Pharmacological Characterization of SDX-7320/Evexomostat: A Novel Methionine Aminopeptidase Type 2 Inhibitor with Anti-tumor and Anti-metastatic Activity.

Methionine aminopeptidase type 2 (METAP2) is a ubiquitous, evolutionarily conserved metalloprotease fundamental to protein biosynthesis which catalyzes removal of the N-terminal methionine residue from nascent polypeptides. METAP2 is an attractive target for cancer therapeutics based upon its over-expression in multiple human cancers, the importance of METAP2-specific substrates whose biological activity may be altered following METAP2 inhibition, and additionally, that METAP2 was identified as the target for the anti-angiogenic natural product, fumagillin. Irreversible inhibition of METAP2 using fumagillin analogues has established the anti-angiogenic and anti-tumor characteristics of these derivatives; however, their full clinical potential has not been realized due to a combination of poor drug-like properties and dose-limiting central nervous system (CNS) toxicity. This report describes the physicochemical and pharmacological characterization of SDX-7320 (evexomostat), a polymer-drug conjugate of the novel METAP2 inhibitor (METAP2i) SDX-7539. In vitro binding, enzyme, and cell-based assays demonstrated that SDX-7539 is a potent and selective METAP2 inhibitor. In utilizing a high molecular weight, water-soluble polymer to conjugate the novel fumagillol-derived, cathepsin-released, METAP2i SDX-7539, limitations observed with prior generation, small molecule fumagillol derivatives were ameliorated including reduced CNS exposure of the METAP2i, and prolonged half-life enabling convenient administration. Multiple xenograft and syngeneic cancer models were utilized to demonstrate the anti-tumor and anti-metastatic profile of SDX-7320. Unlike polymer-drug conjugates in general, reductions in small molecule-equivalent efficacious doses following polymer conjugation were observed. SDX-7320 has completed a phase I clinical safety study in patients with late-stage cancer and is currently being evaluated in multiple phase Ib/II clinical studies in patients with advanced solid tumors.

Sequential CRISPR screening reveals partial NatB inhibition as a strategy to mitigate alpha-synuclein levels in human neurons.

Alpha-synuclein (αSyn) protein levels correlate with the risk and severity of Parkinson's disease and related neurodegenerative diseases. Lowering αSyn is being actively investigated as a therapeutic modality. Here, we systematically map the regulatory network that controls endogenous αSyn using sequential CRISPR-knockout and -interference screens in an αSyn gene (SNCA)-tagged cell line and induced pluripotent stem cell-derived neurons (iNeurons). We uncover αSyn modifiers at multiple regulatory layers, with amino-terminal acetyltransferase B (NatB) enzymes being the most potent endogenous αSyn modifiers in both cell lines. Amino-terminal acetylation protects the cytosolic αSyn from rapid degradation by the proteasome in a Ube2w-dependent manner. Moreover, we show that pharmacological inhibition of methionyl-aminopeptidase 2, a regulator of NatB complex formation, attenuates endogenous αSyn in iNeurons carrying SNCA triplication. Together, our study reveals several gene networks that control endogenous αSyn, identifies mechanisms mediating the degradation of nonacetylated αSyn, and illustrates potential therapeutic pathways for decreasing αSyn levels in synucleinopathies.

Structural insights into N-terminal methionine cleavage by the human mitochondrial methionine aminopeptidase, MetAP1D.

Isozymes are enzymes that catalyze identical biological reactions, yet exhibit slight variations in structures and catalytic efficiency, which enables the precise adjustment of metabolism to fulfill the specific requirements of a particular tissue or stage of development. Methionine aminopeptidase (MetAP) isozymes function a critical role in cleaving N-terminal methionine from nascent proteins to generate functional proteins. In humans, two distinct MetAP types I and II have been identified, with type I further categorized into cytosolic (MetAP1) and mitochondrial (MetAP1D) variants. However, despite extensive structural studies on both bacterial and human cytosolic MetAPs, the structural information remains unavailable for human mitochondrial MetAP. This study was aimed to elucidate the high-resolution structures of human mitochondrial MetAP1D in its apo-, cobalt-, and methionine-bound states. Through a comprehensive analysis of the determined structures and a docking simulation model with mitochondrial substrate peptides, we present mechanistic insights into the cleavage process of the initiator methionine from mitochondrial proteins. Notably, despite the shared features at the active site between the cytosolic and mitochondrial MetAP type I isozymes, we identified distinct structural disparities within the active-site pocket primarily contributed by two specific loops that could play a role in accommodating specific substrates. These structural insights offer a basis for the further exploration of MetAP isozymes as critical players in cellular processes and potential therapeutic applications.

