Downsizing human, bacterial, and viral proteins to short water-stable alpha helices that maintain biological potency

Abstract

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...

Identification of High-Affinity PB1-Derived Peptides with Enhanced Affinity to the PA Protein of Influenza A Virus Polymerase

“…Furthermore, the binding affinity could not be enhanced by substitution of aa 6 with either Y (PB1 5-11 T6Y) or W (PB1 5-11 T6W), two previously described affinity-enhancing amino acid substitutions (21). In contrast, peptide PB1 [1][2][3][4][5][6][7][8][9][10][11] , harboring the four N-terminal amino acids, regained binding affinity, although the IC 50 was 39-fold higher than with PB1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] .…”

“…To determine the minimal requirements of the core PA-binding domain of PB1 (PB1 5-11 ), which was previously proposed by structural analysis (8, 13), we measured the 50% inhibitory concentration (IC 50 ) of this peptide in a competition ELISA (21). No detectable binding was observed with PB1 [5][6][7][8][9][10][11] (Table 1). Furthermore, the binding affinity could not be enhanced by substitution of aa 6 with either Y (PB1 5-11 T6Y) or W (PB1 5-11 T6W), two previously described affinity-enhancing amino acid substitutions (21).…”

“…We then further demonstrated that binding to PA was preserved with peptides of 15 aa in length (PB1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] ) and that an enhanced binding affinity of a PB1-derived peptide correlates with increased antiviral activity (22). However, direct proof that the core PA-binding domain of PB1 (PB1 [5][6][7][8][9][10][11] ) can bind to PA efficiently is missing. Consequently, we wanted to clarify (i) whether peptides only comprising the core PA-binding domain can be used as lead peptides for drug development and (ii) whether we could further improve the binding affinity of the PB1-derived peptide to the PA protein.…”

“…Based on a comprehensive structure-affinity-relationship analysis, we now show that the described core-binding region of PB1 (PB1 [5][6][7][8][9][10][11] ) is not sufficient for PA binding and should be extended to aa 2 to 12. Importantly, our data suggest that increased PA binding of PB1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] can be achieved by affinity enhancing mutations.…”

The Prophylaxis and Treatment with Antiviral Agents of Respiratory Syncytial Virus Infections

“…A collection of publications relating to the use of peptides as inhibitors of RSV fusion has appeared over the past 4 years [40][41][42][43][44]. Perhaps the most interesting is the report by Harrison et al [45] that relates to the discovery of short, water-stable a-helices as potential anti-infectives. Mimicking F483-V495 of the RSV fusion protein heptad repeat, a short 12-residue peptide was synthesized with two 5-residue lactams via K-D linkages.…”