Exploration of a Large Virtual Chemical Space: Identification of Potent Inhibitors of Lactate Dehydrogenase-A against Pancreatic Cancer.

It is imperative to explore the gigantic available chemical space to identify new scaffolds for drug lead discovery. Identifying potent hits from virtual screening of large chemical databases is challenging and computationally demanding. Rather than the traditional two-dimensional (2D)/three-dimensional (3D) approaches on smaller chemical libraries of a few hundred thousand compounds, we screened a ZINC library of 15 million compounds using multiple computational methods. Here, we present the successful application of a virtual screening methodology that identifies several chemotypes as starting hits against lactate dehydrogenase-A (LDHA). From 29 compounds identified from virtual screening, 17 (58%) showed IC50 values < 63 µM, two showed single-digit micromolar inhibition, and the most potent hit compound had IC50 down to 117 nM. We enriched the database and employed an ensemble approach by combining 2D fingerprint similarity searches, pharmacophore modeling, molecular docking, and molecular dynamics. WaterMap calculations were carried out to explore the thermodynamics of surface water molecules and gain insights into the LDHA binding pocket. The present work has led to the discovery of two new chemical classes, including compounds with a succinic acid monoamide moiety or a hydroxy pyrimidinone ring system. Selected hits block lactate production in cells and inhibit pancreatic cancer cell lines with cytotoxicity IC50 down to 12.26 µM against MIAPaCa-2 cells and 14.64 µM against PANC-1, which, under normoxic conditions, is already comparable or more potent than most currently available known LDHA inhibitors.

Novel pyrazolo[4,3-d]pyrimidine microtubule targeting agents (MTAs): Synthesis, structure-activity relationship, in vitro and in vivo evaluation as antitumor agents.

The design, synthesis, and biological evaluation of a series novel N1­methyl pyrazolo[4,3-d]pyrimidines as inhibitors of tubulin polymerization and colchicine binding were described here. Synthesis of target compounds involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, characterized and identified with 1H NMR and NOESY spectroscopy. All compounds, except 10, inhibited [3H]colchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9 and 11-13 strongly inhibited the polymerization of tubulin, with IC50 values of 0.45, 0.42, 0.49 and 0.42 µM, respectively. Compounds 14-16 inhibited the polymerization of tubulin with IC50s near ∼1 µM. Compounds 9, 12, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented ßIII-tubulin mediated cancer cell resistance to taxanes and other MTAs, and compounds 9-17 circumvented Pgp-mediated drug resistance. In the standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI50 values (≤10 nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was significantly (P < 0.0001) better than paclitaxel at reducing MCF-7 TUBB3 (ßIII-tubulin overexpressing) tumors in a mouse xenograft model. Collectively, these studies support the further preclinical development of the pyrazolo[4,3-d]pyrimidine scaffold as a new generation of tubulin inhibitors and 9 as an anticancer agent with advantages over paclitaxel.

Potential of substituted quinazolines to interact with multiple targets in the treatment of cancer.

The efficacy of quinazoline-based antiglioma agents has been attributed to their effects on microtubule dynamics.1,2 The design, synthesis and biological evaluation of quinazolines as potent inhibitors of multiple intracellular targets, including microtubules and multiple RTKs, is described. In addition to the known ability of quinazolines 1 and 2 to cause microtubule depolymerization, they were found to be low nanomolar inhibitors of EGFR, VEGFR-2 and PDGFR-ß. Low nanomolar inhibition of EGFR was observed for 1-3 and 9-10. Compounds 1 and 4 inhibited VEGFR-2 kinase with activity better than or equal to that of sunitinib. In addition, compounds 1 and 2 had similar potency to sunitinib in the CAM angiogenesis assay. Multitarget activities of compounds in the present study demonstrates that the quinazolines can affect multiple pathways and could lead to these agents having antitumor potential caused by their activity against multiple targets.

The 3-D conformational shape of N-naphthyl-cyclopenta[d]pyrimidines affects their potency as microtubule targeting agents and their antitumor activity.

