

The Proviral Integration of Molony murine leukemia virus (PIM)-1 protein contributes to the solid cancers and hematologic malignancies, cell growth, proliferation, differentiation, migration, and other life activities. Many studies have related these functions to its molecular structure, subcellular localization and expression level. However, recognition of specific active sites and their effects on the activity of this constitutively active kinase is still a challenge. Based on the close relationship between its molecular structure and functional activity, this review covers the specific residues involved in the binding of ATP and different substrates in its catalytic domain. This review then elaborates on the relevant changes in protein conformation and cell functions after PIM-1 binds to different substrates. Therefore, this intensive study can improve the understanding of PIM-1-regulated signaling pathways by facilitating the discovery of its potential phosphorylation substrates.

Optimization of 4,6-Disubstituted Pyrido[3,2-d]pyrimidines as Dual MNK/PIM Inhibitors to Inhibit Leukemia Cell Growth.

Mitogen-activated protein kinase-interacting kinases (MNKs) and provirus integration in maloney murine leukemia virus kinases (PIMs) are downstream enzymes of cell proliferation signaling pathways associated with the resistance of tyrosine kinase inhibitors. MNKs and PIMs have complementary effects to regulate cap-dependent translation of oncoproteins. Dual inhibitors of MNKs and PIMs have not been developed. We developed a novel 4,6-disubstituted pyrido[3,2-d]pyrimidine compound 21o with selective inhibition of MNKs and PIMs. The IC50's of 21o to inhibit MNK1 and MNK2 are 1 and 7 nM and those to inhibit PIM1, PIM2, and PIM3 are 43, 232, and 774 nM, respectively. 21o inhibits the growth of myeloid leukemia K562 and MOLM-13 cells with GI50's of 2.1 and 1.2 µM, respectively. 21o decreases the levels of p-eIF4E and p-4EBP1, the downstream products of MNKs and PIMs, as well as cap-dependent proteins c-myc, cyclin D1, and Mcl-1. 21o inhibits the growth of MOLM-13 cell xenografts without causing evident toxicity. 21o represents an innovative dual MNK/PIM inhibitor with a good pharmacokinetic profile.

New Quinoxaline Derivatives as Dual Pim-1/2 Kinase Inhibitors: Design, Synthesis and Biological Evaluation.

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure-activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.

Thiosemicarbazone(s)-anchored water soluble mono- and bimetallic Cu(ii) complexes: enzyme-like activities, biomolecular interactions, anticancer property and real-time live cytotoxicity.

The reactions of CuCl2·2H2O with chromone thiosemicarbazone ligands containing a -H or -CH3 substituent on terminal N yielded monometallic Cu(ii) complexes [Cu(HL1)Cl2] (1) and [Cu(HL2)Cl2] (2), whereas bimetallic Cu(ii) complexes [Cu(µ-Cl)(HL3)]2Cl2 (3), [Cu(µ-Cl)(HL4)]2Cl2 (4) and [Cu(µ-Cl)(L5)]2 (5) were obtained when a -C2H5, -C6H11 or -C6H5 substituent was present, respectively, in the ligands. The complexes were characterized using elemental analyses, UV-Vis, FT-IR, EPR, mass and TGA studies. The structures of neutral monometallic and dicationic bimetallic complexes were confirmed by single crystal X-ray diffraction, and they exhibited a distorted square pyramidal geometry around Cu(ii) ions. The catecholase-mimicking activity of complexes 1-5 was examined spectrophotometrically, and the results revealed that all the complexes except 5 had the ability to oxidize 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylquinone (3,5-DTBQ) under aerobic conditions with moderate turnover numbers. In order to find the possible complex-substrate intermediates, a mass spectrometry study was carried out for complexes 1-4 in the presence of 3,5-DTBC. The phosphatase-like activity of 1-5 was also investigated using 4-nitrophenylphosphate (4-NPP) as a model substrate. All the complexes exhibited excellent phosphatase activity in DMF-H2O medium. The complexes displayed significant biomolecular interactions and antioxidant potential. Complex 3 showed good interaction with apoptotic CASP3 protein, VEGFR2 and PIM-1 kinase receptors as revealed by a molecular docking study. Complexes (3-5) exhibited promising cytotoxicity against HeLa-cervical cancer cells with IC50 values of 2.24 (3), 2.25 (4) and 3.77 (5) µM, respectively, and showed a two-fold higher activity than cisplatin. The active complex 3 showed complete inhibition of colony formation at 10 µM concentration. In addition, the acridine orange (AO)/ethidium bromide (EB) staining and real-time live cell imaging results confirmed that complex 3 induced cell death in HeLa cells.

A new method for estimating the relative binding free energy, derived from a free energy variational principle for the Pim-1-kinase-ligand and FKBP-ligand systems.

