Structural plasticity of a transmembrane peptide allows self-assembly into biologically active nanoparticles.

Significant efforts have been devoted to the development of nanoparticular delivering systems targeting tumors. However, clinical application of nanoparticles is hampered by insufficient size homogeneity, difficulties in reproducible synthesis and manufacturing, frequent high uptake in the liver, systemic toxicity of the carriers (particularly for inorganic nanoparticles), and insufficient selectivity for tumor cells. We have found that properly modified synthetic analogs of transmembrane domains of membrane proteins can self-assemble into remarkably uniform spherical nanoparticles with innate biological activity. Self-assembly is driven by a structural transition of the peptide that adopts predominantly a beta-hairpin conformation in aqueous solutions, but folds into an alpha-helix upon spontaneous fusion of the nanoparticles with cell membrane. A 24-amino acid peptide corresponding to the second transmembrane helix of the CXCR4 forms self-assembled particles that inhibit CXCR4 function in vitro and hamper CXCR4-dependent tumor metastasis in vivo. Furthermore, such nanoparticles can encapsulate hydrophobic drugs, thus providing a delivery system with the potential for dual biological activity.

Normalization of proliferation and tight junction formation in bladder epithelial cells from patients with interstitial cystitis/painful bladder syndrome by d-proline and d-pipecolic acid derivatives of antiproliferative factor.

Interstitial cystitis/painful bladder syndrome is a chronic bladder disorder with epithelial thinning or ulceration, pain, urinary frequency and urgency, for which there is no reliably effective therapy. We previously reported that interstitial cystitis/painful bladder syndrome bladder epithelial cells make a glycopeptide antiproliferative factor or 'APF' (Neu5Acα2-3Galß1-3GalNAcα-O-TVPAAVVVA) that induces abnormalities in normal cells similar to those in interstitial cystitis/painful bladder syndrome cells in vitro, including decreased proliferation, decreased tight junction formation, and increased paracellular permeability. We screened inactive APF derivatives for their ability to block antiproliferative activity of asialylated-APF ('as-APF') in normal bladder cells and determined the ability of as-APF-blocking derivatives to normalize tight junction protein expression, paracellular permeability, and/or proliferation of interstitial cystitis/painful bladder syndrome cells. Only two of these derivatives [Galß1-3GalNAcα-O-TV-(d-pipecolic acid)-AAVVVA and Galß1-3GalNAcα-O-TV-(d-proline)-AAVVVA] blocked as-APF antiproliferative activity in normal cells (p < 0.001 for both). Both of these antagonists also 1) significantly increased mRNA expression of ZO-1, occludin, and claudins 1, 4, 8, and 12 in interstitial cystitis/painful bladder syndrome cells by qRT-PCR; 2) normalized interstitial cystitis/painful bladder syndrome epithelial cell tight junction protein expression and tight junction formation by confocal immunofluorescence microscopy; and 3) decreased paracellular permeability of (14) C-mannitol and (3) H-inulin between confluent interstitial cystitis/painful bladder syndrome epithelial cells on Transwell plates, suggesting that these potent APF antagonists may be useful for the development as interstitial cystitis/painful bladder syndrome therapies.

Development of antiproliferative phenylmaleimides that activate the unfolded protein response.

The current paper presents the synthesis and evaluation of a series of maleimides that were designed to inhibit the Cdc25 phosphatase by alkylation of catalytically essential cysteine residues. Although in HepB3 cell culture assays the analogues did exhibit antiproliferative IC(50) values ranging from sub-micromolar to greater than 100 microM, inhibition of Cdc25 through cysteine alkylation could not be demonstrated. It was also found that analysis using fluorescence activated cell sorting (FACS) following treatment with the most potent analogue (1t) did not provide data consistent with inhibition at one specific point in the cell cycle, as would be expected if Cdc25A were inhibited. Further studies with a subset of analogues resulted in a correlation of antiproliferative potencies with activation of the unfolded protein response (UPR). The UPR is a regulatory pathway that temporarily suspends protein production when misfolding of proteins occurs within the endoplastic reticulum (ER). In addition, ER chaperones that promote proper refolding become up-regulated. If cellular damage cannot be resolved by these mechanisms, then the UPR can initiate apoptosis. The current study indicates that these maleimide analogues lead to UPR activation, which is predictive of the selective antiproliferative activity of the series.

