Roberto D'alessio

Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death

VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.

Cdc7 Kinase Inhibitors : Pyrrolopyridinones as Potential Antitumor Agents. 1. Synthesis and Structure-Activity Relationships

Cdc7 kinase is an essential protein that promotes DNA replication in eukaryotic organisms. Genetic evidence indicates that Cdc7 inhibition can cause selective tumor-cell death in a p53-independent manner, supporting the rationale for developing Cdc7 small-molecule inhibitors for the treatment of cancers. In this paper, the synthesis and structure-activity relationships of 2-heteroaryl-pyrrolopyridinones, the first potent Cdc7 kinase inhibitors, are described. Starting from 2-pyridin-4-yl-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one, progress toward a simple scaffold, tailored for Cdc7 inhibition, is reported.

Identification of N,1,4,4-Tetramethyl-8-{[4-(4-Methylpiperazin-1-Yl)Phenyl]Amino}-4,5-Dihydro-1H-Pyrazolo[4,3-H]Quinazoline-3-Carboxamide (Pha-848125), a Potent, Orally Available Cyclin Dependent Kinase Inhibitor.

The discovery of a novel class of inhibitors of cyclin dependent kinases (CDKs) is described. Starting from compound 1, showing good potency as inhibitor of CDKs but being poorly selective against a panel of serine-threonine and tyrosine kinases, new analogues were synthesized. Enhancement in selectivity, antiproliferative activity against A2780 human ovarian carcinoma cells, and optimization of the physical properties and pharmacokinetic profile led to the identification of highly potent and orally available compounds. Compound 28 (PHA-848125), which in the preclinical xenograft A2780 human ovarian carcinoma model showed good efficacy and was well tolerated upon repeated daily treatments, was identified as a drug candidate for further development. Compound 28 is currently undergoing phase I and phase II clinical trials.

First Cdc7 kinase inhibitors: pyrrolopyridinones as potent and orally active antitumor agents. 2. Lead discovery.

Cdc7 kinase is a key regulator of the S-phase of the cell cycle, known to promote the activation of DNA replication origins in eukaryotic organisms. Cdc7 inhibition can cause tumor-cell death in a p53-independent manner, supporting the rationale for developing Cdc7 inhibitors for the treatment of cancer. In this paper, we conclude the structure-activity relationships study of the 2-heteroaryl-pyrrolopyridinone class of compounds that display potent inhibitory activity against Cdc7 kinase. Furthermore, we also describe the discovery of 89S, [(S)-2-(2-aminopyrimidin-4-yl)-7-(2-fluoro-ethyl)-1,5,6,7-tetrahydropyrrolo[3,2-c]pyridin-4-one], as a potent ATP mimetic inhibitor of Cdc7. Compound 89S has a Ki value of 0.5 nM, inhibits cell proliferation of different tumor cell lines with an IC50 in the submicromolar range, and exhibits in vivo tumor growth inhibition of 68% in the A2780 xenograft model.

Structure-based approaches to improve selectivity: CDK2-GSK3beta binding site analysis.

An evaluation and comparison of two different approaches, GRID/CPCA and GRIND/CPCA (CPCA = consensus principal component analysis; GRIND = GRid-INdependent Descriptors), suitable for visualizing the structural differences between related proteins is presented. Ten crystal structures of CDK2/cyclin A and GSK3beta solved in-house with different inhibitors were compared with the aim of highlighting regions that could be potential sites for gaining selectivity for CDK2 versus GSK3beta. The analyses pointed out remarkable differences in the backs of the CDK2-GSK3beta ATP binding pockets that guided the optimization toward a selective benzodipyrazole CDK2 inhibitor. The gain in selectivity can be associated with the two main differences in the ATP pocket between the enzymes. Phe80 of CDK2, the so-called gatekeeper residue often exploited for the design of kinase selective ligands, is replaced by a leucine in GSK3beta, and Ala144 is replaced by a cysteine. As a consequence of these mutations, CDK2 has a less elongated and less flat buried region at the back of the ATP pocket.

4-aminoandrostenedione derivatives: A novel class of irreversible aromatase inhibitors. Comparison with FCE 24304 and 4-hydroxyandrostenedione

FCE 24928 (4-aminoandrosta-1,4,6-triene-3,17-dione) was selected among a series of 4-aminoandrostenedione derivatives as a novel irreversible aromatase inhibitor. Its in vitro and in vivo properties have been studied and compared to FCE 24304 (6-methylenandrosta-1,4-diene-3,17-dione) and 4-OHA (4-hydroxyandrostenedione). FCE 24928 caused time-dependent inhibition of human placental aromatase with a t1/2 of 4 min and Ki of 59 nM. Enzyme inactivation by FCE 24928 was faster than by FCE 24304 (t1/2 13.9 min). In PMSG-treated rats, microsomal ovarian aromatase activity was reduced 24 h after FCE 24928 dosing by both the s.c. (ED50 1.2 mg/kg) and the oral (ED50 14.1 mg/kg) routes. The compound was more potent than FCE 24304 and 4-OHA (ED50 1.8 and 3.1 mg/kg s.c.). FCE 24928 did not show any interference with 5 alpha-reductase and desmolase activity nor any significant binding affinity for androgen and estrogen receptors. Slight binding affinity for androgen receptor was observed with FCE 24304 and 4-OHA (0.21 and 0.25% of DHT). In immature, castrated rats, FCE 24928 did not show any intrinsic androgenic activity, up to 100 mg/kg/day s.c., in contrast to a slight androgenic activity observed with FCE 24304 at 10 mg/kg s.c.

