Discovery and optimization of 4-pyrazolyl-2-aminopyrimidine derivatives as potent spleen tyrosine kinase (SYK) inhibitors.

Spleen tyrosine kinase (Syk) is a key signal transduction mediator of the B cell receptor (BCR) signaling pathway. Abnormal BCR signaling plays a key role in initiation and development of B-cell-derived hematological malignancies, therefore, Syk represents a potential target for inhibiting the BCR signaling resulting in a therapeutic effect in these cancers. Herein, we describe a novel series of SYK inhibitors with 4-(3'-pyrazolyl)-2-amino-pyrimidine scaffold. Extensive study of structure-activity relationships led to the identification of 1 (NMS-0963), a highly potent Syk inhibitor (IC50 = 3 nM) endowed with high selectivity within a panel of tested kinases and high antiproliferative activity in SYK-dependent BaF3-TEL/SYK cells and in other BCR-dependent hematological tumor cell lines. Additionally, 1 effectively inhibited Syk phosphorylation and downstream signaling mediators of the BCR in treated cells. In in vivo pharmacokinetics studies, 1, displayed good pharmacokinetics properties, with linear exposure with dose and excellent oral bioavailability. These findings suggest that 1 is a promising new Syk inhibitor for treating BCR-dependent hematological cancers.

Extensive severe intraorbital arteriovenous malformations: report of a rare laser complication and successful management.

Arteriovenous malformations (AVMs) are rare congenital errors of vascular morphogenesis, characterised by abnormal direct communications between arteries and veins, bypassing the normal capillary bed. Apart from the central nervous system, typical locations include the head and neck, and less frequently limbs, trunk or internal organs. AVMs of the head and neck often become clinically evident and symptomatic only in later childhood and are characterised by a history of variable growth, sometimes leading to large, deforming, pulsating masses with a propensity to massive haemorrhage. Therapeutic strategy is based on selective embolisation, surgical excision, or a combination of both. Radical surgical excision of local AVMs is the only effective treatment but it may be mutilating, especially for AVMs of the head and neck. Laser therapy represents a good option to treat the cutaneous aspects of AVMs nevertheless, its efficacy is limited. To the date, pharmacological therapy for AVMs is still on debate due to its controversial outcomes as it seems not as effective as other treatments and usually requires a longer course of application. However, pharmacological therapy could be useful in selected patients and for AVMs nonresponsive to traditional treatment, allowing them to obtain acceptable results without serious complications. This paper reports the case of a serious laser complication of extensive intraorbital AVM successfully treated by local reconstruction and topical pharmacological treatment.

Italian recommendations on enzymatic debridement in burn surgery.

INTRODUCTION: Nexobrid®, a bromelain-based type of enzymatic debridement, has become more prevalent in recent years. We present the recommendations on enzymatic debridement (Nexobrid®)'s role based on the practice knowledge of expert Italian users. METHODS: The Italian recommendations, endorsed by SIUST (Italian Society of Burn Surgery), on using enzymatic debridement to remove eschars for burn treatment were defined. The definition followed a process to evaluate the level of agreement (a measure of consensus) among selected experts, representing Italian burn centers, concerning defined clinical aspects of enzymatic debridement. The consensus involved a multi-phase process based on the Delphi method. RESULTS: The consensus panel included experts from Italy with a combined experience of 1068 burn patients treated with enzymatic debridement. At the end of round 3 of the Delphi method, the panel reached 100% consensus on 26 out of 27 statements. The panel achieved full, strong consensus (all respondents strongly agreed on the statement) on 24 out of 27 statements. DISCUSSION: The statements provided by the Italian consensus panel represent a "ready to use" set of recommendations for enzymatic debridement in burn surgery that both draw from and complete the existing scientific literature on the topic. These recommendations are specific to the Italian experience and are neither static nor definitive. As such, they will be updated periodically as further quality evidence becomes available.