In this study, a new method is proposed for calculating the relative binding free energy between a ligand and a protein, derived from a free energy variational principle (FEVP). To address the shortcomings of the method used in our previous study, we incorporate the dynamical fluctuation of a ligand in the FEVP calculation. The present modified method is applied to the Pim-1-kinase-ligand system and also to the FKBP-ligand system as a comparison with our previous work. Any inhibitor of Pim-1 kinase is expected to function as an anti-cancer drug. Some improvements are observed in the results compared to the previous study. The present work also shows comparable or better results than approaches using a standard technique of binding free energy calculations, such as the LIE and the MM-PB/SA methods. The possibility of applying the present method in the drug discovery process is also discussed.

Virtual Screening and Design with Machine Intelligence Applied to Pim-1 Kinase Inhibitors.

Ligand-based virtual screening of large compound collections, combined with fast bioactivity determination, facilitate the discovery of bioactive molecules with desired properties. Here, chemical similarity based machine learning and label-free differential scanning fluorimetry were used to rapidly identify new ligands of the anticancer target Pim-1 kinase. The three-dimensional crystal structure complex of human Pim-1 with ligand bound revealed an ATP-competitive binding mode. Generative de novo design with a recurrent neural network additionally suggested innovative molecular scaffolds. Results corroborate the validity of the chemical similarity principle for rapid ligand prototyping, suggesting the complementarity of similarity-based and generative computational approaches.

Insights into the Interaction Mechanisms of the Proviral Integration Site of Moloney Murine Leukemia Virus (Pim) Kinases with Pan-Pim Inhibitors PIM447 and AZD1208: A Molecular Dynamics Simulation and MM/GBSA Calculation Study.

Based on the up-regulation of the proviral integration site of the Moloney murine leukemia virus (Pim) kinase family (Pim1, 2, and 3) observed in several types of leukemias and lymphomas, the development of pan-Pim inhibitors is an attractive therapeutic strategy. While only PIM447 and AZD1208 have entered the clinical stages. To elucidate the interaction mechanisms of three Pim kinases with PIM447 and AZD1208, six Pim/ligand systems were studied by homology modeling, molecular docking, molecular dynamics (MD) simulation and molecular mechanics/generalized Born surface area (MM/GBSA) binding free energy calculation. The residues of the top group (Leu44, Val52, Ala65, Lys67, and Leu120 in Pim1) dominated the pan-Pim inhibitors binding to Pim kinases. The residues of the bottom group (Gln127, Asp128, and Leu174 in Pim1) were crucial for Pims/PIM447 systems, while the contributions of these residues were decreased sharply for Pims/AZD1208 systems. It is likely that the more potent pan-Pim inhibitors should be bound strongly to the top and bottom groups. The residues of the left, right and loop groups were located in the loop regions of the binding pocket, however, the flexibility of these regions triggered the protein interacting with diverse pan-Pim inhibitors efficiently. We hope this work can provide valuable information for the design of novel pan-Pim inhibitors in the future.

Kinase Chemodiversity from the Arctic: The Breitfussins.

In this work, we demonstrate that the indole-oxazole-pyrrole framework of the breitfussin family of natural products is a promising scaffold for kinase inhibition. Six new halogenated natural products, breitfussin C-H (3 - 8) were isolated and characterized from the Arctic, marine hydrozoan Thuiaria breitfussi. The structures of two of the new natural products were also confirmed by total synthesis. Two of the breitfussins (3 and 4) were found to selectively inhibit the survival of several cancer cell lines, with the lowest IC50 value of 340 nM measured against the drug-resistant triple negative breast cancer cell line MDA-MB-468, while leaving the majority of the tested cell lines not or significantly less affected. When tested against panels of protein kinases, 3 gave IC50 and Kd values as low as 200 and 390 nM against the PIM1 and DRAK1 kinases, respectively. The activity was confirmed to be mediated through ATP competitive binding in the ATP binding pocket of the kinases. Furthermore, evaluation of potential off-target and toxicological effects, as well as relevant in vitro ADME parameters for 3 revealed that the breitfussin scaffold holds promise for the development of selective kinase inhibitors.

Synthesis, and anti-proliferative, Pim-1 kinase inhibitors and molecular docking of thiophenes derived from estrone.

Heterocyclization of steroids were reported to give biologically active products where ring D modification occured. Estrone (1) was used as a template to develop new heterocyclic compounds. Ring D modification of 1 through its reaction with cyanoacetylhydrazine and elemental sulfur gave the thiophene derivative 3. The latter compound reacted with acetophenone derivatives 4a-c to give the hydrazide-hydrazone derivatives 5a-c, respectively. In addition, compound 3 formed thiazole derivatives through its first reaction with phenylisothiocyanate to give the thiourea derivative 9 followed by the reaction of the later with α-halocarbonyl compounds. In the present work a series of novel estrone derivatives were designed, synthesized and evaluated for their in vitro biological activities against c-Met kinase, and six typical cancer cell lines (A549, H460, HT-29, MKN-45, U87MG and SMMC-7721). The most promising compounds 5b, 5c, 11a, 13c, 15b, 15c, 15d, 17a and 17b were further investigated against the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR. Compounds 5b, 15d, 17a and 17b were selected to examine their Pim-1 kinase inhibition activity where compounds 15d and 17b showed high activities. Molecular docking of some of the most potent compounds was demonstrated.