A series of methoxy naphthyl substituted cyclopenta[d]pyrimidine compounds, 4-10, were designed and synthesized to study the influence of the 3-D conformation on microtubule depolymerizing and antiproliferative activities. NOESY studies with the N,2-dimethyl-N-(6'-methoxynaphthyl-1'-amino)-cyclopenta[d]pyrimidin-4-amine (4) showed hindered rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. In contrast, NOESY studies with N,2-dimethyl-N-(5'-methoxynaphthyl-2'-amino)-cyclopenta[d]pyrimidin-4-amine (5) showed free rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. The rotational flexibility and conformational dissimilarity between 4 and 5 led to a significant difference in biological activities. Compound 4 is inactive while 5 is the most potent in this series with potent microtubule depolymerizing effects and low nanomolar IC50 values in vitro against a variety of cancer cell lines. The ability of 5 to inhibit tumor growth in vivo was investigated in a U251 glioma xenograft model. The results show that 5 had better antitumor effects than the positive control temozolomide and have identified 5 as a potential preclinical candidate for further studies. The influence of conformation on the microtubule depolymerizing and antitumor activity forms the basis for the development of conformation-activity relationships for the cyclopenta[d]pyrimidine class of microtubule targeting agents.

Design, synthesis and preclinical evaluation of 5-methyl-N4-aryl-furo[2,3-d]pyrimidines as single agents with combination chemotherapy potential.

The design, synthesis and biological evaluation of 4-substituted 5-methyl-furo[2,3-d]pyrimidines is described. The Ullmann coupling of 5-methyl-furo[2,3-d]pyrimidine with aryl iodides was successfully optimized to synthesize these analogs. Compounds 6-10 showed single-digit nanomolar inhibition of EGFR kinase. Compounds 1 and 6-10 inhibited VEGFR-2 kinase better than or equal to sunitinib. Compounds 1 and 3-10 were more potent inhibitors of PDGFR-ß kinase than sunitinib. In addition, compounds 4-11 had higher potency in the CAM angiogenesis assay than sunitinib. Compound 1 showed in vivo efficacy in an A498 renal xenograft model in mice. Multiple RTK and tubulin inhibitory attributes of 1, 4, 6 and 8 indicates that these compounds may be valuable preclinical single agents targeting multiple intracellular targets.

Interleukin (IL)-6 modulates transforming growth factor-ß receptor I and II (TGF-ßRI and II) function in epidermal keratinocytes.

It been shown that IL-6 modulates TGF-ß1 expression in fibroblasts, however, what role IL-6 plays concerning TGF-ßR expression and function in skin is unknown. Therefore, the aim of this study was to investigate the mechanism by which IL-6 might modulates TGF-ß receptors in skin. Skin from WT, IL-6 over-expressing mice and IL-6 treated keratinocyte cultures was analysed for TGF-ßRI and TGF-ßRII expression via histology, PCR and flow cytometry. Receptor function was assessed by cell migration, bromodeoxyuridine (BrdU) proliferation assays, and Smad7 expression and Smad2/3 phosphorylation. Receptor localization within the membrane was determined by co-immunoprecipitation. IL-6 overexpression and treatment increased TGF-ßRII expression in the epidermis. IL-6 treatment of keratinocytes induced TGF-ßRI and II expression and augmented TGF-ß1-induced function as demonstrated through increased migration and decreased proliferation. Additionally, IL-6 treatment of keratinocytes altered receptor activity as indicated by altered Smad2/3 phosphorylation and increased Smad7 and membrane localization. These results suggest that IL-6 regulates keratinocyte function by modulating TGF-ßRI and II expression and signal transduction via trafficking of the receptor to lipid raft pools.

Synthesis and evaluation of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines as receptor tyrosine kinase and thymidylate synthase inhibitors and as antitumor agents.