Structure-Activity Studies on Antiproliferative Factor (APF) Glycooctapeptide Derivatives.

Antiproliferative factor (APF), a sialylated glycopeptide secreted by explanted bladder epithelial cells from interstitial cystitis/painful bladder syndrome (IC/PBS) patients, and its unsialylated analogue (as-APF) significantly decrease proliferation of bladder epithelial cells and/or certain carcinoma cell lines in vitro. We recently reported a structure-activity relationship profile for the peptide portion of as-APF and revealed that truncation of the C-terminal alanine did not significantly affect antiproliferative activity. To better understand the structural basis for the maintenance of activity of this truncated eight amino acid as-APF (as-APF8), we synthesized several amino acid-substituted derivatives and studied their ability to inhibit bladder epithelial cell proliferation in vitro as well as their solution conformations by CD and NMR spectroscopy. While single amino acid changes to as-APF8 often strongly reduced activity, full potency was retained when the trivaline tail was replaced with three alanines. The Ala(6-8) derivative 9 is the simplest, fully potent APF analogue synthesized to date.

Growth inhibition of hepatocellular carcinoma cells in vitro and in vivo by the 8-methoxy analog of WMC79.

HKH40A (RTA 502), an optimized 8-methoxy analog of the unsymmetrical bifunctional antitumor agent WMC79, was found to be potently active against liver cancer cell growth in vitro and in vivo. Studies on selected human hepatocellular carcinoma (HCC) cell lines with differing p53 status (HepG2, Hep3B, and PLC/PRF/5), revealed that drug-mediated growth inhibition was independent of p53 status. FACS analysis showed an accumulation of cells in S-phase within 24 h of treatment with 100 nM HKH40A. Subsequent incubation of cells, either in the presence of drug or without, caused cell cycle block at the S and G2/M checkpoints, which was consistent with the observed up-regulation of p21, cyclin A, cyclin B1, sustained phosphorylation of Cdk1, and down-regulation of Cdc6, Cdc7, and RRM2. This irreversible growth arrest eventually led to apoptosis. HKH40A completely suppressed growth of the rat transplantable HCC cell line (JM-1) in an orthotopic model in Fisher 344 rats in vivo, without evidence of toxicity. HKH40A may be a useful agent for new therapeutic strategies focusing on inhibition of HCC cell proliferation.

Structure-activity relationship studies for the peptide portion of the bladder epithelial cell antiproliferative factor from interstitial cystitis patients.

We performed comprehensive structure-activity relationship (SAR) studies on the peptide portion of antiproliferative factor (APF), a sialylated frizzled-8 related glycopeptide that inhibits normal bladder epithelial and urothelial carcinoma cell proliferation. Glycopeptide derivatives were synthesized by solid-phase methods using standard Fmoc chemistry and purified by RP-HPLC; all intermediate and final products were verified by HPLC-MS and NMR analyses. Antiproliferative activity of each derivative was determined by inhibition of (3)H-thymidine incorporation in primary normal human bladder epithelial cells. Structural components of the peptide segment of APF that proved to be important for biological activity included the presence of at least eight of the nine N-terminal amino acids, a negative charge in the C-terminal amino acid, a free amino group at the N-terminus, maintenance of a specific amino acid sequence in the C-terminal tail, and trans conformation for the peptide bonds. These data provide critical guidelines for optimization of structure in design of APF analogues as potential therapeutic agents.

Bombesin marine toxin conjugates inhibit the growth of lung cancer cells.