Targeting Pim Kinases and DAPK3 to Control Hypertension.

Sustained vascular smooth muscle hypercontractility promotes hypertension and cardiovascular disease. The etiology of hypercontractility is not completely understood. New therapeutic targets remain vitally important for drug discovery. Here we report that Pim kinases, in combination with DAPK3, regulate contractility and control hypertension. Using a co-crystal structure of lead molecule (HS38) in complex with DAPK3, a dual Pim/DAPK3 inhibitor (HS56) and selective DAPK3 inhibitors (HS94 and HS148) were developed to provide mechanistic insight into the polypharmacology of hypertension. In vitro and ex vivo studies indicated that Pim kinases directly phosphorylate smooth muscle targets and that Pim/DAPK3 inhibition, unlike selective DAPK3 inhibition, significantly reduces contractility. In vivo, HS56 decreased blood pressure in spontaneously hypertensive mice in a dose-dependent manner without affecting heart rate. These findings suggest including Pim kinase inhibition within a multi-target engagement strategy for hypertension management. HS56 represents a significant step in the development of molecularly targeted antihypertensive medications.

Identification and molecular characterization of the Pim1 serine/threonine kinase homolog in Litopenaeus vannamei.

The Pim1 serine/threonine kinase is associated with multiple cellular functions including proliferation, survival, differentiation, apoptosis, tumorigenesis, immune regulation and inflammation in vertebrates. However, little is known about the role of Pim1 in invertebrate immunity. In this study, we identified and characterized for the first time, a Pim1 (LvPim1) gene in Litopenaeus vannamei, with a full-length cDNA of 2352 bp and a 1119 bp open reading frame (ORF) encoding a putative protein of 372 amino acids, which contains a typical serine/threonine kinase domain. Sequence and phylogenetic analysis revealed that LvPim1 shared a close evolutionary relationship with Pim1 from vertebrates. Real-time qPCR analysis showed that LvPim1 was widely expressed in all tissues tested; with its transcript level induced in hepatopancreas and hemocytes upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), thus, suggesting its probable involvement in shrimp immune response. Moreover, knockdown of LvPim1 resulted in increased hemocytes apoptosis; shown by high caspase3/7 activity, coupled with increase in pro-apoptotic LvCaspase3 and LvCytochrome C, and decrease in pro-survival LvBcl2, LvIAP1, and LvIAP2 mRNA expression in hemocytes. Finally, LvPim1 knockdown renders shrimps more susceptible to V. parahaemolyticus infection. Taken together, our present data strongly suggest that LvPim1 is involved in modulating shrimp resistance to pathogen infection, promote hemocytes survival, and therefore plays a role in shrimp immune response.

Theoretical Analysis of Activity Cliffs among Benzofuranone-Class Pim1 Inhibitors Using the Fragment Molecular Orbital Method with Molecular Mechanics Poisson-Boltzmann Surface Area (FMO+MM-PBSA) Approach.

Significant activity changes due to small structural changes (i.e., activity cliffs) of serine/threonine kinase Pim1 inhibitors were studied theoretically using the fragment molecular orbital method with molecular mechanics Poisson-Boltzmann surface area (FMO+MM-PBSA) approach. This methodology enables quantum-chemical calculations for large biomolecules with solvation. In the course of drug discovery targeting Pim1, six benzofuranone-class inhibitors were found to differ only in the position of the indole-ring nitrogen atom. By comparing the various qualities of complex structures based on X-ray, classical molecular mechanics (MM)-optimized, and quantum/molecular mechanics (QM/MM)-optimized structures, we found that the QM/MM-optimized structures provided the best correlation (R2 = 0.85) between pIC50 and the calculated FMO+MM-PBSA binding energy. Combining the classical solvation energy with the QM binding energy was important to increase the correlation. In addition, decomposition of the interaction energy into various physicochemical components by pair interaction energy decomposition analysis suggested that CH-π and electrostatic interactions mainly caused the activity differences.

Structural analysis of PIM1 kinase complexes with ATP-competitive inhibitors.

PIM1 is an oncogenic kinase overexpressed in a number of cancers where it correlates with poor prognosis. Several studies demonstrated that inhibition of PIM1 activity is an attractive strategy in fighting overexpressing cancers, while distinct structural features of ATP binding pocket make PIM1 an inviting target for the design of selective inhibitors. To facilitate development of specific PIM1 inhibitors, in this study we report three crystal structures of ATP-competitive inhibitors at the ATP binding pocket of PIM1. Two of the reported structures (CX-4945 and Ro-3306) explain the off-target effect on PIM1 of respectively casein kinase 2 and cyclin-dependent kinase 1 dedicated inhibitors. In turn, the structure with CX-6258 demonstrates a binding mode of a potent, selective inhibitor of PIM1, PIM2, PIM3 and Flt-3 kinases. The consequences of our findings for future inhibitor development are discussed.