Conrad Kunick

Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4.

Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such compounds may provide mechanistic insight into the reprogramming process.

Cyclin-dependent kinases: initial approaches to exploit a novel therapeutic target.

Cyclin-dependent kinases (CDKs) have been recognized as key regulators of cell cycle progression. Alteration and deregulation of CDK activity are pathogenic hallmarks of neoplasia. Therefore, inhibitors or modulators would be of interest to explore as novel therapeutic agents in cancer, as well as other hyperproliferative disorders. Flavopiridol is a semisynthetic flavonoid that emerged from an empirical screening program as a potent antiproliferative agent that mechanistic studies demonstrated to directly inhibit CDKs 1, 2, and 4 as a competitive ATP site antagonist. Initial clinical trials have shown that concentrations that inhibit cell proliferation and CDK activity in vitro can be safely achieved in humans, and additional clinical trials will establish its clinical potential. To address the need for additional chemotypes that may serve as lead structures for drugs that would not have the toxicities associated with flavopiridol, compounds with a similar pattern of cell growth inhibitory activity in the National Cancer Institutes in vitro anticancer drug screen have been recognized by the computer-assisted pattern recognition algorithm COMPARE and then screened for anti-CDK activity in a biochemical screen. The benzodiazepine derivative NSC 664704 (7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one) was revealed by that approach as a moderately potent (IC50 0.4 microM) inhibitor of CDK2, which in initial experiments shows evidence of causing cell cycle redistribution in living cells. NSC 664704 is, therefore, a candidate for further structural optimization, guided in part by understanding of the ATP-binding site in CDK2. This approach represents one way of combining empirical screening information with structure-based design to derive novel candidate therapeutic agents directed against an important cellular target.

9-Cyano-1-azapaullone (Cazpaullone), a Glycogen Synthase Kinase-3 (GSK-3) Inhibitor Activating Pancreatic β Cell Protection and Replication

Recently, the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) emerged as a regulator of pancreatic beta cell growth and survival. On the basis of the previous observation that GSK-3 inhibitors like 1-azakenpaullone promote beta cell protection and replication, paullone derivatives were synthesized including 1-aza-, 2-aza-, and 12-oxapaullone scaffolds. In enzymatic assays distinct 1-azapaullones were found to exhibit selective GSK-3 inhibitory activity. Within the series of 1-azapaullones, three derivatives stimulated INS-1E beta cell replication and protected INS-1E cells against glucolipotoxicity induced cell death. Cazpaullone (9-cyano-1-azapaullone), the most active compound in the protection assays, also stimulated the replication of primary beta cells in isolated rat islets. Furthermore, cazpaullone showed a pronounced transient stimulation of the mRNA expression of the beta cell transcription factor Pax4, an important regulator of beta cell development and growth. These features distinguish cazpaullone as a unique starting point for the development of beta cell regenerative agents which might be useful in the treatment of diabetes.

2-Substituted paullones: CDK1/cyclin B-inhibiting property and in vitro antiproliferative activity

9-Trifluoromethyl-paullones with a carbon chain in the 2-position were synthesized by palladium-catalyzed coupling reactions of a 2-iodoprecursor with terminal alkenes or alkynes, respectively. The introduction of a 2-cyanoethyl substituent led to a significant enhancement of CDK1/cyclin B inhibiting property and in vitro antiproliferative activity.

2-(3-Aryl-3-oxopropen-1-yl)-9-tert-butyl-paullones: A New Antileishmanial Chemotype

A screening program directed to find new agents against Leishmania donovani, the parasite causing visceral leishmaniasis, revealed that paullones attenuate the proliferation of axenic amastigotes. Because these structures were not active in a test system involving infected macrophages, a structure optimization campaign was carried out. Concomitant introduction of an unsaturated side chain into the 2-position and a tert-butyl substituent into the 9-position of the parent scaffold led to compounds inhibiting also parasites dwelling in macrophages. By inclusion of the so elaborated scaffold into a chalcone substructure, the toxicity against uninfected host cells was significantly reduced. For the synthesis of this new compound class, a novel modification of the Heck-type palladium-catalyzed C,C-cross coupling strategy was used, employing a ketone Mannich base as precursor for the alkene reactant. The so-prepared compounds exhibited improved antileishmanial activity both on axenic amastigotes (GI50 < 1 microM) as well as on parasites in infected macrophages.