Novel phenyl nitrogen mustard and half-mustard derivatives of distamycin A

Abstract The design, synthesis, in vitro and in vivo activities of novel benzoyl and cinnamoyl nitrogen mustard and half-mustard derivatives of distamycin A are described and structure-activity relationships are discussed. The equipotent activities of N-ethyl-N-chloroethyl half-mustards and N,N-dichloroethyl mustards and the superior activities of cinnamoyl derivatives are the most relevant features of the series.

Molecular recognition of T:G mismatched base pairs in DNA as studied by electrospray ionization mass spectrometry.

Postreplicative mismatch repair (MMR) is a cellular system involved in the recognition and correction of DNA polymerase errors that escape detection in proofreading. Of the various mismatched bases, T:G pairing in DNA is one of the more common mutations leading to the formation of tumors in humans. In addition, the absence of the MMR system can generate resistance to several chemotherapeutic agents, particularly DNA‐damaging substances. The main purpose of this study was the setup and validation of an electrospray ionization (ESI) mass spectrometry method for the identification of small molecules that are able to recognize T:G mismatches in DNA targets. These findings could be useful for the discovery of new antitumor drugs. The analytical method is based on the ability of electrospray to preserve the noncovalent adducts present in solution and transfer them to the gas phase. Lexitropsin derivatives (polyimidazole compounds) have been previously described as selective for T:G mismatch binding by NMR and ITC studies. We synthesized and tested various polyimidazole derivatives, one of which in particular (NMS‐057) showed a higher affinity for an oligonucleotide DNA sequence containing a T:G mismatched base pair. To rationalize these findings, molecular docking studies were performed using available NMR structures. Moreover, ESI‐MS experiments, performed on an orbitrap mass spectrometer, highlighted the formation of heterodimeric complexes between DNA sequences, distamycin A, and polyimidazole compounds. Our results confirm that this ESI method could be a valuable tool for the identification of new molecules able to specifically recognize T:G mismatched base pairs.

Abstract 2940: Identification of potent VCP/p97/CDC48 inhibitors with distinct biochemical mechanisms including a reversible, allosteric inhibitor that activates the unfolded protein response, induce autophagy and cancer cell death

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Valosin Containing Protein (VCP), also called p97 in mammals and cdc48 in yeast, is an ubiquitously expressed and essential AAA ATPase important in specific cellular processes including Endoplasmic Reticulum Associated Degradation (ERAD), Golgi reformation, membrane fusion and autophagy. VCP is a hexameric complex formed by six identical protomers, each composed of three domains: an N-terminal domain responsible for the interaction with co-factors and adaptor proteins, and two AAA ATPase domains, D1 and D2. VCP acts as a protein-directed molecular machine that converts energy derived from ATP hydrolysis into mechanical force to cause disassembly of multiprotein complexes or extraction of molecules from the membrane to be delivered to the proteasome for degradation. VCP also affects autophagy and aggresome formation processes. Overall, VCP plays a key role in cellular homeostasis. The clinical success of proteasome inhibitors, and recent advances in the preclinical development of molecules that interfere with protein folding and degradation has highlighted that cancer cells can be extremely sensitive to perturbation of protein homeostasis. The availability of small molecules that specifically inhibit VCP function would help to clarify whether VCP is a valid target for cancer therapy. We found that VCP silencing by siRNA induces cancer cell death in a variety of tumor cell lines, and examined the main cellular pathways modulated upon VCP ablation, thus identifying biomarkers suitable for characterizing the cellular activity of VCP inhibitors. We then performed a High Throughput Screening campaign using recombinant VCP and identified multiple compound classes that inhibit VCP function with distinct biochemical mechanisms. Initial hits included a compound that covalently modifies VCP, representatives of two classes of ATP-sensitive inhibitors, and an inhibitor class characterized by a novel allosteric mechanism of action. Chemical expansion of initial hits resulted in improvement of biochemical potency, yielding highly active derivatives for each class, suggesting that VCP is a druggable target. Notably, the binding site of the allosteric class of inhibitors was identified by photo-affinity labeling in combination with available structural data. We will present data on a potent (30 nM) and specific compound emerging from this class that displays anti-proliferative activity related to the modulation of direct VCP biomarkers, activation of the Unfolded Protein Response (UPR) and perturbation of autophagy, which ultimately result in cancer cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2940. doi:1538-7445.AM2012-2940