S-trityl-L-cysteine Research Articles

Abstract C108: A novel mitotic kinesin Eg5 inhibitor exerts the growth-inhibitory effect in cancer cells in a manner independent of induction of monoastral formation but dependent of KRAS mutation status

Abstract Mitotic kinesin Eg5 plays an important role in mitosis, as it is critical for proper bipolar spindle assembly. After the discovery of the first Eg5 inhibitor monastrol, a number of Eg5 inhibitors have been developed as anticancer drugs. We have previously synthesized a series of S-trityl-L-cysteine (STLC) derivatives as Eg5 inhibitors and demonstrated potent inhibitory effect on Eg5 ATPase activity and induction of mitotic arrest with characteristic monoastral spindles in HeLa cells. In this study, we evaluated the growth-inhibitory acitivities of novel STLC derivatives, Compound1, Compound2, and Compound3 by MTT assay in a wide range of human cancer cell lines derived from gastric (MKN1, MKN45, MKN74, NUGC3, NUGC4, NCI-N87), colon (C170, DLD1, HCT15, COLO205), pancreatic (AsPC1, BxPC3, SUIT2) and lung (PC-9, PC-14, H1975, H441, A549) cancers and investigated possible predictive biomarkers. Among the derivatives, Compound3 showed the most potent growth-inhibitory effect and sensitivity between cell lines showed marked differences, with IC50 values ranging from 0.134 to 7.14 M. Pancreatic cancer cell line AsPC1 and non-small cell lung cancer (NSCLC) cell lines H441 and A549 which harbor KRAS mutation showed relatively high IC50 values compared to other cell lines without KRAS mutation. Compound3 induced G2/M arrest in all cell lines as early as 8 hours after the treatment by flowcytometry, while the increase of the apoptotic sub-G1 fraction was not observed in these AsPC1, H441 and A549 cells harboring KRAS mutation. These results suggested that differential sensitivity to Compound3 and attenuated induction of apoptosis by Compound3 were attributed to KRAS mutation. Induction of monoastral spindles formation by Compound3 was observed as early as 8 hours after the treatment not only in sensitive cell lines but in refractory cell lines such as AsPC1 and H441 cells by immunocytochemistry, suggesting the induction of monoastral spindles formation does not work as a predictive biomarker for clinical development. There was no correlation observed between the growth-inhibitory effect and the basal Eg5 expression level of each cell line by western blot analysis. No significant difference was observed in the induction of a spindle checkpoint kinase BubR1 phosphorylation among the cells, a possible predictive biomarker in response to the Eg5 inhibitor treatment by western blot analysis. Taken together, a novel mitotic kinesin Eg5 inhibitor Compound3 should be considered for further exploration and development and KRAS mutation status was suggested to be a candidate for a predictive biomarker of poor response to Eg5 inhibitors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C108.

Isobaric Peptide Termini Labeling for MS/MS-Based Quantitative Proteomics

Since its introduction, isobaric peptide labeling has played an important role in relative quantitative comparisons of proteomes. This paper describes isobaric peptide termini labeling (IPTL), a novel approach for the identification and quantification of two differentially labeled states using MS/MS spectra. After endoproteinase Lys-C digestion, peptides were labeled at C-terminal lysine residues with either 2-methoxy-4,5-dihydro-1H-imidazole (MDHI) or with tetradeuterated MDHI-d(4). Subsequently, their N-termini were derivatized either with tetradeuterated succinic anhydride (SA-d(4)) or with SA. The mixed isotopic labeling results in isobaric masses and provided several quantification data points per peptide. The suitability of this approach is demonstrated with MS and MS/MS analyses of Lys-C digests of standard proteins. A conceptually simple quantification strategy with a dynamic range of 25 is achieved through the use of Mascot score ratios. The utility of IPTL for the analysis of proteomes was verified by comparing the well-characterized effect of the antimitotic inhibitor S-Trityl-l-Cysteine (STLC) on HeLa cells that were treated for either 24 or 48 h with the inhibitor. Many apoptosis-linked proteins were identified as being differentially regulated, confirming the suitability of IPTL for the analysis of complex proteomes.

