Recombinant proteins are important therapeutics due to potent, highly specific, and nontoxic actions in vivo. However, they are expensive medicines to manufacture, chemically unstable, and difficult to administer with low patient uptake and compliance. Small molecule drugs are cheaper and more bioavailable, but less target-specific in vivo and often have associated side effects. Here we combine some advantages of proteins and small molecules by taking short amino acid sequences that confer potency and selectivity to proteins, and fixing them as small constrained molecules that are chemically and structurally stable and easy to make. Proteins often use short α-helices of just 1-4 helical turns (4-15 amino acids) to interact with biological targets, but peptides this short usually have negligible α-helicity in water. Here we show that short peptides, corresponding to helical epitopes from viral, bacterial, or human proteins, can be strategically fixed in highly α-helical structures in water. These helix-constrained compounds have similar biological potencies as proteins that bear the same helical sequences. Examples are (i) a picomolar inhibitor of Respiratory Syncytial Virus F protein mediated fusion with host cells, (ii) a nanomolar inhibitor of RNA binding to the transporter protein HIV-Rev, (iii) a submicromolar inhibitor of Streptococcus pneumoniae growth induced by quorum sensing pheromone Competence Stimulating Peptide, and (iv) a picomolar agonist of the GPCR pain receptor opioid receptor like receptor ORL-1. This approach can be generally applicable to downsizing helical regions of proteins with broad applications to biology and medicine.helix | inhibitor | structure | mimetic | anti-infective P roteins are key functional components that define life, aging, disease, and death. Their uses in medicine, science, and industry are however limited by their complexity, high costs, chemical instability, and low bioavailability. Consequently, new chemical technology is needed to create simpler, smaller, cheaper, more stable, and bioavailable molecules that can execute or inhibit selected functions of proteins. If generic approaches could be developed to stabilize or recreate new molecular shapes that mimic structural components of proteins (e.g., helices, turns, strands, and combinations), the resulting molecules could potentially be extremely valuable for interrogating biological systems and for exploring prospective applications such as new pharmaceuticals, diagnostics, vaccines, and nanomaterials.Some proteins elicit biological function through a single short α-helical segment (usually 4-15 amino acids in 1-4 helical turns) that interacts with nucleic acids or proteins (1-3). However, short synthetic peptides of this length are usually not thermodynamically stable helices in water and adopt only random structures (4). Techniques developed as generalized strategies for stabilising or mimicking short peptide α-helices have been limited by lack of helical structure in water, by sequence or context dependent helici...

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Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of N-acryloyllysine piperazides as irreversible inhibitors of TGase 2. Systematic structural modifications on 54 new compounds were performed with a major focus on fluorine-bearing substituents due to the potential of such compounds to serve as radiotracer candidates for positron emission tomography. The determined inhibitory activities ranged from 100 to 10 000 M s, which resulted in comprehensive structure-activity relationships. Structure-activity correlations using various substituent parameters accompanied by covalent docking studies provide an advanced understanding of the molecular recognition for this inhibitor class within the active site of TGase 2. Selectivity profiling of selected compounds for other transglutaminases demonstrated an excellent selectivity toward transglutaminase 2. Furthermore, an initial pharmacokinetic profiling of selected inhibitors was performed, including the assessment of potential membrane permeability and liver microsomal stability.

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Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity. Here we combine structural stability and target specificity of proteins, with low cost and rapid synthesis of small molecules, towards meeting the significant challenge of binding coiled coil proteins in transcriptional regulation. By iteratively truncating a Jun-based peptide from 37 to 22 residues, strategically incorporating i→i+4 helix-inducing constraints, and positioning unnatural amino acids, we have produced short, water-stable, α-helical peptides that bind cFos. A three-dimensional NMR-derived structure for one peptide (24) confirmed a highly stable α-helix which was resistant to proteolytic degradation in serum. These short structured peptides are entropically pre-organized for binding with high affinity and specificity to cFos, a key component of the oncogenic transcriptional regulator Activator Protein-1 (AP-1). They competitively antagonized the cJun–cFos coiled-coil interaction. Truncating a Jun-based peptide from 37 to 22 residues decreased the binding enthalpy for cJun by ∼9 kcal/mol, but this was compensated by increased conformational entropy (TΔS ≤7.5 kcal/mol). This study demonstrates that rational design of short peptides constrained by α-helical cyclic pentapeptide modules is able to retain parental high helicity, as well as high affinity and specificity for cFos. These are important steps towards small antagonists of the cJun-cFos interaction that mediates gene transcription in cancer and inflammatory diseases.