Hemiasterlin (Hem) and dolastatin (Dol) are marine natural products which are cytotoxic for cancer cells. Hem, a tripeptide, and Dol, a hexapeptide, were conjugated with linkers (L) to the universal BB agonist DPhe-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2(BA1) and the effects of the Hem-BB and Dol-BB conjugates investigated on NCI-H1299 lung cancer cells. Hem-LA-BA1 and Hem-LB-BA1 inhibited specific (125I-Tyr4)BB binding to NCI-H1299 cells, which have BB2 receptors (R), with IC50 values of 15 and 25 nM, respectively. Addition of Hem-LA-BA1 and Hem-LB-BA1 to Fura-2 AM loaded cells containing BB2R, caused elevated cytosolic Ca2+. In a growth assay, Hem-LA-BA1 and Hem-LB-BA1 inhibited the proliferation of NCI-H1299 cells. Dol-succinamide (Dols)-LD-BA1 and Dols-LE-BA1 bound with high affinity to NCI-H1299 cells and elevated cytosolic Ca2+, but did not inhibit the proliferation of NCI-H1299 cells. Also, Hem-LA-BA1 inhibited 125I-DTyr-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2 (BA2) binding to Balb/3T3 cells transfected with BB1R or BB2R as well as with BRS-3 with IC50 values of 130, 8, and 540 nM, respectively. These results show that Hem-BB conjugates are cytotoxic for cancer cells containing BB2R.

Rationally designed inhibitors identify STAT3 N-domain as a promising anticancer drug target.

Activation of the signal transducer and activator of transcription 3 (STAT3) is frequently detected in many cancer types. Activated STAT3 may participate in oncogenesis by stimulating cell proliferation and resisting apoptosis, as well as promoting tumor angiogenesis, invasion, and migration. Many STAT3-dependent cellular responses are mediated through interactions with other proteins, and the amino-terminal domain (N-domain) of STAT3 was proposed to be responsible for this. Our NMR studies revealed that synthetic analogs of the STAT4 second alpha-helix bind to the N-domain and perturb its structure. Structural data available for the STAT4 N-domain was used for the rational design of STAT3 helix 2 analogs with enhanced biological activity. Cell-permeable derivatives of the STAT3 second helix were found to directly and specifically bind to STAT3 but not STAT1 as determined by FRET analysis in cells expressing GFP-STAT3 and GFP-STAT1. Furthermore, they potently induced apoptotic death in breast cancer cells but not normal breast cells or STAT3-deficient fibroblasts. The inhibitors caused significant changes in the mitochondrial potential of cancer cells, leading to cell death. These compounds not only are promising drug candidates but also offer a convenient tool for studying the mechanisms of action of STAT transcription factors and have facilitated our understanding of the crucial role of the N-domain in STAT3 function.

Design and synthesis of novel and potent inhibitors of the type II transmembrane serine protease, matriptase, based upon the sunflower trypsin inhibitor-1.

Matriptase, initially isolated from human breast cancer cells in culture, is a member of the emerging class of type II transmembrane serine proteases. Matriptase blockade could potentially modulate tumorigenesis and metastasis in vivo. Sunflower trypsin inhibitor-1 (1, SFTI-1), isolated from sunflower seeds, exhibits very potent matriptase inhibitory activity. On the basis of these findings, we designed and synthesized 13 analogues of the naturally occurring peptide 1 with the intention to explore the structure-activity relationships of this type of bicyclic peptides and to improve inhibitory selectivity and metabolic stability of the disulfide-bridge-containing peptide 1. We discovered that the methylenedithioether-bridged compound 14 demonstrates very potent binding affinity to matriptase. Compound 8 exhibits much better selectivity for inhibition of matriptase versus thrombin, whereas compound 2 becomes a more potent thrombin inhibitor, which can be potentially used as an anticoagulant for prophylaxis and therapy of thromboembolism.

Optimization of naphthalimide-imidazoacridone with potent antitumor activity leading to clinical candidate (HKH40A, RTA 502).

Unsymmetrical bifunctional antitumor agent WMC79 was further optimized to generate compound 7b that not only inhibited the growth of many tumor cell lines, but caused rapid apoptosis. Unlike the parent compound, 7b is toxic to both p53 positive and negative cancer cells. It has potent in vivo activity against xenografts of human colon and pancreatic tumors in athymic mice.