In an effort to optimize the structural requirements for combined cytostatic and cytotoxic effects in single agents, a series of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines 3-7 were synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs) as well as thymidylate synthase (TS). The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo/5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate aryl thiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six-step sequence. Biological evaluation of these compounds indicated dual activity in RTKs and human TS (hTS). In the VEGFR-2 assay, compound 5 was equipotent to the standard compound semaxanib and was better than standard TS inhibitor pemetrexed, in the hTS assay. Compounds 3, 6 and 7 were nanomolar inhibitors of hTS and were several fold better than pemetrexed.

Discovery and preclinical evaluation of 7-benzyl-N-(substituted)-pyrrolo[3,2-d]pyrimidin-4-amines as single agents with microtubule targeting effects along with triple-acting angiokinase inhibition as antitumor agents.

The utility of cytostatic antiangiogenic agents (AA) in cancer chemotherapy lies in their combination with cytotoxic chemotherapeutic agents. Clinical combinations of AA with microtubule targeting agents (MTAs) have been particularly successful. The discovery, synthesis and biological evaluations of a series of 7-benzyl-N-substituted-pyrrolo[3,2-d]pyrimidin-4-amines are reported. Novel compounds which inhibit proangiogenic receptor tyrosine kinases (RTKs) including vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor-ß (PDGFR-ß) and epidermal growth factor receptor (EGFR), along with microtubule targeting in single molecules are described. These compounds also inhibited blood vessel formation in the chicken chorioallantoic membrane (CAM) assay, and some potently inhibited tubulin assembly (with activity comparable to that of combretastatin A-4 (CA)). In addition, some of the analogs circumvent the most clinically relevant tumor resistance mechanisms (P-glycoprotein and ß-III tubulin expression) to microtubule targeting agents (MTA). These MTAs bind at the colchicine site on tubulin. Two analogs displayed two to three digit nanomolar GI50 values across the entire NCI 60 tumor cell panel and one of these, compound 7, freely water soluble as its HCl salt, afforded excellent in vivo antitumor activity against an orthotopic triple negative 4T1 breast cancer model and was superior to doxorubicin.

A small molecule with anticancer and antimetastatic activities induces rapid mitochondrial-associated necrosis in breast cancer.

Therapy for treatment-resistant breast cancer provides limited options and the response rates are low. Therefore, the development of therapies with alternative chemotherapeutic strategies is necessary. AG311 (5-[(4-methylphenyl)thio]-9H-pyrimido[4,5-b]indole-2,4-diamine), a small molecule, is being investigated in preclinical and mechanistic studies for treatment of resistant breast cancer through necrosis, an alternative cell death mechanism. In vitro, AG311 induces rapid necrosis in numerous cancer cell lines as evidenced by loss of membrane integrity, ATP depletion, HMGB1 (high-mobility group protein B1) translocation, nuclear swelling, and stable membrane blebbing in breast cancer cells. Within minutes, exposure to AG311 also results in mitochondrial depolarization, superoxide production, and increased intracellular calcium levels. Additionally, upregulation of mitochondrial oxidative phosphorylation results in sensitization to AG311. This AG311-induced cell death can be partially prevented by treatment with the mitochondrial calcium uniporter inhibitor, Ru360 [(µ)[(HCO2)(NH3)4Ru]2OCl3], or an antioxidant, lipoic acid. Additionally, AG311 does not increase apoptotic markers such as cleavage of poly (ADP-ribose) polymerase (PARP) or caspase-3 and -7 activity. Importantly, in vivo studies in two orthotopic breast cancer mouse models (xenograft and allograft) demonstrate that AG311 retards tumor growth and reduces lung metastases better than clinically used agents and has no gross or histopathological toxicity. Together, these data suggest that AG311 is a first-in-class antitumor and antimetastatic agent inducing necrosis in breast cancer tumors, likely through the mitochondria.

A new anti-glioma therapy, AG119: pre-clinical assessment in a mouse GL261 glioma model.