Anti-Tuberculosis Potential of OJT008 against Active and Multi-Drug-Resistant Mycobacterium Tuberculosis: In Silico and In Vitro Inhibition of Methionine Aminopeptidase.

Despite the recent progress in the diagnosis of tuberculosis (TB), the chemotherapeutic management of TB continues to be challenging. Mycobacterium tuberculosis (Mtb), the etiological agent of TB, is classified as the 13th leading cause of death globally. In addition, 450,000 people were reported to develop multi-drug-resistant TB globally. The current project focuses on targeting methionine aminopeptidase (MetAP), an essential protein for the viability of Mtb. MetAP is a metalloprotease that catalyzes the excision of the N-terminal methionine (NME) during protein synthesis, allowing the enzyme to be an auspicious target for the development of novel therapeutic agents for the treatment of TB. Mtb possesses two MetAP1 isoforms, MtMetAP1a and MtMetAP1c, which are vital for Mtb viability and, hence, a promising chemotherapeutic target for Mtb therapy. In this study, we cloned and overexpressed recombinant MtMetAP1c. We investigated the in vitro inhibitory effect of the novel MetAP inhibitor, OJT008, on the cobalt ion- and nickel ion-activated MtMetAP1c, and the mechanism of action was elucidated through an in silico approach. The compound's potency against replicating and multi-drug-resistant (MDR) Mtb strains was also investigated. The induction of the overexpressed recombinant MtMetAP1c was optimized at 8 h with a final concentration of 1 mM Isopropyl ß-D-1-thiogalactopyranoside. The average yield from 1 L of Escherichia coli culture for MtMetAP1c was 4.65 mg. A preliminary MtMetAP1c metal dependency screen showed optimum activation with nickel and cobalt ions occurred at 100 µM. The half-maximal inhibitory concentration (IC50) values of OJT008 against MtMetAP1c activated with CoCl2 and NiCl2 were 11 µM and 40 µM, respectively. The in silico study showed OJT008 strongly binds to both metal-activated MtMetAP1c, as evidenced by strong molecular interactions and a higher binding score, thereby corroborating our result. This in silico study validated the pharmacophore's metal specificity. The potency of OJT008 against both active and MDR Mtb was <0.063 µg/mL. Our study reports OJT008 as an inhibitor of MtMetAP1c, which is potent at low micromolar concentrations against both active susceptible and MDR Mtb. These results suggest OJT008 is a potential lead compound for the development of novel small molecules for the therapeutic management of TB.

The aminopeptidase LAP3 suppression accelerates myogenic differentiation via the AKT-TFE3 pathway in C2C12 myoblasts.

Skeletal muscle maintenance depends largely on muscle stem cells (satellite cells) that supply myoblasts required for muscle regeneration and growth. The ubiquitin-proteasome system is the major intracellular protein degradation pathway. We previously reported that proteasome dysfunction in skeletal muscle significantly impairs muscle growth and development. Furthermore, the inhibition of aminopeptidase, a proteolytic enzyme that removes amino acids from the termini of peptides derived from proteasomal proteolysis, impairs the proliferation and differentiation ability of C2C12 myoblasts. However, no evidence has been reported on the role of aminopeptidases with different substrate specificities on myogenesis. In this study, therefore, we investigated whether the knockdown of aminopeptidases in differentiating C2C12 myoblasts affects myogenesis. The knockdown of the X-prolyl aminopeptidase 1, aspartyl aminopeptidase, leucyl-cystinyl aminopeptidase, methionyl aminopeptidase 1, methionyl aminopeptidase 2, puromycine-sensitive aminopeptidase, and arginyl aminopeptidase like 1 gene in C2C12 myoblasts resulted in defective myogenic differentiation. Surprisingly, the knockdown of leucine aminopeptidase 3 (LAP3) in C2C12 myoblasts promoted myogenic differentiation. We also found that suppression of LAP3 expression in C2C12 myoblasts resulted in the inhibition of proteasomal proteolysis, decreased intracellular branched-chain amino acid levels, and enhanced mTORC2-mediated AKT phosphorylation (S473). Furthermore, phosphorylated AKT induced the translocation of TFE3 from the nucleus to the cytoplasm, promoting myogenic differentiation through increased expression of myogenin. Overall, our study highlights the association of aminopeptidases with myogenic differentiation.