Design and synthesis of paclitaxel conjugated with an ErbB2-recognizing peptide, EC-1.

The selective delivery of therapeutic agents to receptors overexpressed in cancer cells without harming the rest of the body is a major challenge in clinical oncology today. In this study, we report the design and synthesis of paclitaxel (PTX) conjugated with an erbB2-recognizing peptide (EC-1). The cyclic peptide EC-1 specifically binds to the extracellular domain of ErbB2 and selectively inhibits proliferation of breast cancer cells overexpressing ErbB2. PTX is a potent antitumor agent commonly used in the treatment of advanced metastatic breast cancer, yet patients have to suffer some side effects caused by its systemic toxicity. The aim of our conjugate is to specifically deliver antitumor agent PTX to breast cancer cells that overexpress oncogenic ErbB2 with the purpose to reduce toxicity and enhance selective killing of cancer cells. In this study, a concise and efficient synthetic route for the preparation of the PTX-EC-1 conjugate has been developed in 6% overall yield. This synthetic approach provides a general method for conjugating a highly functionalized and disulfide-bridge containing cyclopeptide to Taxol or other antitumor agents.

Synthesis, biological activity, and crystal structure of potent nonnucleoside inhibitors of HIV-1 reverse transcriptase that retain activity against mutant forms of the enzyme.

In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 = 0.2 microM, EC50 = 0.44 microM, and TC50 >/= 100 against WT). This paper describes how substitution on the benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A 7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants.

Synthesis and biological activity of 5-aza-ellipticine derivatives.

Novel 5-aza-ellipticine derivatives were synthesized and tested as antitumor agents. The new compounds were prepared more readily than the analogous ellipticine derivatives, which are known to be potent anti-tumor agents Although the novel 5-aza-ellipticine derivatives are not as biologically active as their corresponding ellipticine analogues, the new compounds represent a new, readily accessible class of heteroaromatic catalytic inhibitors of topoisomerase II and possible anti-tumor agents.

CKAP4/p63 is a receptor for the frizzled-8 protein-related antiproliferative factor from interstitial cystitis patients.

Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF. The antiproliferative activity of APF is effectively inhibited by preincubation with anti-CKAP4/p63-specific antibodies, as well as by short interfering RNA knockdown of CKAP4/p63. Immunofluorescent confocal microscopy showed co-localization of anti-CKAP4/p63 and rhodamine-labeled synthetic APF binding in both cell membrane and perinuclear areas. APF also inhibits the proliferation of HeLa cervical carcinoma cells that are known to express CKAP4/p63. These data indicate that CKAP4/p63 is an important epithelial cell receptor for APF.

PM-20, a novel inhibitor of Cdc25A, induces extracellular signal-regulated kinase 1/2 phosphorylation and inhibits hepatocellular carcinoma growth in vitro and in vivo.

We have synthesized several new phenyl maleimide compounds, which are potent growth inhibitors of several human tumor cell lines. Among these, PM-20 was the most potent with an IC50 of 700 nmol/L for Hep3B human hepatoma cell growth. Two other derivatives, PM-26 and PM-38, did not inhibit Hep3B cell growth even at 100 micromol/L. Interestingly, under identical experimental conditions, PM-20 inhibited DNA synthesis of primary cultures of normal hepatocytes at a 10-fold higher concentration than that needed to inhibit the DNA synthesis of the Hep3B hepatoma cells. PM-20 affected two cellular signaling pathways in Hep3B cells: Cdc25 phosphatase and extracellular signal-regulated kinase (ERK) 1/2. It competitively inhibited the activity of Cdc25 (preferentially Cdc25A) by binding to the active site, likely through the catalytic cysteine, but did not inhibit PTP1B, CD45, or MKP-1 phosphatases. As a result of its action, tyrosine phosphorylation of the cellular Cdc25A substrates Cdk2 and Cdk4 was induced. It also induced strong and persistent phosphorylation of the Cdc25A substrate ERK1/2. Hep3B cell lysates were found to contain ERK2 phosphatase(s) activity, which was inhibited by the actions of PM-20. However, activity of exogenous dual-specificity ERK2 phosphatase MKP1 was not inhibited. Induction of ERK1/2 phosphorylation correlated with the potency of growth inhibition in tumor cell lines and inhibition of ERK1/2 phosphorylation by the mitogen-activated protein kinase (MAPK)/ERK kinase 1/2 inhibitor U0126 or overexpression of the cdc25A gene in Hep3B cells antagonized the growth inhibitory actions of PM-20. Growth of transplantable rat hepatoma cells in vivo was also inhibited by PM-20 action with a concomitant induction of pERK in the tumors. The mechanism(s) of growth inhibition of Hep3B hepatoma cells by the phenyl maleimide PM-20 involves prolonged ERK1/2 phosphorylation, likely resulting from inhibition of the ERK phosphatase Cdc25A. PM-20 thus represents a novel class of tumor growth inhibitor that inhibits mainly Cdc25A, is dependent on ERK activation, and has a considerable margin of selectivity for tumor cells compared with normal cells.

