This Account provides an overview and examples of function-oriented synthesis (FOS) and its increasingly important role in producing therapeutic leads that can be made in a step-economical fashion. Biologically active natural product leads often suffer from several deficiencies. Many are scarce or difficult to obtain from natural sources. Often, they are highly complex molecules and thus not amenable to a practical synthesis that would impact supply. Most are not optimally suitable for human therapeutic use. The central principle of FOS is that the function of a biologically active lead structure can be recapitulated, tuned, or greatly enhanced with simpler scaffolds designed for ease of synthesis and also synthetic innovation. This approach can provide practical access to new (designed) structures with novel activities while at the same time allowing for synthetic innovation by target design. This FOS approach has been applied to a number of therapeutically important natural product leads. For example, bryostatin is a unique natural product anticancer lead that restores apoptosis in cancer cells, reverses multidrug resistance, and bolsters the immune system. Remarkably, it also improves cognition and memory in animals. We have designed and synthesized simplified analogs of bryostatin that can be made in a practical fashion (pilot scale) and are superior to bryostatin in numerous assays including growth inhibition in a variety of human cancer cell lines and in animal models. Laulimalide is another exciting anticancer lead that stabilizes microtubules, like paclitaxel, but unlike paclitaxel, it is effective against multidrug-resistant cell lines. Laulimalide suffers from availability and stability problems, issues that have been addressed using FOS through the design and synthesis of stable and efficacious laulimalide analogs. Another FOS program has been directed at the design and synthesis of drug delivery systems for enabling or enhancing the uptake of drugs or drug candidates into cells and tissue. We have generated improved transporters that can deliver agents in a superior fashion compared with naturally occurring cell-penetrating peptides and that can be synthesized in a practical and step-economical fashion. The use of FOS has allowed for the translation of exciting, biologically active natural product leads into simplified analogs with superior function. This approach enables the development of synthetically innovative strategies while targeting therapeutically novel structures.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

The ability of a drug or probe to cross a biological barrier has historically been viewed to be a function of its intrinsic physical properties. This view has largely restricted drug design and selection to agents within a narrow log P range. Molecular transporters offer a strategy to circumvent these restrictions. In the case of guanidinium-rich transporters (GRTs), a typically highly water-soluble conjugate is found to readily pass through the non-polar membrane of a cell and for some across tissue barriers. This activity opens a field of opportunities for the use of GRTs to enable delivery of polar and nonpolar drugs or probes as well as to enhance uptake of those of intermediate polarity. The field of transporter enabled or enhanced uptake has grown dramatically in the last decade. Some GRT drug conjugates have been advanced into clinical trials. This review will provide an overview of recent work pertinent to the design and mechanism of uptake of GRTs.

Many cancer therapeutic agents elicit resistance that renders them ineffective and often produces cross-resistance to other drugs. One of the most common mechanisms of resistance involves P-glycoprotein (Pgp)-mediated drug efflux. To address this problem, new agents have been sought that are less prone to inducing resistance and less likely to serve as substrates for Pgp efflux. An alternative to this approach is to deliver established agents as molecular transporter conjugates into cells through a mechanism that circumvents Pgp-mediated efflux and allows for release of free drug only after cell entry. Here we report that the widely used chemotherapeutic agent Taxol, ineffective against Taxol-resistant human ovarian cancer cell lines, can be incorporated into a releasable octaarginine conjugate that is effective against the same Taxolresistant cell lines. It is significant that the ability of the Taxol conjugates to overcome Taxol resistance is observed both in cell culture and in animal models of ovarian cancer. The generality and mechanistic basis for this effect were also explored with coelenterazine, a Pgp substrate. Although coelenterazine itself does not enter cells because of Pgp efflux, its octaarginine conjugate does so readily. This approach shows generality for overcoming the multidrug resistance elicited by small-molecule cancer chemotherapeutics and could improve the prognosis for many patients with cancer and fundamentally alter search strategies for novel therapeutic agents that are effective against resistant disease.ovarian cancer ͉ Pgp ͉ Taxol ͉ imaging ͉ luciferase

With over 20 antibody-drug conjugates in clinical trials as well as a recently FDA-approved drug, it is clear that this is becoming an important and viable approach for selectively delivering highly cytotoxic agents to tumor cells while sparing normal tissue. This review discusses the critical aspects for this approach with an emphasis on the properties of the linker between the antibody and the cytotoxic payload that are required for an effective antibody-drug conjugate. Different linkers are illustrated with attention focused on (i) the specifics of attachment to the antibody, (ii) the polarity of the linker, (iii) the trigger on the linker that initiates cleavage from the drug, and (iv) the self-immolative spacer that liberates the active payload. Future directions in the field are proposed.

The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico‐chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE‐987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader‐antibody conjugate by attaching GNE‐987 to an anti‐CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen‐specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.

A novel bioconjugation strategy utilizes a disulfide bond to site-specifically couple a small molecule drug to an antibody, enabling both high circulation stability and quick intracellular release.

The biological and medicinal impacts of proteolysis-targeting chimeras (PROTACs) and related chimeric molecules that effect intracellular degradation of target proteins via ubiquitin ligase-mediated ubiquitination continue to grow. However, these chimeric entities are relatively large compounds that often possess molecular characteristics, which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. We therefore explored the conjugation of such molecules to monoclonal antibodies using technologies originally developed for cytotoxic payloads so as to provide alternate delivery options for these novel agents. In this report, we describe the first phase of our systematic development of antibody–drug conjugates (ADCs) derived from bromodomain-containing protein 4 (BRD4)-targeting chimeric degrader entities. We demonstrate the antigen-dependent delivery of the degrader payloads to PC3-S1 prostate cancer cells along with related impacts on MYC transcription and intracellular BRD4 levels. These experiments culminate with the identification of one degrader conjugate, which exhibits antigen-dependent antiproliferation effects in LNCaP prostate cancer cells.