NAC controls cotranslational N-terminal methionine excision in eukaryotes.

N-terminal methionine excision from newly synthesized proteins, catalyzed cotranslationally by methionine aminopeptidases (METAPs), is an essential and universally conserved process that plays a key role in cell homeostasis and protein biogenesis. However, how METAPs interact with ribosomes and how their cleavage specificity is ensured is unknown. We discovered that in eukaryotes the nascent polypeptide-associated complex (NAC) controls ribosome binding of METAP1. NAC recruits METAP1 using a long, flexible tail and provides a platform for the formation of an active methionine excision complex at the ribosomal tunnel exit. This mode of interaction ensures the efficient excision of methionine from cytosolic proteins, whereas proteins targeted to the endoplasmic reticulum are spared. Our results suggest a broader mechanism for how access of protein biogenesis factors to translating ribosomes is controlled.

Neuroprotective Effect of Dexmedetomidine on Cerebral Ischemia-Reperfusion Injury in Rats.

Background: The number of patients having ischemic stroke is increasing year on year. The anesthetic adjuvant dexmedetomidine is neuroprotective in rats and has potential for use in the treatment of ischemic stroke. Objective: The neuroprotective mechanism of dexmedetomidine in cerebral ischemia-reperfusion injury was studied in relation to its regulation of the oxidative stress response, astrocyte response, microglia overactivation, and apoptosis-related protein expression. Methods: We randomly and equally divided 25 male Sprague-Dawley rats into 5 groups: a sham-operation group, an ischemia-reperfusion injury group, and low-, medium-, and high-dose dexmedetomidine groups. A rat model of focal cerebral ischemia-reperfusion injury was established by embolization of the right middle cerebral artery for 60 minutes and reperfusion for 2 hours. The volume of cerebral infarction was calculated by triphenyl tetrazolium chloride staining. The protein expression levels of caspase-3, methionyl aminopeptidase 2 (MetAP2 or MAP2), glial fibrillary acidic protein, and allograft inflammatory factor 1 (AIF-1) in the cerebral cortex were determined by Western blot and immunohistochemistry. Results: The volume of cerebral infarction in rats decreased with increasing dose of dexmedetomidine (P = .039, 95% CI = .027 to .044). The expression levels of caspase-3, glial fibrillary acidic protein, and allograft inflammatory factor 1 and the amount of 4-hydroxynonenal decreased with increasing doses of dexmedetomidine (P = .033, 95% CI = .021 to .037). Methionyl aminopeptidase 2 (MetAP2 or MAP2) expression increased with increasing doses of dexmedetomidine (P = .023, 95% CI = .011 to .028). Conclusion: Dexmedetomidine has a dose-dependent protective effect on cerebral ischemic injury in rats. The neuroprotective effects of dexmedetomidine are achieved, in part, by reducing the oxidative stress response, inhibiting glial overactivation, and inhibiting expression levels of apoptosis-related proteins.

Novel inhibitors of Rickettsia prowazekii methionine aminopeptidase from the Malaria Box.

