Novel Psilocin Prodrugs with Altered Pharmacological Properties as Candidate Therapies for Treatment-Resistant Anxiety Disorders.

The psychedelic prodrug psilocybin has shown therapeutic benefits for the treatment of numerous psychiatric conditions. Despite positive clinical end points targeting depression and anxiety, concerns regarding the duration of the psychedelic experience produced by psilocybin, associated with enduring systemic exposure to the active metabolite psilocin, pose a barrier to its therapeutic application. Our objective was to create a novel prodrug of psilocin with similar therapeutic benefits but a reduced duration of psychedelic effects compared with psilocybin. Here, we report the synthesis and functional screening of 28 new chemical entities. Our strategy was to introduce a diversity of cleavable groups at the 4-hydroxy position of the core indole moiety to modulate metabolic processing. We identified several novel prodrugs of psilocin with altered pharmacokinetic profiles and reduced pharmacological exposure compared with psilocybin. These candidate prodrugs have the potential to maintain the long-term benefits of psilocybin therapy while attenuating the duration of psychedelic effects.

Synthesis and preliminary evaluation of octreotate conjugates of bioactive synthetic amatoxins for targeting somatostatin receptor (sstr2) expressing cells.

Targeted cancer therapy represents a paradigm-shifting approach that aims to deliver a toxic payload selectively to target-expressing cells thereby sparing normal tissues the off-target effects associated with traditional chemotherapeutics. Since most targeted constructs rely on standard microtubule inhibitors or DNA-reactive molecules as payloads, new toxins that inhibit other intracellular targets are needed to realize the full potential of targeted therapy. Among these new payloads, α-amanitin has gained attraction as a payload in targeted therapy. Here, we conjugate two synthetic amanitins at different sites to demonstrate their utility as payloads in peptide drug conjugates (PDCs). As an exemplary targeting agent, we chose octreotate, a well-studied somatostatin receptor (sstr2) peptide agonist for the conjugation to synthetic amatoxins via three tailor-built linkers. The linker chemistry permitted the evaluation of one non-cleavable and two cleavable self-immolative conjugates. The immolating linkers were chosen to take advantage of either the reducing potential of the intracellular environment or the high levels of lysosomal proteases in tumor cells to trigger toxin release. Cell-based assays on target-positive Ar42J cells revealed target-specific reduction in viability with up to 1000-fold enhancement in bioactivity compared to the untargeted amatoxins. Altogether, this preliminary study enabled the development of a highly modular synthetic platform for the construction of amanitin-based conjugates that can be readily extended to various targeting moieties.

Synthesis and evaluation of "Ama-Flash", a photocaged amatoxin prodrug for light-activated RNA Pol II inhibition and cell death.

Amanitin is used extensively as a research tool to inhibit RNA Pol II thereby implicating its role in mRNA transcription. Recently, amanitin has gained traction as a toxic payload for targeted therapy. Here we report the first-ever photocaged amanitin analog, that is non-toxic and can be pre-loaded into cells. Light provides a means to inhibit RNA Pol II and provoke cell death on-demand.

Design, Synthesis, and Biochemical Evaluation of Alpha-Amanitin Derivatives Containing Analogs of the trans-Hydroxyproline Residue for Potential Use in Antibody-Drug Conjugates.

Alpha-amanitin, an extremely toxic bicyclic octapeptide extracted from the death-cap mushroom, Amanita phalloides, is a highly selective allosteric inhibitor of RNA polymerase II. Following on growing interest in using this toxin as a payload in antibody-drug conjugates, herein we report the synthesis and biochemical evaluation of several new derivatives of this toxin to probe the role of the trans-hydroxyproline (Hyp), which is known to be critical for toxicity. This structure activity relationship (SAR) study represents the first of its kind to use various Hyp-analogs to alter the conformational and H-bonding properties of Hyp in amanitin.

Scaling Amatoxin Synthesis with an Improved Route to (2S,3R,4R)-Dihydroxyisoleucine Exemplified by a Toxic, Clickable α-Amanitin Analogue.

Here we report a scalable synthesis of the key amino acid residue, (2S,3R,4R)-4,5-dihydroxyisoleucine (DHIle) in α-amanitin, that in turn enables the scalable synthesis of an equipotent analogue, Asn(N-ethylazide)-S,6'-dideoxy-α-amanitin, suitable for CuAAC conjugation to empower studies on therapeutic antibody-drug conjugates.

Meeting key synthetic challenges in amanitin synthesis with a new cytotoxic analog: 5'-hydroxy-6'-deoxy-amanitin.

Appreciating the need to access synthetic analogs of amanitin, here we report the synthesis of 5'-hydroxy-6'-deoxy-amanitin, a novel, rationally-designed bioactive analog and constitutional isomer of α-amanitin, that is anticipated to be used as a payload for antibody drug conjugates. In completing this synthesis, we meet the challenge of diastereoselective sulfoxidation by presenting two high-yielding and diastereoselective sulfoxidation approaches to afford the more toxic (R)-sulfoxide.

Synthesis of the Death-Cap Mushroom Toxin α-Amanitin.

α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, Amanita phalloides. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody-drug conjugates. For over 60 years, A. phalloides has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-tryptathionine sulfoxide bridge, enantioselective synthesis of (2 S,3 R,4 R)-4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation.

Synthesis of a Cytotoxic Amanitin for Biorthogonal Conjugation.

Alpha-amanitin is an exceedingly toxic, naturally occurring, bicyclic octapeptide that inhibits RNA polymerase and results in cellular and organismal death. Here we report the straightforward synthesis of an amanitin analogue that exhibited near-native toxicity. A pendant alkyne was readily installed to enable copper-catalyzed alkyne-azide cycloaddition (CuAAC) to azido-rhodamine and two azide-bearing versions of the RGD peptide. The fluorescent toxin analogue entered cells and provoked morphological changes consistent with cell death. The latter two conjugates are as toxic as the parent alkyne precursor, which demonstrates that conjugation does not diminish toxicity. In addition, we showed that toxicity depends on a single diastereomer of the unnatural amino acid, dihydroxyisoleucine (DHIle), at position 3. The convenient synthesis of a heptapeptide precursor now provides access to bioactive amanitin analogues that may be readily conjugated to biomolecules of interest.