WMC-79, a potent agent against colon cancers, induces apoptosis through a p53-dependent pathway.

WMC-79 is a synthetic agent with potent activity against colon and hematopoietic tumors. In vitro, the agent is most potent against colon cancer cells that carry the wild-type p53 tumor suppressor gene (HCT-116 and RKO cells: GI50<1 nmol/L, LC50 approximately 40 nmol/L). Growth arrest of HCT-116 and RKO cells occurs at the G1 and G2-M check points at sublethal concentrations (10 nmol/L) but the entire cell population was killed at 100 nmol/L. WMC-79 is localized to the nucleus where it binds to DNA. We hypothesized that WMC-79 binding to DNA is recognized as an unrepairable damage in the tumor cells, which results in p53 activation. This triggers transcriptional up-regulation of p53-dependent genes involved in replication, cell cycle progression, growth arrest, and apoptosis as evidenced by DNA microarrays. The change in the transcriptional profile of HCT-116 cells is followed by a change in the levels of cell cycle regulatory proteins and apoptosis. The recruitment of the p53-dependent apoptosis pathway was suggested by the up-regulation of p53, p21, Bax, DR-4, DR-5, and p53 phosphorylated on Ser15; down-regulation of Bcl-2; and activation of caspase-8, -9, -7, and -3 in cells treated with 100 nmol/L WMC-79. Apoptosis was also evident from the flow cytometric studies of drug-treated HCT-116 cells as well as from the appearance of nuclear fragmentation. However, whereas this pathway is important in wild-type p53 colon tumors, other pathways are also in operation because colon cancer cell lines in which the p53 gene is mutated are also affected by higher concentrations of WMC-79.

A new synthetic agent with potent but selective cytotoxic activity against cancer.

The synthesis of novel unsymmetrical bifunctional antitumor agents was accomplished by linking an imidazoacridone moiety to another polycyclic heteroaromatic moiety via linkers of various length and rigidity. These compounds bind to cellular DNA, but it is hypothesized that biological effects become manifested when the drug-DNA complexes interact with critical DNA binding proteins that are involved in repair and transcription. The most promising compound of the series, 4ad (WMC79), consists of an imidazoacridone linked to a 3-nitronaphthalimide moiety via a 1,4-dipropanopiperazine linker. It was found to be potently, but selectively, cytotoxic against colon cancers (GI(50) = 0.5 nM, LC(50) = 32 nM) and leukemias (GI(50) = 3.5 nM, LC(50) = 33 nM). Compound 4ad, which appears to be a candidate for further development as an anticancer drug, kills sensitive cells by induction of apoptosis. It also showed significant in vivo activity against HCT-116 colon cancer xenografts in nude mice. Other compounds in the series also exhibited antitumor properties, but they were significantly lower than that of 4ad.

Transmembrane inhibitors of P-glycoprotein, an ABC transporter.