Alsterpaullone, a novel cyclin-dependent kinase inhibitor, induces apoptosis by activation of caspase-9 due to perturbation in mitochondrial membrane potential†

The majority of human neoplasms have aberrations in the retinoblastoma pathway due to hyperactivation of cyclin‐dependent kinases (CDK). Based on this observation, novel small molecules, such as flavopiridol and UCN‐01, are being developed and are currently being tested in the clinic. Efforts to develop CDK modulators led us to the discovery of a novel class of CDK inhibitors, the paullones [Cancer Res 1999;59:2566]. Initial studies demonstrated that paullones inhibit CDKs in vitro, thereby blocking cell‐cycle progression. However, the exact mechanism for the antiproliferative effects of paullones was never explored. In this report, we demonstrate for the first time that the most potent paullone, alsterpaullone (Alp), induced apoptosis and promoted loss in clonogenicity in the Jurkat cell line. Alp caused early activation of both caspase‐8 and ‐9, leading to cleavage of caspase‐3 and poly(ADP‐ribose) polymerase (PARP). Moreover, apoptosis by Alp was not associated with loss in anti‐apoptotic proteins such as XIAP or BCL‐XL. Pre‐incubation with cell‐permeable inhibitors z‐Asp(OMe)‐Glu(OMe)‐Val‐Asp(Ome)‐fluoromethylketone and benzyloxycarbonyl‐Val‐Ala‐Asp (OMe)‐fluoromethylketone (ZVAD) blocked Alp‐induced apoptosis. Moreover, the general caspase inhibitor ZVAD blocked the cleavage and activation of most caspases tested except caspase‐9. Studies of mitochondrial membrane potential also demonstrated that Alp is able to disrupt mitochondrial potential in the presence of ZVAD, suggesting that the activation of caspase‐9 by Alp follows mitochondrial perturbation. Pre‐incubation of Jurkat cells with ZVAD did not prevent the depletion of cyclin D3, loss of CDK, or cell‐cycle arrest by Alp. In summary, these experiments suggest that Alp activates caspase‐9 via mitochondrial perturbation. Active caspase‐9 cleaves and activates caspase‐8 and caspase‐3, leading to apoptosis. In the presence of the general caspase inhibitor ZVAD, the cell‐cycle effects of Alp are unaltered while apoptosis is blocked, suggesting that the CDK effects of Alp are not sufficient for Alp‐induced apoptosis. Additional studies with paullones are warranted to further characterize their preclinical effects and to explore their potential use in the clinical setting. Published 2003 Wiley‐Liss, Inc.

Structure‐Aided Optimization of Kinase Inhibitors Derived from Alsterpaullone

In order to perform computer‐aided design of novel alsterpaullone derivatives, the vicinity of the entrance to the ATP‐binding site was scanned for areas that could be useful as anchoring points for additional protein–ligand interactions. Based on the alignment of alsterpaullone in a CDK1/cyclin B homology model, substituents were attached to the 2‐position of the parent scaffold to enable contacts within the identified areas. Synthesis of the designed structures revealed three derivatives (3–5) with kinase‐inhibitory activity similar to alsterpaullone. The novel 2‐cyanoethylalsterpaullone (7) proved to be the most potent paullone described so far, exhibiting inhibitory concentrations for CDK1/ cyclin B and GSK‐3β in the picomolar range.

Inhibitors of the RET tyrosine kinase based on a 2-(alkylsulfanyl)-4-(3-thienyl)nicotinonitrile scaffold.

In an approach to optimize 2-(4-fluorobenzylsulfanyl)-4-(2-thienyl)-5,6,7,8-tetrahydroquinoline-3-carbonitrile (1a), a weak inhibitor of the cancer-related tyrosine kinase RET originating from a screening campaign, analogues with 3-thienyl substitution were prepared. Among the novel derivatives, 2-amino-6-{[2-(4-chlorophenyl)-2-oxoethyl]sulfanyl}-4-(3-thienyl)pyridine-3,5-dicarbonitrile (13 g) was identified as a submicromolar RET inhibitor, displaying 3- and 100-fold selectivity versus ALK and ABL kinases, respectively. The novel inhibitor exhibited antiproliferative activity in the micromolar concentration range against both RET-dependent and RET-independent cancer cell lines. Docking experiments suggest a binding mode of the new inhibitors in the ATP binding pocket of the target kinase, explaining the observed structure-activity relationships.

Homology Model of the CDK1/cyclin B Complex

Abstract We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.

10-Iodo-11H-indolo[3,2-c]quinoline-6-carboxylic Acids Are Selective Inhibitors of DYRK1A.

The protein kinase DYRK1A has been suggested to act as one of the intracellular regulators contributing to neurological alterations found in individuals with Down syndrome. For an assessment of the role of DYRK1A, selective synthetic inhibitors are valuable pharmacological tools. However, the DYRK1A inhibitors described in the literature so far either are not sufficiently selective or have not been tested against closely related kinases from the DYRK and the CLK protein kinase families. The aim of this study was the identification of DYRK1A inhibitors exhibiting selectivity versus the structurally and functionally closely related DYRK and CLK isoforms. Structure modification of the screening hit 11H-indolo[3,2-c]quinoline-6-carboxylic acid revealed structure–activity relationships for kinase inhibition and enabled the design of 10-iodo-substituted derivatives as very potent DYRK1A inhibitors with considerable selectivity against CLKs. X-ray structure determination of three 11H-indolo[3,2-c]quinoline-6-carboxylic acids cocrystallized with DYRK1A confirmed the predicted binding mode within the ATP binding site.