A biosensor for detecting proteolytic cleavage activity of two proteases, coronavirus main protease (M.sup.pro) and papain-like protease (PL.sup.pro) is provided. The biosensor utilizes Bioluminescence Resonance Energy Transfer (BRET) between a donor and two acceptor moieties.

The invention is directed to compositions, e.g., cell-based and multiplexed platforms, to screen for small molecule drugs that inhibit enzymes such as proteases, e.g., viral proteases, e.g., HIV proteases; and methods for making and using these compositions. The invention provides compositions and methods for identifying compositions, e.g., drug molecules, that can inhibit proteases, e.g., viral proteases such as HIV proteases. In alternative embodiments, the invention provides cell-based platforms or assays to screen for compositions, e.g., small molecules or drugs, that inhibit or modify the activity of enzymes such as calcium-dependent protein convertases involved in HIV envelope protein processing, including cleavage of the HIV gp160 envelope precursor, resulting in gp120 and gp41 envelope products. In one embodiment, the invention provides a cell-based or multiplexed platform for monitoring the activity of enzymes, e.g., proteases such as viral proteases.

The invention is directed to compositions, e.g., cell-based and multiplexed platforms, to screen for small molecule drugs that inhibit enzymes such as proteases, e.g., viral proteases, e.g., HIV proteases; and methods for making and using these compositions. The invention provides compositions and methods for identifying compositions, e.g., drug molecules, that can inhibit proteases, e.g., viral proteases such as HIV proteases. In alternative embodiments, the invention provides cell-based platforms or assays to screen for compositions, e.g., small molecules or drugs, that inhibit or modify the activity of enzymes such as calcium-dependent protein convertases involved in HIV envelope protein processing, including cleavage of the HIV gp160 envelope precursor, resulting in gp120 and gp41 envelope products. In one embodiment, the invention provides a cell-based or multiplexed platform for monitoring the activity of enzymes, e.g., proteases such as viral proteases.

The invention is directed to compositions to screen for small molecule drugs that inhibit proteases, such as viral proteases, e.g., HIV proteases; and methods for making and using these compositions. The invention provides compositions and methods for identifying compositions, e.g., drug molecules, that can inhibit proteases, e.g., HIV proteases. In alternative embodiments, the invention provides cell-based assays to screen for compositions, e.g., small molecules or drugs, that inhibit or modify the activity of enzymes such as calcium-dependent protein convertases involved in HIV envelop protein processing, including cleavage of the HIV gp160 envelope precursor, resulting in gp120 and gp41 envelope products.

The present application provides a synthetic molecule and a method for producing the same, and also provides methods for determining a disease or condition in a subject. The method comprises introducing a synthetic molecule comprising a synthetic polymer and a linker, wherein the linker comprises an organic molecule, a spacer sequence, and a reporter, wherein the reporter is cleaved by an agent present in the environment. Diseases and conditions that can be determined by the method are also described in the present application.

In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for detecting proteases including detecting proteases in a biological sample. In alternative embodiments, products of manufacture, formulations, mixtures or kits are used for stabilizing (or substantially stabilizing) nanoparticles in a reversible aggregate, and, detecting the presence of a protease in a fluid, wherein optionally the products formulations or mixtures can aggregate or assemble into nanoparticles such that the nanoparticles undergo plasmonic coupling, and the plasmonic coupling is reversible (or substantially reversible) leading to monodisperse nanoparticles when a chemical cue or signal is added.