BACKGROUND: High grade gliomas (HGGs; grades III and IV) are the most common primary brain tumors in adults, and their malignant nature ranks them fourth in incidence of cancer death. Standard treatment for glioblastomas (GBM), involving surgical resection followed by radiation and chemotherapy with temozolomide (TMZ) and the anti-angiogenic therapy bevacizumab, have not substantially improved overall survival. New therapeutic agents are desperately needed for this devastating disease. Here we study the potential therapeutic agent AG119 in a pre-clinical model for gliomas. AG119 possesses both anti-angiogenic (RTK inhibition) and antimicrotubule cytotoxic activity in a single molecule. METHODS: GL261 glioma-bearing mice were either treated with AG119, anti-VEGF (vascular endothelial growth factor) antibody, anti c-Met antibody or TMZ, and compared to untreated tumor-bearing mice. Animal survival was assessed, and tumor volumes and vascular alterations were monitored with morphological magnetic resonance imaging (MRI) and perfusion-weighted imaging, respectively. RESULTS: Percent survival of GL261 HGG-bearing mice treated with AG119 was significantly higher (p < 0.001) compared to untreated tumors. Tumor volumes (21-31 days following intracerebral implantation of GL261 cells) were found to be significantly lower for AG119 (p < 0.001), anti-VEGF (p < 0.05) and anti-c-Met (p < 0.001) antibody treatments, and TMZ-treated (p < 0.05) mice, compared to untreated controls. Perfusion data indicated that both AG119 and TMZ were able to reduce the effect of decreasing perfusion rates significantly (p < 0.05 for both), when compared to untreated tumors. It was also found that IC50 values for AG119 were much lower than those for TMZ in T98G and U251 cells. CONCLUSIONS: These data support further exploration of the anticancer activity AG119 in HGG, as this compound was able to increase animal survival and decrease tumor volumes in a mouse GL261 glioma model, and that AG119 is also not subject to methyl guanine transferase (MGMT) mediated resistance, as is the case with TMZ, indicating that AG119 may be potentially useful in treating resistant gliomas.

Identification of novel drugs to target dormant micrometastases.

BACKGROUND: Cancer-specific survival has changed remarkably little over the past half century, mainly because metastases that are occult at diagnosis and generally resistant to chemotherapy subsequently develop months, years or even decades following definitive therapy. Targeting the dormant micrometastases responsible for these delayed or occult metastases would represent a major new tool in cancer patient management. Our hypothesis is that these metastases develop from micrometastatic cells that are suppressed by normal extracellular matrix (ECM). METHODS: A new screening method was developed that compared the effect of drugs on the proliferation of cells grown on a normal ECM gel (small intestine submucosa, SISgel) to cells grown on plastic cell culture plates. The desired endpoint was that cells on SISgel were more sensitive than the same cells grown as monolayers. Known cancer chemotherapeutic agents show the opposite pattern. RESULTS: Screening 13,000 compounds identified two leads with low toxicity in mice and EC50 values in the range of 3-30 µM, depending on the cell line, and another two leads that were too toxic to mice to be useful. In a novel flank xenograft method of suppressed/dormant cells co-injected with SISgel into the flank, the lead compounds significantly eliminated the suppressed cells, whereas conventional chemotherapeutics were ineffective. Using a 4T1 triple negative breast cancer model, modified for physiological metastatic progression, as predicted, both lead compounds reduced the number of large micrometastases/macrometastases in the lung. One of the compounds also targeted cancer stem cells (CSC) isolated from the parental line. The CSC also retained their stemness on SISgel. Mechanistic studies showed a mild, late apoptotic response and depending on the compound, a mild arrest either at S or G2/M in the cell cycle. CONCLUSIONS: In summary we describe a novel, first in class set of compounds that target micrometastatic cells and prevent their reactivation to form recurrent tumors/macrometastases.

The design, synthesis and biological evaluation of conformationally restricted 4-substituted-2,6-dimethylfuro[2,3-d]pyrimidines as multi-targeted receptor tyrosine kinase and microtubule inhibitors as potential antitumor agents.

