Unravelling the Allosteric Targeting of PHGDH at the ACT-Binding Domain with a Photoactivatable Diazirine Probe and Mass Spectrometry Experiments

Abstract

The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among attractive PHGDH inhibitors are the α-ketothioamides. In previous work, we have demonstrated their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action of this series, which would help the rational design of new inhibitors, remained undefined. In the present study, the detailed mechanism-of-action of a representative α-ketothioamide inhibitor is reported using several complementary experimental techniques. Strikingly, our work led to the identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass spectrometry experiments and an original α-ketothioamide diazirine-based photoaffinity probe, we identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of α-ketothioamides. Mutagenesis experiments further documented the specificity of our compound at this allosteric site. Our results thus pave the way for the development of new anticancer drugs using a completely novel mechanism-of-action.

Nucleophilic sulfur controlled efficient ketothioamide synthesis from tribromomethyl carbinols

“…It is worth mentioning that α-ketothioamides 3aa and 3ha prepared by the presented protocol are known to inhibit the activity of phosphoglycerate dehydrogenase (PHGDH), thereby preventing malignant tumour cell growth. 14…”

“…It is worth mentioning that α-ketothioamides 3aa and 3ha prepared by the presented protocol are known to inhibit the activity of phosphoglycerate dehydrogenase (PHGDH), thereby preventing malignant tumour cell growth. 14 To explore the practical utility of the controlled synthesis of α-ketothioamides, a diverse array of amines, such as primary and secondary amines, were reacted with the substrate using the presented protocol (Scheme 2).…”

Efficient Synthesis of α‐Ketothioamides From α‐Nitroketones, Amines or DMF and Elemental Sulfur Under Oxidant‐Free Conditions

“…They have been used as valuable building blocks to construct sulfur‐containing compounds and natural products [10] . Besides synthetic chemistry, they have also proven themselves as promising candidates in medicinal chemistry [8,11] . For example, they have been frequently used in the field of cancer treatments.…”

Direct Synthesis of α-Ketothioamide Derivatives through a One-Pot Reaction of Sulfur Ylides, Nitrosobenzenes, and Thioacetic Acid

“…5,8a Consequently, the synthesis of α-ketothioamide derivatives has attracted great interest among organic and pharmaceutical chemists in recent years. 2,3 However, the conventional methodologies employed for synthesizing thioamides, such as utilizing thionating reagents like Lawesson's reagent on the corresponding amides, are not applicable to the synthesis of α-ketothioamides. This limitation stems from the concern regarding the site selectivity of the two carbonyl groups in the α-ketoamide scaffold.…”

“…Additionally, thioamides display dissimilarities in bond lengths and bond rotation properties when compared to their amide counterparts . These unique attributes make thioamides a significant class of compounds in the realms of natural products, bioactive substances, and polymers. − Among the derivatives of thioamides, α-ketothioamides hold substantial value due to their diverse biological activities and potential for further chemical transformations. , The α-ketothioamide scaffold can be found in various natural or bioactive compounds with a wide range of medicinal properties, such as anticancer, antifungal, and antibacterial properties (Figure S1; see the Supporting Information (SI)). , Consequently, the synthesis of α-ketothioamide derivatives has attracted great interest among organic and pharmaceutical chemists in recent years. , …”