Drug resistance mediated by ABC transporters such as P-glycoprotein (P-gp) continues to be a major impediment to effective cancer chemotherapy. We have developed a panel of highly specific peptide inhibitors of P-gp based on the structure of the transmembrane domains of the transporter. These peptides are thought to exert their inhibitory action by disrupting the proper assembly of P-gp. A novel 96-well-plate assay based on the efflux of fluorescent P-gp substrate DiOC2 (3-ethyl-2-[3-(3-ethyl-2(3H)-benzoxazolylidene)-1-propenyl]benzoxazolium iodide) was developed and used for structure-functional characterization of transporter inhibitors. The studies strongly suggest that potent and selective inhibitors of ABC transporters can now be developed solely on the basis of the primary structures of the target proteins. The inhibition of P-gp with transmembrane peptides was shown to be chirality-independent. A 25-residue long retroinverso D-analogue of transmembrane domain 5 inhibited the efflux of the fluorescent P-gp substrate with an IC50 of 500 nM. Transmembrane peptides effectively sensitized resistant cancer cells to doxorubicin in vitro without demonstrating any cell toxicity of their own. The newly synthesized P-gp antagonists appear to be promising nontoxic drug resistance inhibitors that merit further development.

The development of VIP-ellipticine conjugates.

The mechanism by which vasoactive intestinal peptide (VIP)-ellipticine (E) conjugates are cytotoxic for human lung cancer cells was investigated. VIP-alanyl-leucyl-alanyl-leucyl-alanine (ALALA)-E and VIP-leucyl-alanyl-leucyl-alanine (LALA)-E inhibited (125)I-VIP binding to NCI-H1299 cells with an IC50 values of 0.5 and 0.1 microM, respectively. VIP-ALALA-E and VIP-LALA-E caused elevation of cAMP in NCI-H1299 cells with ED50 values of 0.7 and 0.1 microM. Radiolabeled VIP-LALA-E was internalized at 37 degrees C and delivered the cytotoxic E into NCI-H1299 cells. VIP-LALA-E inhibited the growth of NCI-H1299 cells in vitro. Three days after the addition of VIP-LALA-E to NCI-H1299 cells, cell viability decreased based on trypan blue exclusion and reduced 3H-thymidine uptake. These results suggest that VIP-E conjugates are internalized in lung cancer cells as a result of VPAC1 receptor-mediated endocytosis.

Caspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells.

Irofulven (hydroxymethylacylfulvene) is a novel antitumor drug, which acts by alkylating cellular macromolecular targets. The drug is a potent inducer of apoptosis in various types of tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to irofulven involving mitochondrial dysfunction and leading to death of prostate tumor LNCaP-Pro5 cells. Irofulven caused early (2-5 hours) translocation of the proapoptotic Bax from cytosol to mitochondria followed by the dissipation of mitochondrial membrane potential and cytochrome c release at 4 to 12 hours. These effects preceded caspase activation and during the first 6 hours were not affected by caspase inhibitors. Processing of caspase-9 initiated the caspase cascade at approximately 6 hours and progressed over time. The activation of the caspase cascade provided a positive feedback loop that enhanced Bcl-2-independent translocation and cytochrome c release. General and specific caspase inhibitors abrogated irofulven-induced apoptotic DNA fragmentation with the following order of potency: pan-caspase > or = caspase-9 > caspase-8/6 > caspase-2 > caspase-3/7 > caspase-1/4. Abrogation of caspase-mediated DNA fragmentation failed to salvage irofulven-treated cells from growth inhibition and loss of viability, demonstrating a substantial contribution of a caspase-independent cell death. Monobromobimane, an inhibitor of alternative caspase-independent apoptotic pathway that is mediated by mitochondrial permeability transition, antagonized both apoptosis, measured as phosphatidylserine externalization, and cytotoxicity of irofulven. Collectively, the results indicate that irofulven-induced signaling is integrated at the level of mitochondrial dysfunction. The induction of both caspase-dependent and caspase-independent death pathways is consistent with pleiotropic effects of irofulven, which include targeting of cellular DNA and proteins.