A series of eleven conformationally restricted, 4-substituted 2,6-dimethylfuro[2,3-d]pyrimidines was designed to explore the bioactive conformation required for dual inhibition of microtubule assembly and receptor tyrosine kinases (RTKs), and their biological activities are reported. All three rotatable single bonds in the lead compound 1 were sequentially restricted to address the role of each in SAR for microtubule and RTK inhibitory effects. Compounds 2, 3, 7 and 10 showed microtubule depolymerizing activity comparable to or better than the lead 1, some with nanomolar EC50 values. While compound 8 had no effect on microtubules, 8 and 10 both showed potent RTK inhibition with nanomolar IC50s. These compounds confirm that the bioactive conformation for RTK inhibition is different from that for tubulin inhibition. The tetrahydroquinoline analog 10 showed the most potent dual tubulin and RTK inhibitory activities (low nanomolar inhibition of EGFR, VEGFR2 and PDGFR-ß). Compound 10 has highly potent activity against many NCI cancer cell lines, including several chemo-resistant cell lines, and could serve as a lead for further preclinical studies.

The design and discovery of water soluble 4-substituted-2,6-dimethylfuro[2,3-d]pyrimidines as multitargeted receptor tyrosine kinase inhibitors and microtubule targeting antitumor agents.

The design, synthesis and biological evaluations of fourteen 4-substituted 2,6-dimethylfuro[2,3-d]pyrimidines are reported. Four compounds (11-13, 15) inhibit vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor ß (PDGFR-ß), and target tubulin leading to cytotoxicity. Compound 11 has nanomolar potency, comparable to sunitinib and semaxinib, against tumor cell lines overexpressing VEGFR-2 and PDGFR-ß. Further, 11 binds at the colchicine site on tubulin, depolymerizes cellular microtubules and inhibits purified tubulin assembly and overcomes both ßIII-tubulin and P-glycoprotein-mediated drug resistance, and initiates mitotic arrest leading to apoptosis. In vivo, its HCl salt, 21, reduced tumor size and vascularity in xenograft and allograft murine models and was superior to docetaxel and sunitinib, without overt toxicity. Thus 21 affords potential combination chemotherapy in a single agent.

Synthesis and biological characterization of an orally bioavailable lactate dehydrogenase-A inhibitor against pancreatic cancer.

Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice.

Evaluation of 99mTc-probestin for imaging APN expressing tumors by SPECT.

Aminopeptidase N (APN) is known to play important roles in tumor angiogenesis, tumor cell invasion, and metastasis. Thus, APN is an attractive biomarker for imaging tumor angiogenesis. Here we report results obtained from biodistribution and single photon emission computed tomography (SPECT) imaging studies of a technetium-99m labeled probestin (a potent APN inhibitor) conjugate containing a tripeptide, Asp-DAP-Cys (DAP=2,3-diaminopropionic acid), chelator and a 8-amino-3,6-dioxaoctanoic acid (PEG2) linker conducted in nude mice xenografted with HT-1080 human fibrosarcoma tumors (APN-positive tumors). These results collectively demonstrate that (99m)Tc-probestin uptake by tumors and other APN expressing tissues in vivo is specific and validate the use of probestin as a vector for targeting APN in vivo.

Solid phase synthesis and biological evaluation of probestin as an angiogenesis inhibitor.

Probestin is a potent aminopeptidase N (APN) inhibitor originally isolated from the bacterial culture broth. Here, we report probestin synthesis by solid phase peptide synthesis (SPPS) method and evaluated its activity to inhibit angiogenesis using a chicken embryo chorioallantoic membrane (CAM) assay and a CAM tumor xenograft model. Results from these studies demonstrate that probestin inhibits the angiogenic activity and tumor growth.

N2-Trimethylacetyl substituted and unsubstituted-N4-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines: design, cellular receptor tyrosine kinase inhibitory activities and in vivo evaluation as antiangiogenic, antimetastatic and antitumor agents.

