Pseudoirreversible inhibition of prostate-specific membrane antigen by phosphoramidate peptidomimetics

The mode of inhibition for phosphoramidate peptidomimetic inhibitors of prostate-specific membrane antigen was determined by inhibition reversibility experiments. The results revealed that these inhibitors can be classified into three types: pseudoirreversible (compounds 1-3), moderately reversible (compounds 4-9), and rapidly reversible inhibitors (compounds 10 and 11). Representative compounds from each class were further evaluated for their ability to induce cellular internalization of PSMA. Results from these experiments revealed that the pseudoirreversible inhibitor 1 induced the greatest PSMA internalization. The discovery of pseudoirreversible PSMA inhibitors is expected to provide a new avenue of investigation and therapeutic applications for prostate cancer and neurological disorders.     

Substrate specificity of prostate-specific membrane antigen

A series of putative dipeptide substrates of prostate-specific membrane antigen (PSMA) was prepared that explored alpha- and beta/gamma-linked acidic residues at the P1 position and various chromophores at the P2 position, while keeping the P1' residue constant as L-Glu. Four chromophores were examined, including 4-phenylazobenzoyl, 1-pyrenebutyryl, 9-anthracenylcarboxyl-gamma-aminobutyryl, and 4-nitrophenylbutyryl. When evaluating these chromophores, it was found that a substrate containing 4-phenylazobenzoyl at the P2 position was consumed most efficiently. Substitution at the P1 position with acidic residues showed that only gamma-linked L-Glu and D-Glu were recognized by the enzyme, with the former being more readily proteolyzed. Lastly, binding modes of endogenous substrates and our best synthetic substrate (4-phenylazobenzoyl-Glu-gamma-Glu) were proposed by computational docking studies into an X-ray crystal structure of the PSMA extracellular domain.     

Optimizing phenylethylphosphonamidates for the inhibition of prostate-specific membrane antigen

A series of eight N-2-phenylethylphosphonyl derivatives of glutamic acid was prepared to determine if the inhibitory potency of a phenylethylphosphonyl derivative of glutamic acid against prostate-specific membrane antigen (PSMA) could be improved through rational substitutions on the phenyl ring. The design of these eight analogs was based upon the Topliss batchwise approach. Of the inhibitors from the first generation, the 3,4-dichlorophenyl analog exhibited the greatest improvement over the lead compound which was an unsubstituted phenyl derivative, while the 4-methoxyphenyl analog was essentially void of inhibitory potency against PSMA in single-dose studies. From the potency ranking order of the first generation, the parameter most important to the pharmacophore was determined to be pi + sigma. Attempts to optimize further the potency of inhibitors by preparing a second generation of compounds did not result in structures with greater potency than that of the 3,4-dichlorophenyl analog from the first generation. Based upon K(i) values, the 3,4-dichlorophenyl analog represented a potency improvement of nearly one order of magnitude. These results confirm further the usefulness of the Topliss approach to analog development when large library synthesis cannot be achieved readily.     

Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates

To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved K(i) values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n=5 and 1g, n=6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA.     

Phosphoramidate derivatives of hydroxysteroids as inhibitors of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a membrane-bound cell surface peptidase which is over-expressed in prostate cancer cells. The enzymatic activities of PSMA are understood but the role of the enzyme in prostate cancer remains conjectural. We previously confirmed the existence of a hydrophobic binding site remote from the enzyme's catalytic center. To explore the specificity and accommodation of this binding site, we prepared a series of six glutamate-containing phosphoramidate derivatives of various hydroxysteroids (1a-1f). The inhibitory potencies of the individual compounds of the series were comparable to a simple phenylalkyl analog (8), and in all cases IC50 values were sub-micromolar. Molecular docking was used to develop a binding model for these inhibitors and to understand their relative inhibitory potencies against PSMA.     

Purification of prostate-specific membrane antigen using conformational epitope-specific antibody-affinity chromatography

Prostate-specific membrane antigen (PSMA) is a type II membrane protein that has attracted significant attention as a target for immunioscintigraphic and radioimmunotherapeutic applications for prostate cancer. However, definitive studies on its substrate and inhibitor specificity as well as protein-protein interactions have been somewhat limited by difficulties in the purification of native PSMA. In this study, we optimized the purification of native PSMA from LNCaP cells using conformational epitope-specific antibody-affinity chromatography. Western blot analysis and an HPLC-based enzymatic activity assay were used to compare the yield and activity of PSMA purified by different methods. The ratio of purified PSMA in a native and active conformation was determined by quantifying the amount of non-native PSMA not retained in a second antibody-affinity isolation. The addition of both a neutralization step and the inclusion of Zn(2+) to the equilibration buffer in desalting step provides considerable enhancement in the yield of active PSMA from LNCaP cells.     