Methionine aminopeptidases (MetAp) are dinuclear metalloenzymes found in both prokaryotes and eukaryotes that catalyze the hydrolysis of the N-terminal methionine residue from nascent proteins, an important post-translational modification, which makes it an attractive target for drug discovery. Rickettsia prowazekii (Rp) is an obligate pathogen and causative agent of epidemic typhus and typhus fever. In our ongoing search for anti-rickettsial agents we screened 400 compounds from the Malaria Box for inhibition of RpMetAp1 and discovered 12 compounds that inhibited the enzyme with IC50 values ranging from 800 nM to 22 µM. These inhibitors are from eleven different chemical series and represent leads that can be used to discover more potent and efficacious anti-rickettsial agents.

A single amino acid difference between archaeal and human type 2 methionine aminopeptidases differentiates their affinity towards ovalicin.

In almost all living cells, methionine aminopeptidase (MetAP) co-translationally cleaves the initiator methionine in at least 70% of the newly synthesized polypeptides. MetAPs are typically classified into Type 1 and Type 2. While prokaryotes and archaea contain only either Type 1 or Type 2 MetAPs respectively, eukaryotes contain both types of enzymes. Almost all MetAPs published till date cleave only methionine from the amino terminus of the substrate peptides. Earlier experiments on crude Type 2a MetAP isolated from Pyrococcus furiosus (PfuMetAP2a) cosmid protein library was shown to cleave leucine in addition to methionine. Authors in that study have ruled out the PfuMetAP2a activity against leucine substrates and assumed it to be a background reaction contributed by other contaminating proteases. In the current paper, using the pure recombinant enzyme, we report that indeed activity against leucine is directly carried out by the PfuMetAP2a. In addition, the natural product ovalicin which is a specific covalent inhibitor of Type 2 MetAPs does not show efficient inhibition against the PfuMetAP2a. Bioinformatic analysis suggested that a glycine in eukaryotic MetAP2s (G222 in human MetAP2b) and asparagine (N53 in PfuMetAP2a) in archaeal MetAP2s positioned at the analogous position. N53 side chain forms a hydrogen bond with a conserved histidine (H62) at the entrance of the active site and alters its orientation to accommodate the ovalicin. This slight orientational difference of the H62, reduces affinity of the ovalicin by 300,000-fold when compared with the HsMetAP2b inhibition. This difference in the activity is partly reduced in the case of N53G mutation of the PfuMetAP2a.

The Potential Role of the Methionine Aminopeptidase Gene PxMetAP1 in a Cosmopolitan Pest for Bacillus thuringiensis Toxin Tolerance.

Methionine aminopeptidases (MetAPs) catalyze the cleavage of the N-terminal initiator methionine (iMet) in new peptide chains and arylamides, which is essential for protein and peptide synthesis. MetAP is differentially expressed in two diamondback moth (DBM; Plutella xylostella) strains: the G88 susceptible strain and the Cry1S1000 strain, which are resistant to the Bt toxin Cry1Ac, implicating that MetAP expression might be associated with Bt resistance. In this study, we identified and cloned a MetAP gene from DBMs, named PxMetAP1, which has a CDS of 1140 bp and encodes a 379 amino acid protein. The relative expression of PxMetAP1 was found to be ~2.2-fold lower in the Cry1S1000 strain compared to that in the G88 strain. PxMetAP1 presents a stage- and tissue-specific expression pattern, with higher levels in the eggs, adults, integument, and fatbody of DBMs. The linkage between PxMetAP1 and Cry1Ac resistance is verified by genetic linkage analysis. The knockout of PxMetAP1 in G88 by CRISPR/Cas9 leads to a ~5.6-fold decrease in sensitivity to the Cry1Ac toxin, further supporting the association between the PxMetAP1 gene and Bt tolerance. Our research sheds light on the role of MetAP genes in the development of Bt tolerance in P. xylostella and enriches the knowledge for the management of such a cosmopolitan pest.

Methionine aminopeptidase­2 is a pivotal regulator of vasculogenic mimicry.