Six novel N(4)-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines and their N(2)-trimethylacetyl substituted analogs were synthesized as receptor tyrosine kinase (RTK) inhibitors. A microwave-mediated Sonogashira reaction was used as a key step for the synthesis of these compounds. Biological evaluation, in whole cell assays, showed that some analogs had remarkable inhibitory activity against a variety of RTKs and in particular cytotoxic activity against A431 tumor cells in culture. The inhibitory data against RTKs in this study demonstrated that variation of the 4-anilino substituents of these analogs dictates both potency and specificity of inhibitory activity against various RTKs. The study also supported the hypothesis that interaction of substituents on the 2-amino group with hydrophobic site-II provides an increase in potency. Compound 8 of this series was selected for evaluation in vivo in a B16-F10 syngeneic mouse tumor model and exhibited significant reduction in tumor growth rate, in tumor vascular density and in metastases to the lung compared to the control.

Synthesis and biological activity of 5-chloro-N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamines as vascular endothelial growth factor receptor-2 inhibitors and antiangiogenic agents.

Inhibition of receptor tyrosine kinase (RTK) signaling pathways is an important area for the development of novel anticancer agents. Numerous multikinase inhibitors (MKIs) have been recently approved for the treatment of cancer. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the principal mediator of tumor angiogenesis. In an effort to develop ATP-competitive VEGFR-2 selective inhibitors the 5-chloro-N(4)-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold was designed. The synthesis of the target compounds involved N-(4,5-dichloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethylpropanamide) as a common intermediate. A nucleophilic displacement of the 4-chloro group of the common intermediate by appropriately substituted anilines afforded the target compounds. Biological evaluation indicated that compound 5 is a potent and selective VEGFR-2 inhibitor comparable to sunitinib and semaxinib.

Precision nephrotoxicity testing using 3D in vitro models.

Nephrotoxicity is a significant concern during the development of new drugs or when assessing the safety of chemicals in consumer products. Traditional methods for testing nephrotoxicity involve animal models or 2D in vitro cell cultures, the latter of which lack the complexity and functionality of the human kidney. 3D in vitro models are created by culturing human primary kidney cells derived from urine in a 3D microenvironment that mimics the fluid shear stresses of the kidney. Thus, 3D in vitro models provide more accurate and reliable predictions of human nephrotoxicity compared to existing 2D models. In this review, we focus on precision nephrotoxicity testing using 3D in vitro models with human autologous urine-derived kidney cells as a promising approach for evaluating drug safety.

Synthesis and evaluation of novel Tc-99m labeled probestin conjugates for imaging APN/CD13 expression in vivo.

The enzyme aminopeptidase N (APN, also known as CD13) is known to play an important role in tumor proliferation, attachment, angiogenesis, and tumor invasion. In this study, we hypothesized that a radiolabeled high affinity APN inhibitor could be potentially useful for imaging APN expression in vivo. Here, we report synthesis, radiolabeling, and biological evaluation of new probestin conjugates containing a tripeptide, N,N-dimethylglycyl-l-lysinyl-l-cysteinylamide (N(3)S), chelator. New probestin conjugates were synthesized by solid-phase peptide synthesis method, purified by reversed-phase HPLC, and characterized by electrospray mass spectrometry. The conjugates were complexed with Re(V) and (99m)Tc(V) by transmetalation using corresponding Re(V) or (99m)Tc(V) gluconate synthon. The mass spectral analyses of ReO-N(3)S-Probestin conjugates were consistent with the formation of neutral Re(V)O-N(3)S complexes. Initial biological activity of ReO-N(3)S-Probestin conjugates determined by performing an in vitro APN enzyme assay using intact HT-1080 cells demonstrated higher inhibition of APN enzyme activity than bestatin. In vivo biodistribution and whole body planar imaging studies of (99m)TcO-N(3)S-PEG(2)-Probestin performed in nude mice xenografted with human fibrosarcoma tumors derived from HT-1080 cells demonstrated a tumor uptake value of 2.88 ± 0.64%ID/g with tumor-to-blood and tumor-to-muscle ratios of 4.8 and 5.3, respectively, at 1 h postinjection (p.i.). Tumors were clearly visible in whole body planar image obtained at 1 h p.i., but not when the APN was competitively blocked with a coinjection of excess nonradioactive ReO-N(3)S-PEG(2)-Probestin conjugate. These results demonstrate the feasibility of using high affinity APN inhibitor conjugates as targeting vectors for in vivo targeting of APN.