Efficient and highly stereoselective synthesis of a beta-lactam inhibitor of the serine protease prostate-specific antigen

An efficient synthesis of a beta-lactam precursor of the serine protease, prostate-specific antigen inhibitor 1 has been accomplished. The synthesis relies on two completely stereoselective reactions that allow the introduction of the stereocenters at C-3 and C-4 of the azetidinone ring in a predictable manner.     

The proteolytic activity of human prostate-specific antigen

Human prostate-specific antigen has been found to exhibit a mild activity of protease at neutral pH. This finding is based on two observations: a proteolytic activity was always associated with the antigen fractions during purification, and the proteolytic activity and the antigen were precipitated with specific antibody to the antigen. In comparison with physico-chemical and catalytic properties of known proteases, human prostate-specific antigen is a distinct neutral protease.     

Apical transport and folding of prostate-specific membrane antigen occurs independent of glycan processing

Prostate-specific membrane antigen (PSMA) is an integral cell-surface membrane glycoprotein that is overexpressed in prostate carcinomas rendering it an appropriate target for antibody-based therapeutic strategies. The biosynthesis of PSMA in transfected COS-1 cells reveals a slow conversion of mannose-rich to complex glycosylated PSMA compatible with slow transport kinetics from the endoplasmic reticulum to the Golgi. Importantly, mannose-rich PSMA persists as a trypsin-sensitive protein throughout its entire life cycle, and only Golgi-located PSMA glycoforms acquire trypsin resistance. This resistance, used here as a tool to examine correct folding, does not depend on the type of glycosylation, because different PSMA glycoforms generated in the presence of inhibitors of carbohydrate processing in the Golgi are also trypsin resistant. The conformational transition of PSMA to a correctly folded molecule is likely to occur in the Golgi and does not implicate ER molecular chaperones, such as BiP. We show here that PSMA is not only heavily N-but also O-glycosylated. The question arising is whether glycans, which do not play a role in folding of PSMA, are implicated in its transport to the cell surface. Neither the cell-surface expression of PSMA nor its efficient apical sorting in polarized Madin-Darby canine kidney cells are influenced by modulators of N- and O-glycosylation. The acquisition of folding determinants in the Golgi, therefore, is an essential prerequisite for protein trafficking and sorting of PSMA and suggests that altered or aberrant glycosylation often occurring during tumorigenesis has no regulatory effect on the cell-surface expression of PSMA.     

A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.     

The chymotrypsin-like activity of human prostate-specific antigen, gamma-seminoprotein

gamma-Seminoprotein (gamma-Sm) is a human prostate-specific antigen and a serine protease judging from the complete amino acid sequence which shows extensive homology with the kallikrein family. The enzymatic activity of gamma-Sm was defined as a chymotrypsin-like activity using reduced and S-3-(trimethylated amino)propylated lysozyme and insulin-oxidized A and B chains as substrates. The -Leu/Ser- peptide bond of lysozyme was rapidly hydrolyzed by gamma-Sm. gamma-Sm also hydrolyzed the -Phe/Glu- of lysozyme and the -Leu/Cys(SO3H)- of insulin B chain. Insulin A chain and arginyl- or lysyl-linkage of these proteins were not hydrolyzed by gamma-Sm at all.     

High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.     

Rational truncation of an RNA aptamer to prostate-specific membrane antigen using computational structural modeling

RNA aptamers represent an emerging class of pharmaceuticals with great potential for targeted cancer diagnostics and therapy. Several RNA aptamers that bind cancer cell-surface antigens with high affinity and specificity have been described. However, their clinical potential has yet to be realized. A significant obstacle to the clinical adoption of RNA aptamers is the high cost of manufacturing long RNA sequences through chemical synthesis. Therapeutic aptamers are often truncated postselection by using a trial-and-error process, which is time consuming and inefficient. Here, we used a "rational truncation" approach guided by RNA structural prediction and protein/RNA docking algorithms that enabled us to substantially truncateA9, an RNA aptamer to prostate-specific membrane antigen (PSMA),with great potential for targeted therapeutics. This truncated PSMA aptamer (A9L; 41mer) retains binding activity, functionality, and is amenable to large-scale chemical synthesis for future clinical applications. In addition, the modeled RNA tertiary structure and protein/RNA docking predictions revealed key nucleotides within the aptamer critical for binding to PSMA and inhibiting its enzymatic activity. Finally, this work highlights the utility of existing RNA structural prediction and protein docking techniques that may be generally applicable to developing RNA aptamers optimized for therapeutic use.     

Thiophilic interaction chromatography of prostate-specific antigen

Prostate-specific antigen (PSA) protein and complexes of PSA with α₁-antichymotrypsin (PSA-ACT) or α₂-macroglobulin (PSA-A₂M) prepared in vitro, have strong affinity for different thiophilic gels (T-gel). Free PSA, and these PSA complexes can be isolated due to their affinity for T-gels. The average recovery of PSA from several of the T-gels, based upon ELISA measurements, was 84 to 94%. The data suggest that T-gel affinity can be explored for the purification of free and complexed PSA from various biologic fluids.