Prostate specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase overexpressed in many forms of prostate cancer. Our laboratory has recently disclosed a class of small molecules, called ARM-Ps (antibody-recruiting molecule targeting prostate cancer) that are capable of enhancing antibody-mediated immune recognition of prostate cancer cells. Interestingly, during the course of these studies, we found ARM-Ps to exhibit extraordinarily high potencies toward PSMA, compared to previously reported inhibitors. Here, we report in-depth biochemical, crystallographic, and computational investigations which elucidate the origin of the observed affinity enhancement. These studies reveal a previously unreported arene-binding site on PSMA, which we believe participates in an aromatic stacking interaction with ARMs. Although this site is composed of only a few amino acid residues, it drastically enhances small molecule binding affinity. These results provide critical insights into the design of PSMA-targeted small molecules for prostate cancer diagnosis and treatment; more broadly, the presence of similar arene-binding sites throughout the proteome could prove widely enabling in the optimization of small-molecule-protein interactions.

Prostate cancer is the second leading cause of cancer-related death among the American male population, and society is in dire need of new approaches to treat this disease. Here we report the design, synthesis, and biological evaluation of a class of bifunctional small molecules, called antibody-recruiting molecules targeting prostate-cancer (ARM-Ps), that enhance the recognition of prostate cancer cells by the human immune system. ARM-P derivatives were designed rationally via the computational analysis of crystallographic data, and we demonstrate here that these materials are able to: (1) bind PSMA with high affinity (high pM to low nM), (2) template the formation of ternary complexes between anti-DNP antibodies, ARM-P, and LNCaP human prostate cancer cells, and (3) mediate the antibody-dependent killing of LNCaP cells in the presence of human effector cells. This manuscript describes the application of fundamental chemical principles to the design of a novel class of molecules with high therapeutic potential. We believe that this general small-molecule-based strategy could give rise to novel directions in treating cancer and other diseases.Prostate cancer is the second leading cause of cancer-related death among the American male population, and it has been predicted that one out of every six American men will develop prostate cancer during their lifetime. 1 Available treatment options, including chemical/surgical castration, radiation therapy, and chemotherapy, are often ineffective against advanced disease, and are also associated with severe side effects. 2 Thus, new approaches to treat prostate cancer are highly desirable. To this end, monoclonal antibody therapies have shown promise; 2 however no such agent has yet successfully obtained FDA approval for treating prostate cancer. Further, antibody drugs are limited by severe side effects, lack of oral bioavailabiliy, and high cost. 3 Here we describe a novel technology for prostate cancer treatment that we believe could address many of the limitations of currently available therapies, and combines advantages of both small-molecule-based and antibody-based strategies. Figure 1). As shown, ARMs are composed of an antibody-binding terminus (ABT), a cell surface binding terminus (CBT), and a linker region. In this manuscript, it is demonstrated that ternary complexes formed between ARM-Ps, human prostate cancer cells (LNCaP cells), and antibodies recognizing the 2,4-dinitrophenyl (DNP) group lead to targeted cell-mediated cytotoxicity of LNCaP cells. The power of this approach derives from the observation that anti-DNP antibodies are already found in the human bloodstream in a high percentage of the human population, 4 and are competent to mediate target cell killing. 5,6 Several approaches have appeared that utilize bifunctional materials to recruit antibodies to human pathogens, 7 but ARM-Ps are the first class of antibodyrecruiting small molecules that target prostate cancer. The general strategy reported herein has the potential to initiate novel...

Staphylococcus aureus (S. aureus) is a Gram-positive bacterial pathogen that has emerged as a major public health threat. Here we report that the cell wall of S. aureus can be covalently reengineered to contain non-native small molecules. This process makes use of endogenous levels of the bacterial enzyme sortase A (SrtA), which ordinarily functions to incorporate proteins into the bacterial cell wall. Thus, incubation of wild-type bacteria with rationally designed SrtA substrates results in covalent incorporation of functional molecular handles (fluorescein, biotin, and azide) into cell wall peptidoglycan. These conclusions are supported by data obtained through a variety of experimental techniques (epifluorescence and electron microscopy, biochemical extraction, and mass spectrometry), and azide incorporation was exploited as a chemical handle to perform an azide-alkyne cycloaddition reaction on the bacterial cell surface. This report represents the first example of cell wall engineering of S. aureus or any other pathogenic Gram-positive bacteria, and has the potential for widespread utility.Staphylococcus aureus (S. aureus) is a Gram-positive bacterial pathogen that has become a major public health threat. Most hospital isolates of S. aureus are resistant to many if not all available treatments,(1) and recent reports suggest that more Americans die every year as a result of S. aureus infection than due to HIV/AIDS, Parkinson's Disease, or emphysema. (2,3) The virulence of this organism is mediated in part by proteins in its cell wall, which enable it to interact with animal cells and tissues, and to evade the human immune system. (4,5) Flexible strategies for modulating the molecular composition of the S. aureus cell wall would therefore be highly desirable, and could enable both fundamental and therapeutic applications.Here we demonstrate for the first time that the cell surface of wild-type S. aureus can be reengineered biosynthetically to incorporate non-native small molecules. Exposure of wildtype bacteria to rationally designed, low molecular weight substrates for the enzyme sortase A (SrtA)(6) leads to covalent incorporation of functional small molecules (fluorescein, biotin, or azide) into the S. aureus cell wall (Figure 1). Diverse experimental techniques are employed to support these conclusions including epifluorescence and electron microscopy, flow cytometry, mass spectrometry, and biochemical cell wall extraction. Furthermore, azide incorporation is exploited as a chemical handle to perform an azide-alkyne cycloaddition reaction on the bacterial cell surface. This report represents the first example RESULTS AND DISCUSSION Substrate Design and Incorporation into S. aureusOur design of synthetic SrtA substrates was based upon the enzyme's mechanism of action, which involves recognition of a conserved pentapeptide motif, typically LPETG in S. aureus, near the C-terminus of various secreted proteins.(6) Upon substrate recognition, the enzyme cleaves the threonine-glycine bond, forming an acyl...

h Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of ␣-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC 50 ) being 2.3 M. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC 50 ) being 0.34 M. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC 50 ] ‫؍‬ 25 to 79 M). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact ␣-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically ␣-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection. More than 2 billion people have been infected with hepatitis B virus (HBV) at some time in their lives and up to 350 million remain chronically infected as carriers of HBV (1, 2). Approximately 20% of chronic hepatitis B patients develop liver cirrhosis, leading to hepatic insufficiency and portal hypertension (3). Furthermore, there is a 100-fold higher risk of development of hepatocellular carcinoma in chronic HBV patients than in noncarriers (4). Every year, HBV infection kills more than 500,000 people from cirrhosis, liver failure, and hepatocellular carcinoma (5).The global level of chronic HBV infection still mandates development of new drugs despite the development of excellent vaccines and drugs against the virus. Seven drugs have been approved by the U.S. Food and Drug Administration for treating HBV infection. Interferon alpha and pegylated interferon alpha are immunomodulatory agents. However, the need for subcutaneous administration, the poor long-term responses, the very low cure rates, and the high frequency of adverse side effects make interferon far from an ideal drug (6). The nucleos(t)ide analog drugs lamivudine, adefovir, entecavir, telbivudine, and tenofovir are phosphorylated to their triphosphate derivatives by cellular enzymes and become chain-terminating substrates of the HBV reverse transcr...