Short peptide pharmacophores developed from protein phosphatase-1 disrupting peptides (PDPs).

PP1 is a major phosphoserine/threonine-specific phosphatase that is involved in diseases such as heart insufficiency and diabetes. PP1-disrupting peptides (PDPs) are selective modulators of PP1 activity that release its catalytic subunit, which then dephosphorylates nearby substrates. Recently, PDPs enabled the creation of phosphatase-recruiting chimeras, which are bifunctional molecules that guide PP1 to a kinase to dephosphorylate and inactivate it. However, PDPs are 23mer peptides, which is not optimal for their use in therapy due to potential stability and immunogenicity issues. Therefore, we present here the sequence optimization of the 23mer PDP to a 5mer peptide, involving several attempts considering structure-based virtual screening, high throughput screening and peptide sequence optimization. We provide here a strong pharmacophore as lead structure to enable PP1 targeting in therapy or its use in phosphatase-recruiting chimeras in the future.

Structural basis of Naa20 activity towards a canonical NatB substrate.

N-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

Towards Dissecting the Mechanism of Protein Phosphatase-1 Inhibition by Its C-Terminal Phosphorylation.

Phosphoprotein phosphatase-1 (PP1) is a key player in the regulation of phospho-serine (pSer) and phospho-threonine (pThr) dephosphorylation and is involved in a large fraction of cellular signaling pathways. Aberrant activity of PP1 has been linked to many diseases, including cancer and heart failure. Besides a well-established activity control by regulatory proteins, an inhibitory function for phosphorylation (p) of a Thr residue in the C-terminal intrinsically disordered tail of PP1 has been demonstrated. The associated phenotype of cell-cycle arrest was repeatedly proposed to be due to autoinhibition of PP1 through either conformational changes or substrate competition. Here, we use PP1 variants created by mutations and protein semisynthesis to differentiate between these hypotheses. Our data support the hypothesis that pThr exerts its inhibitory function by mediating protein complex formation rather than by a direct mechanism of structural changes or substrate competition.

Microcystins: Synthesis and structure-activity relationship studies toward PP1 and PP2A.

Microcystins are highly toxic cyanotoxins responsible for plant, animal and human poisoning. Exposure to microcystins, mainly through drinkable water and contaminated food, is a current world health concern. Although it is quite challenging, the synthesis of these potent cyanotoxins, analogs and derivatives helps to evaluate their toxicological properties and to elucidate their binding mechanisms to their main targets Protein Phosphatase-1 (PP1) and -2A (PP2A). This review focuses on synthetic approaches to prepare microcystins and analogs and compiles structure-activity relationship (SAR) studies that describe the unique features of microcystins that make them so potent.

Synthesis of Highly Selective Submicromolar Microcystin-Based Inhibitors of Protein Phosphatase (PP)2A over PP1.

Research and therapeutic targeting of the phosphoserine/threonine phosphatases PP1 and PP2A is hindered by the lack of selective inhibitors. The microcystin (MC) natural toxins target both phosphatases with equal potency, and their complex synthesis has complicated structure-activity relationship studies in the past. We report herein the synthesis and biochemical evaluation of 11 MC analogues, which was accomplished through an efficient strategy combining solid- and solution-phase approaches. Our approach led to the first MC analogue with submicromolar inhibitory potency that is strongly selective for PP2A over PP1 and does not require the complex lipophilic Adda group. Through mutational and structural analyses, we identified a new key element for binding, as well as reasons for the selectivity. This work gives unprecedented insight into how selectivity between these phosphatases can be achieved with MC analogues.

Phosphatases: Their Roles in Cancer and Their Chemical Modulators.

Phosphatases are involved in basically all cellular processes by dephosphorylating cellular components such as proteins, phospholipids and second messengers. They counteract kinases of which many are established oncogenes, and therefore kinases are one of the most important drug targets for targeted cancer therapy. Due to this relationship between kinases and phosphatases, phosphatases are traditionally assumed to be tumour suppressors. However, research findings over the last years prove that this simplification is incorrect, as bona-fide and putative phosphatase oncogenes have been identified. We describe here the role of phosphatases in cancer, tumour suppressors and oncogenes, and their chemical modulators, and discuss new approaches and opportunities for phosphatases as drug targets.

In situ synthesis-gelation at room temperature vs. heating-cooling procedure. Fine tuning of molecular gels derived from succinic acid and L-valine.

HYPOTHESIS: The reaction between succinic anhydride and a diamine derived from L-valine should afford efficiently a molecular gelator. Based on this reaction, it should be feasible to prepare molecular gels at room temperature, avoiding the conventional thermal treatment required for the solubilization of the gelator, by in situ, simultaneous, synthesis and gelation. The gels prepared by in situ and conventional heating-cooling protocols could present important differences relevant for potential practical applications of these materials. EXPERIMENTAL: The gelator was synthesized by reaction of succinic anhydride and a diamine derived from L-valine, affording two new amide bonds. The molecular gels were studied by IR, NMR, electron microscopy, X-ray diffraction and DSC. FINDINGS: The results indicate that different polymorphic fibrillar networks are formed depending on the gel preparation method, highlighting how the properties of molecular gels can be tuned in this way. Significant differences between thermal and in situ gels were found in properties such as thermal stability, thixotropic behavior or release of an entrapped dye. In situ synthesis-gelation has also been shown to provide gels in media such as oleic acid which cannot be jellified by conventional heating-cooling procedures.