Vasculogenic mimicry (VM) is the formation of a blood supply system that confers aggressive and metastatic properties to tumors and correlates with a poor prognosis in cancer patients. Thus, the inhibition of VM is considered an effective approach for cancer treatment, although such a mechanism remains poorly described. In the present study, we examined methionine aminopeptidase­2 (MetAP2), a key factor of angiogenesis, and demonstrated that it is pivotal for VM, using pharmacological and genetic approaches. Fumagillin and TNP­470, angiogenesis inhibitors that target MetAP2, significantly suppressed VM in various human cancer cell lines. We established MetAP2­knockout (KO) human fibrosarcoma HT1080 cells using the CRISPR/Cas9 system and found that VM was attenuated in these cells. Furthermore, re­expression of wild­type MetAP2 restored VM in the MetAP2­KO HT1080 cells, but the substitution of D251, a conserved amino acid in MetAP2, failed to rescue the VM. Collectively, our results demonstrate that MetAP2 is critical for VM in human cancer cells and suggest fumagillin and TNP­470 as potent VM­suppressing agents.

Selective inhibition of Helicobacter pylori methionine aminopeptidase by azaindole hydroxamic acid derivatives: Design, synthesis, in vitro biochemical and structural studies.

Methionine aminopeptidases (MetAPs) are an important class of enzymes that work co-translationally for the removal of initiator methionine. Chemical inhibition or gene knockdown is lethal to the microbes suggesting that they can be used as antibiotic targets. However, sequence and structural similarity between the microbial and host MetAPs has been a challenge in the identification of selective inhibitors. In this study, we have analyzed several thousands of MetAP sequences and established a pattern of variation in the S1 pocket of the enzyme. Based on this knowledge, we have designed a library of 17 azaindole based hydroxamic acid derivatives which selectively inhibited the MetAP from H. pylori compared to the human counterpart. Structural studies provided the molecular basis for the selectivity.

Cellular Control of Protein Turnover via the Modification of the Amino Terminus.

The first amino acid of a protein has an important influence on its metabolic stability. A number of ubiquitin ligases contain binding domains for different amino-terminal residues of their substrates, also known as N-degrons, thereby mediating turnover. This review summarizes, in an exemplary way, both older and more recent findings that unveil how destabilizing amino termini are generated. In most cases, a step of proteolytic cleavage is involved. Among the over 500 proteases encoded in the genome of higher eukaryotes, only a few are known to contribute to the generation of N-degrons. It can, therefore, be expected that many processing paths remain to be discovered.

MetAP2 inhibition modifies hemoglobin S to delay polymerization and improves blood flow in sickle cell disease.

Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and ßS-globin. Kinetic studies with MetAP2 show that ßS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood-derived erythroid progenitor 2 cells shows more extensive modification of α-globin than ß-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on ßS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor-treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2-affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.

Inhibition of the invasion and metastasis of mammary carcinoma cells by NBD peptide targeting S100A4 via the suppression of the Sp1/MMP­14 axis.

A synthetic peptide that blocks the interaction between the metastasis­enhancing calcium­binding protein, S100A4, and its effector protein, methionine aminopeptidase 2 (MetAP2) (the NBD peptide), was previously demonstrated to inhibit the angiogenesis of endothelial cells, leading to the regression of human prostate cancer in a xenograft model. However, the effects of the NBD peptide on the malignant properties of cancer cells that express S100A4 remain to be elucidated. The present study demonstrates that the NBD peptide inhibits the invasiveness and metastasis of highly metastatic human mammary carcinoma cells. The introduction of the peptide into MDA­MB­231 variant cells resulted in the suppression of matrix degradation in a gelatin invadopodia assay and invasiveness in a Matrigel invasion assay. In line with these results, the peptide significantly downregulated the expression of matrix metalloproteinase (MMP)­14 (MT1­MMP). Mechanistic analysis of the downregulation of MMP­14 revealed the suppression of the expression of the transcription factor, specificity protein 1 (Sp1), but not that of nuclear factor (NF)­κB, early growth response 1 (EGR1) or ELK3, all of which were reported to be involved in transcriptional regulation of the MMP­14 gene. At the same time, evidence suggested that the NBD peptide also suppressed Sp1 and MMP­14 expression levels in MDA­MB­468 cells. Importantly, the intravenous administration of the NBD peptide encapsulated in liposomes inhibited pulmonary metastasis from mammary gland tumors in mice with xenograft tumors. These results indicate that the NBD peptide can suppress malignant tumor growth through the suppression of the Sp1/MMP­14 axis. Taken together, these results reveal that the NBD peptide acts on not only endothelial cells, but also on tumor cells in an integrated manner, suggesting that the peptide may prove to be a promising cancer therapeutic peptide drug.