Releasing Burn-Induced Compartment Syndrome by Enzymatic Escharotomy-Debridement: A Case Study.

Burn-induced compartment syndrome represents a serious and acute condition in deep circumferential burns of the extremities which, if left untreated, can cause severe complications. The surgical escharotomy that releases the high subdermal pressure is the therapeutic treatment of choice for burn-induced compartment syndrome. Guidelines for escharotomy indications and timing include pressure greater than 30 mm Hg and 6 Ps (Pain, Pallor, Paresthesia, Paralysis, Pulseless, and Poikilothermia). Nevertheless, despite the need for an early as possible pressure release, escharotomy is often delayed when a capable surgeon is not available, or if the indication is not completely clear to justify potential risks associated with surgical escharotomy. Early treatment of circumferential burns of the extremities with a Bromelain-based enzymatic agent NexoBrid® may represent a less traumatic and invasive procedure to reduce intra-compartmental pressure, replacing surgical escharotomy. This case study of 23 patients describes the variation of compartmental pressure in patients with circumferential burns of the extremities treated with NexoBrid® enzymatic escharotomy-debridement. All the patients were treated with NexoBrid® within 2 to 22 hours post-injury in our Burn Intensive Care Center. The excessive pressure recorded before treatment returned to normal below 30 mm Hg and an approximately 60% reduction of the compartmental pressure was observed in most cases within 1 hour from NXB application. On NexoBrid® removal after 4 hours complete debridement-escharotomy of the burns was achieved. Enzymatic escharotomy-debridement appears to be a useful and safe method to reduce postburn compartmental pressure. Additional randomized, well-controlled powered studies are needed to further support these results.

Molecular Dynamics Simulations of p97 Including Covalent, Allosteric and ATP-competitive Inhibitors.

Binary (nucleotide-protein dimer and hexamer complexes) and ternary (nucleotide-protein-inhibitor complexes) p97 complexes were subjected to molecular dynamics simulations in an attempt to further our understanding of the p97 protein oligomer domain stability and, more importantly, of the recently reported diverse molecular mechanisms of inhibition including allosteric, ATP-competitive and covalent inhibitors. Analysis of stable states following equilibration phases indicated a higher intrinsic stability of the homohexamer as opposed to the dimer, and of N-D1 domains as opposed to the D2 domain. The molecular dynamics of the proposed allosteric binding model reproduced important molecular interactions identified experimentally with high frequency throughout the trajectory. Observed conformational changes occurring in the D2 nucleotide binding site provided a novel bind-rearrange-react hypothesis of stepwise molecular events involved in the specific covalent inhibitor mode of action.

Discovery of 2-(cyclohexylmethylamino)pyrimidines as a new class of reversible valosine containing protein inhibitors.

Valosine-containing protein (VCP), also known as p97 or cdc48 in yeast, is a highly abundant protein belonging to the AAA ATPase family involved in a number of essential cellular functions, including ubiquitin-proteasome mediated protein degradation, Golgi reassembly, transcription activation, and cell cycle control. Altered expression of VCP has been detected in many cancer types sometimes associated with poor prognosis. Furthermore, VCP mutations are causative of some neurodegenerative disorders. In this paper we report the discovery, synthesis, and structure-activity relationships of substituted 2-aminopyrimidines, representing a new class of reversible VCP inhibitors. This class of compounds, identified in a HTS campaign against recombinant VCP, has been progressively expanded and manipulated to increase biochemical potency and gain cellular activity.

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.

Alkylsulfanyl-1,2,4-triazoles, a new class of allosteric valosine containing protein inhibitors. Synthesis and structure-activity relationships.