Crystal structure of HsEg5 in complex with S‐trityl‐L‐cysteine

Eg5, a motor kinesin involved in the formation of the bipolar mitotic spindle, is essential for the successful completion of mitosis. Monastrol and S‐trityl‐L‐cysteine (STC) are two well‐characterized inhibitors of Eg5, with STC being the more potent inhibitor. We have determined the 2.5 Åresolution crystal structure of the Eg5 motor domain in complex with STC and Mg ?ADP. STC interacts with Eg5 via a pocket formed by helices a2, a3 and loop L5, and induces conformational changes within the Eg5 necklinker and switch I and II regions similar to those seen with bound monastrol. However, there are two distinctions between the STC‐Eg5 and monastrol‐Eg5 structures: the nature of the interactions between the allosteric ligand and Eg5 and solvent accessibility of residues in the allosteric binding site. Monastrol contacts with Eg5 are largely mediated by nonpolar drug surfaces. During STC binding, contact between the motor domain and polar surfaces of the drug is increased in relation to that of monastrol by over 20 Å2. STC binding occludes ~80 Å2 more of the Eg5 surface from solvent access than does monastrol. Site‐directed mutagenesis and coupled biochemical assays monitoring ATP hydrolysis were used to test the hypothesis that several residues altered in solvent accessibility upon STC binding to Eg5 were critical to the increased efficacy of inhibition by this small molecule.Supported by Louisiana Board of Regents Grant

Conformational specificity in allosteric signaling: high‐throughput measurement of modular secondary structural changes within human Eg5 kinesin

The motor domain of human Eg5 kinesin (HsEg5) displays an allosteric pocket at the extracellular surface; binding of monastrol or S‐trityl‐L‐cysteine (STC) to this allosteric site, created by the L5 loop, and subsequent distal conformational changes impairs ATP hydrolysis and cellular function. We examined the roles of E116 and E118 in the L5 loop in allosteric signaling. First, there is a positional correlation on the outcome of amino acid substitutions, irrespective of its nature. Single‐site substitution of E118 increased basal ATP hydrolysis rates of Eg5, whereas substitution of E116 resulted in lower ATPase rates. Thus, sequence variation at residues 116 and 118 of kinesin L5 loop can drive upregulation and downregulation of ATP hydrolysis, respectively. From high‐throughput measurements of the secondary structure composition of proteins in solution, vibrational signatures from upregulated proteins were distinct from the net changes measured from downregulated proteins. Moreover, spectral changes found for E116 variants were similar to those of wildtype Eg5 with monastrol or STC. Therefore, allosteric inhibition by amino acid substitution or by small molecules both result in convergent steady‐state changes to the secondary structure of the Eg5 motor domain in solution, and these rapid methods will provide insight into how long‐range structural changes impact motor function.

Crystal Structure of HsEg5 in Complex with S-trityl-L-cysteine

Eg5 is a motor kinesin involved in the formation of the bipolar mitotic spindle which is essential for the completion of mitosis. The discovery of a class of allosteric Eg5 inhibitors has raised the possibility of a novel approach for the treatment of cancer. Monastrol and S-trityl-L-cysteine (STC) are two well-characterized inhibitors of Eg5 known to prevent ADP release by the motor domain, with the latter compound being a more potent inhibitor of Eg5 than the former. We have determined the 2.5 Å resolution crystal structure of the Eg5 motor domain in complex with STC and Mg•ADP. STC interacts with Eg5 via a pocket formed by helices α2, α3 and loop L5, and induces conformational changes within the Eg5 motor domain similar to those seen with bound monastrol. The necklinker is positioned in the “docked” conformation seen in the monastrol-bound Eg5 structures, and the switch I and II regions also adopt conformations similar to those observed for bound monastrol. Moreover, STC contacts with Eg5 differ from those seen in monastrol-bound crystal structures. Monastrol contacts with Eg5 are largely mediated by nonpolar surfaces of the drug. During STC binding, contact between the motor domain and polar surfaces of the drug is increased in relation to that of monastrol by over 20 Å2. STC binding occludes approximately 80 Å2 more of the Eg5 surface from solvent access than does monastrol. Site-directed mutagenesis and coupled biochemical assays monitoring ATP hydrolysis were used to examine whether these residues involved in polar interactions between STC and Eg5, and residues altered in solvent accessibility upon STC binding to Eg5, were critical in the increased efficacy of Eg5 inhibition by this small molecule.