In Vivo Imaging of Methionine Aminopeptidase II for Prostate Cancer Risk Stratification.

Prostate cancer is one of the most common malignancies worldwide, yet limited tools exist for prognostic risk stratification of the disease. Identification of new biomarkers representing intrinsic features of malignant transformation and development of prognostic imaging technologies are critical for improving treatment decisions and patient survival. In this study, we analyzed radical prostatectomy specimens from 422 patients with localized disease to define the expression pattern of methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that has been identified as a druggable target in cancer. MetAP2 was highly expressed in 54% of low-grade and 59% of high-grade cancers. Elevated levels of MetAP2 at diagnosis were associated with shorter time to recurrence. Controlled self-assembly of a synthetic small molecule enabled design of the first MetAP2-activated PET imaging tracer for monitoring MetAP2 activity in vivo. The nanoparticles assembled upon MetAP2 activation were imaged in single prostate cancer cells with post-click fluorescence labeling. The fluorine-18-labeled tracers successfully differentiated MetAP2 activity in both MetAP2-knockdown and inhibitor-treated human prostate cancer xenografts by micro-PET/CT scanning. This highly sensitive imaging technology may provide a new tool for noninvasive early-risk stratification of prostate cancer and monitoring the therapeutic effect of MetAP2 inhibitors as anticancer drugs. SIGNIFICANCE: This study defines MetAP2 as an early-risk stratifier for molecular imaging of aggressive prostate cancer and describes a MetAP2-activated self-assembly small-molecule PET tracer for imaging MetAP2 activity in vivo.

Methionine aminopeptidases with short sequence inserts within the catalytic domain are differentially inhibited: Structural and biochemical studies of three proteins from Vibrio spp.

Methionine aminopeptidases (MetAPs) have been recognized as drug targets and have been extensively studied for discovery of selective inhibitors. MetAPs are essential enzymes in all living cells. While most prokaryotes contain a single gene, some prokaryotes and all eukaryotes including human have redundancy. Due to the similarity in the active sites of the MetAP enzyme between the pathogens and human limited the success of discovering selective inhibitors. We recently have discovered that MetAPs with small inserts within the catalytic domain to have different susceptibilities against some inhibitors compared to those that do not have. Using this clue we used bioinformatic tools to identify new variants of MetAPs with inserts in pathogenic species. Two new isoforms were identified in Vibrio species with two and three inserts in addition to an isoform without any insert. Multiple sequence alignment suggested that inserts are conserved in several of the Vibrio species. Two of the three inserts are common between two and three insert isoforms. One of the inserts is identified to have "NNKNN" motif that is similar to well-characterized quorum sensing peptide, "NNWNN". Another insert is predicted to have a posttranslational modification site. Three Vibrio proteins were cloned, expressed, purified, enzyme kinetics established and inhibitor screening has been performed. Several of the pyridinylpyrimidine derivatives selectively inhibited MetAPs with inserts compared to those that do not have, including the human enzyme. Crystal structure and molecular modeling studies provide the molecular basis for selective inhibition.

Structure-based optimisation of orally active & reversible MetAP-2 inhibitors maintaining a tight 'molecular budget'.

Structure-based led optimisation of orally active reversible Methionine Aminopeptidase-2 (MetAP-2) inhibitors utilising a 'molecular budget' medicinal chemistry strategy is described. The key physicochemical parameters of target molecules (cLogP, molecular size and H-bond donor count) were monitored through straightforward and intuitive use of atom count and distribution. The balance between structure-based design and an awareness of the physicochemical properties of the compounds synthesised enabled the rapid identification of a potent molecule with good oral pharmacokinetic (PK) characteristics by making fewer, higher quality compounds. The resulting candidate quality molecule was validated in a mechanistic cellular assay and a rodent secondary immunisation model.