Valosine containing protein (VCP), also known as p97, is a member of AAA ATPase family that is involved in several biological processes and plays a central role in the ubiquitin-mediated degradation of misfolded proteins. VCP is an ubiquitously expressed, highly abundant protein and has been found overexpressed in many tumor types, sometimes associated with poor prognosis. In this respect, VCP has recently received a great deal of attention as a potential new target for cancer therapy. In this paper, the discovery and structure-activity relationships of alkylsulfanyl-1,2,4-triazoles, a new class of potent, allosteric VCP inhibitors, are described. Medicinal chemistry manipulation of compound 1, identified via HTS, led to the discovery of potent and selective inhibitors with submicromolar activity in cells and clear mechanism of action at consistent doses. This represents a first step toward a new class of potential anticancer agents.

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.

Development of biochemical assays for the identification of eIF4E-specific inhibitors.

Control of mRNA translation plays a critical role in cell growth, proliferation, and differentiation and is tightly regulated by AKT and RAS oncogenic pathways. A key player in the regulation of this process is the mRNA 5' cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E). eIF4E contributes to malignancy by selectively enabling the translation of a limited pool of mRNAs that generally encode key proteins involved in cell cycle progression, angiogenesis, and metastasis. Several data indicate that the inhibition of eIF4E in tumor cell lines and xenograft models impairs tumor growth and induces apoptosis; eIF4E, therefore, can be considered a valuable target for cancer therapy. Targeting the cap-binding pocket of eIF4E should represent a way to inhibit all the eIF4E cellular functions. We present here the development and validation of different biochemical assays based on fluorescence polarization and surface plasmon resonance techniques. These assays could support high-throughput screening, further refinement, and characterization of eIF4E inhibitors, as well as selectivity assessment against CBP80/CBP20, the other major cap-binding complex of eukaryotic cells, overall providing a robust roadmap for development of eIF4E-specific inhibitors.

Cdc7 kinase inhibitors: 5-heteroaryl-3-carboxamido-2-aryl pyrroles as potential antitumor agents. 1. Lead finding.

Cdc7 serine/threonine kinase is a key regulator of DNA synthesis in eukaryotic organisms. Cdc7 inhibition through siRNA or prototype small molecules causes p53 independent apoptosis in tumor cells while reversibly arresting cell cycle progression in primary fibroblasts. This implies that Cdc7 kinase could be considered a potential target for anticancer therapy. We previously reported that pyrrolopyridinones (e.g., 1) are potent and selective inhibitors of Cdc7 kinase, with good cellular potency and in vitro ADME properties but with suboptimal pharmacokinetic profiles. Here we report on a new chemical class of 5-heteroaryl-3-carboxamido-2-substituted pyrroles (1A) that offers advantages of chemistry diversification and synthetic simplification. This work led to the identification of compound 18, with biochemical data and ADME profile similar to those of compound 1 but characterized by superior efficacy in an in vivo model. Derivative 18 represents a new lead compound worthy of further investigation toward the ultimate goal of identifying a clinical candidate.

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.

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.

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.

Benzodipyrazoles: a new class of potent CDK2 inhibitors.

The synthesis and the preliminary expansion of this new class of CDK2 inhibitors are presented. The synthesis was accomplished using a solution-phase protocol amenable to rapid parallel expansion and suitable to be scaled-up in view of possible lead development. Following a medicinal chemistry program aimed at improving cell permeability and selectivity, a series of compounds with nanomolar activity in the biochemical assay and able to efficiently inhibit tumor cell proliferation has been obtained.

Catecholic flavonoids acting as telomerase inhibitors.

In recent years telomerase has been identified as a new promising target in oncology and consequently new telomerase inhibitors have been intensely explored as anticancer agents. Focused screening of several polyhydroxylated flavonoids has allowed us to identify 7,8,3',4'-tetrahydroxyflavone 1 as a new telomerase inhibitor with an interesting in vitro activity in a Flash-Plate assay (IC50 = 0.2 microM) that has been confirmed in the classical TRAP assay. Starting from this compound, we developed a medicinal chemistry program to optimize our lead, and in particular to replace one of the two catechols with potential bioisosteres. From this study, new structural analogues characterized by submicromolar potencies have been obtained. Their synthesis and biological activity are described.