Conformational Specificity in Allosteric Signaling: High-throughput Measurement of Modular Secondary Structural Changes within Human Eg5 Kinesin

Communication within allosteric enzymes requires long-range interactions. The ATPase activity of human Eg5 kinesin (HsEg5) is allosterically inhibited upon noncovalent binding of monastrol or S-trityl-L-cysteine (STC) with the L5 loop and subsequent distal conformation changes. Our hypothesis is that the E116 and E118 carboxylates, flanking an isolated β-bridge in the L5 loop, are required for allosteric signaling during small chemical inhibition. Surprisingly, assessment by molecular and biochemical methods revealed that substitution of these carboxylates, irrespective of its nature, has a positional dependence on HsEg5 ATPase rates. Single-site substitution of E118 increased basal ATP hydrolysis rates of Eg5, whereas substitution of E116 resulted in lower ATPase rates. Thus, sequence variation at residues 116 and 118 of the L5 loop can drive upregulation and downregulation of ATP hydrolysis in vitro, respectively. We conducted high-throughput infrared measurements of the secondary structure composition of the upregulated and downregulated proteins in solution. Vibrational signatures from upregulated proteins were distinct from the net changes measured from downregulated proteins. Compared to control samples, in HsEg5 motors with E118 substitutions, amide I′ components between 1642-1620 cm−1 decreased in normalized area, whereas proteins with alterations of E116 exhibited an increased contribution in the amide I′ area for modes between 1684 and 1646 cm−1. Moreover, the spectral changes exhibited by E116 variants were similar to those of wildtype HsEg5 during allosteric inhibition by monastrol or STC. We conclude that allosteric inhibition by amino acid substitution or by small molecules both result in convergent steady-state changes to the secondary structure of the HsEg5 motor domain in solution, and these rapid methods will provide insight into how long-range structural changes impact motor function.

Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly

Bipolar spindle assembly critically depends on the microtubule plus-end-directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus-end-directed motor dynein, or the dynein-binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5-dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus-end-tracking protein CLIP-170. This function of CLIP-170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP-115, a highly homologous protein that lacks the dynein-dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP-170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.

Structure–Activity Relationship of S-Trityl-l-Cysteine Analogues as Inhibitors of the Human Mitotic Kinesin Eg5

The human kinesin Eg5 is a potential drug target for cancer chemotherapy. Eg5 specific inhibitors cause cells to block in mitosis with a characteristic monoastral spindle phenotype. Prolonged metaphase block eventually leads to apoptotic cell death. S-trityl-L-cysteine (STLC) is a tight-binding inhibitor of Eg5 that prevents mitotic progression. It has proven antitumor activity as shown in the NCI 60 tumor cell line screen. It is of considerable interest to define the minimum chemical structure that is essential for Eg5 inhibition and to develop more potent STLC analogues. An initial structure-activity relationship study on a series of STLC analogues reveals the minimal skeleton necessary for Eg5 inhibition as well as indications of how to obtain more potent analogues. The most effective compounds investigated with substitutions at the para-position of one phenyl ring have an estimated K i (app) of 100 nM in vitro and induce mitotic arrest with an EC 50 of 200 nM.

Proteome analysis of apoptosis signaling by S‐trityl‐L‐cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5

Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S-trityl-L-cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-alpha-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68 kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes.

Mps1 Phosphorylates Borealin to Control Aurora B Activity and Chromosome Alignment

Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.

Synthesis and biological evaluation of l-cysteine derivatives as mitotic kinesin Eg5 inhibitors

Inhibition of Eg5 represents a novel approach for the treatment of cancer. Here, we report the synthesis and structure-activity relationship of S-trityl- l-cysteine (STLC) derivatives as Eg5 inhibitors. Some of these derivatives such as 4f demonstrated enhanced inhibitory activity against Eg5 and induced mitotic arrest with characteristic monoastral spindles in HeLa cells.

S-Trityl-L-cysteine Is a Reversible, Tight Binding Inhibitor of the Human Kinesin Eg5 That Specifically Blocks Mitotic Progression

Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-L-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-L-cysteine does not prevent cell cycle progression at the S or G(2) phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-L-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-L-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-trityl-L-cysteine is a tight binding inhibitor (estimation of K(i,app) <150 nm at 300 mm NaCl and 600 nm at 25 mm KCl). S-trityl-L-cysteine binds more tightly than monastrol because it has both an approximately 8-fold faster association rate and approximately 4-fold slower release rate (6.1 microM(-1) s(-1) and 3.6 s(-1) for S-trityl-L-cysteine versus 0.78 microM(-1) s(-1) and 15 s(-1) for monastrol). S-trityl-L-cysteine inhibits Eg5-driven microtubule sliding velocity in a reversible fashion with an IC(50) of 500 nm. The S and D-enantiomers of S-tritylcysteine are nearly equally potent, indicating that there is no significant stereospecificity. Among nine different human kinesins tested, S-trityl-L-cysteine is specific for Eg5. The results presented here together with the proven effect on human tumor cell line growth make S-trityl-L-cysteine a very attractive starting point for the development of more potent mitotic inhibitors.