Synthesis, molecular modeling, in vivo study and anticancer activity against prostate cancer of (+) (S)-naproxen derivatives.

In this study, (S)-naproxen thiosemicarbazides (3a-d), 1,2,4-triazoles (4a-c), triazole-thioether hybride compounds (5a-p) were synthesized and their structures (3a, 3d, 4a and 5a-p) were confirmed by FT-IR, 1H NMR,13C NMR, HR-Mass spectra and elemental analysis. These compounds are designed to inhibit methionine amino peptidase-2 (MetAP2) enzyme in prostate cancer. These compounds (3d, 5a-p) evaluated against androgen-independent prostate adenocarcinoma (PC-3, DU-145) and androgen-dependent prostate adenocarcinoma (LNCaP) cell lines by using MTS method. Compounds 5a, 5b, 5d and 5e showed 14.2, 5.8, 10.8 and 8.4 µM anticancer activity against PC-3 cell lines, compounds 5e, 5g and 5n presented anticancer activity against DU-145 cell lines 18.8, 12.25 and 10.2 µM, and compounds 5g, 5m and 5n exhibited anticancer activity against LNCaP cell lines 12.25, 22.76 and 2.21 µM, respectively. Consequently, of these results, compounds 5e and 5n showed the highest activities against androgen dependent and independent prostate cancer cell lines, so these compounds could be potent small molecules against prostate cancer. Furthermore, mitogen-activated protein kinase (MAPK) pathway activation, AKT (protein kinase B) phosphorylation and androgen receptor activation of compound 5n (SGK636) were investigated in LNCaP cells by using Western blot method. Compound 5n (SGK636) was also tested against mRNA expression analysis of the Bax, Bcl-2, Caspase 3, Caspase 9 by using real-time PCR analysis. Compound 5n was given to nude male mice with cancer in comparison to the control group. Compound 5n was found to reverse the malignant phenotype in the nude male mice, whereas the prostate cancer progressed in the control group. Analysis of some blood parameters in the study showed that they were within the normal values with respect to the control. The blood values of the animals treated according to the control group also exhibited compliance with the blood limit values. Molecular docking and dynamics simulation of compound 5n binding to Methionine Aminopeptidase 2 (MetAP2) enzyme rationalized its potential activity. In addition, inhibition assay MetAP2 enzyme of compound 5n was evaluated. Taken together, we suggest compound 5n to be a potential candidate for prostate cancer therapy.

CHD1L promotes EOC cell invasiveness and metastasis via the regulation of METAP2.

Chromodomain helicase DNA binding protein 1-like (CHD1L) gene has been proposed to play an oncogenic role in human hepatocellular carcinoma. Previously we reported that CHD1L overexpression is significantly associated with the metastasis proceeding of epithelial ovarian cancer (EOC), and may predict a poor prognosis in EOC patients. However, the potential oncogenic mechanisms by which CHD1L acts in EOC remain unclear. To elucidate the oncogenic function of CHD1L, we carried out a series of in vitro assays, with effects of CHD1L ectogenic overexpression and silencing being determined in EOC cell lines (HO8910, A2780 and ES2). Real-time PCR and Western blotting analyses were used to identify potential downstream targets of CHD1L in the process of EOC invasion and metastasis. In ovarian carcinoma HO8910 cell lines, ectopic overexpression of CHD1L substantially induced the invasive and metastasis ability of the cancer cells in vitro. In contrast, knockdown of CHD1L using shRNA inhibited cell invasion in vitro in ovarian carcinoma A2780 and ES2 cell lines. We also demonstrated that methionyl aminopeptidase 2 (METAP2) was a downstream target of CHD1L in EOC, and we found a significant, positive correlation between the expression of CHD1L and METAP2 in EOC tissues (P<0.05). Our findings indicate that CHD1L plays a potential role in the inducement of EOC cancer cell invasion and/or metastasis via the regulation of METAP2 expression and suggests that CHD1L inhibition may provide a potential target for therapeutic intervention in human EOC.