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Cooperman, B. S.

Publications and source records attributed to Cooperman, B. S..

5 recordsLinked to original sources

Mechanism-Based Discovery of New TRIDs Targeting Ribosome Pockets

Approximately 10% of all human pathogenic mutations are nonsense mutations which give rise to premature termination codon (PTC) diseases, leading to an estimated 35 million people worldwide who harbor such mutations. To date, no approaches leading to medical treatments which increase PTC readthrough have received FDA approval. However, one well-tolerated translation readthrough inducing drug (TRID), ataluren, was conditionally approved for 11 years by the European Medicines Agency for treatment of nonsense mutation Duchenne Muscular Dystrophy. In recent work we have shown that ataluren increases readthrough indirectly, by exclusively inhibiting termination without directly affecting readthrough by suppressor aminoacyl-tRNAs (sup-aa-tRNAs). Based on the idea that this mode of action (MoA) might be the reason for the low toxicity of ataluren, we have discovered potential new TRIDs, which target pockets within the ribosome to which the termination factor eRF1 binds but sup-aa-tRNAs do not. These new TRIDs, dipeptide derivatives of eRF1 and some Sartans, mimic the MoA of ataluren. Three Sartans, which are angiotensin II receptor blockers and FDA-approved for treatment of hypertension, are significantly more potent than ataluren in our reconstituted in vitro readthrough assay. These compounds might be more effective than ataluren in treating some PTC diseases. We anticipate that further development of these lead compounds will lead to more general effectiveness, used both alone and in combination with other classes of readthrough inducers.

biochemistry↗

Suppression of nonsense mutations by small, cyclic peptides

Premature termination codons in mRNAs result from nonsense mutations and hinder the translation of full-length, functional proteins. Nonsense mutations cause numerous serious genetic diseases, including cystic fibrosis and Duchenne muscular dystrophy. Several small-molecule drugs have been reported that could potentially ameliorate these diseases by promoting translational readthrough at the premature termination codon. However, utilization of many of these molecules faces problems such as limited efficacy or high cellular toxicity. Using a selection strategy in Saccharomyces cerevisiae coupling suppression of endogenous nonsense mutations to cell survival, we identified ten readthrough-promoting cyclic peptides from a DNA-encoded library. The selected cyclic peptides suppress nonsense mutations in various reporter genes, and the candidates inducing the highest readthrough levels display no observable cytotoxicity in yeast. Mutational analysis of the most promising cyclic peptide demonstrate that most amino acid side chains contribute to the readthrough-stimulating activity. Importantly, this cyclic peptide appears to bind directly to the eukaryotic core translation machinery and promotes readthrough in vitro by interfering with ribosomal decoding. Our results suggest that small, cyclic peptides selected in vivo could represent a novel drug type to treat the many incurable human genetic diseases that are caused by nonsense mutations.

molecular biology↗

Mechanism-based approach in designing patient-specific combination therapies for nonsense mutation diseases

Premature termination codon (PTC) diseases, arising as a consequence of nonsense mutations in a patients DNA, account for approximately 12% of all human disease mutations. Currently there are no FDA approved treatments for increasing PTC readthrough in nonsense mutation diseases, although one translational readthrough inducing drug, ataluren, has had conditional approval for treatment of Duchenne muscular dystrophy in Europe and elsewhere for 10 years. Ataluren displays consistent low toxicity in clinical trials for treatment of several different PTC diseases, but its therapeutic effects on such diseases are inconsistent. The identity of the stop codon and its sequence context are major determinants of PTC readthrough efficiency in both the absence and presence of nonsense suppressors. Previously we have shown that ataluren stimulates readthrough exclusively by competitively inhibiting release factor complex (RFC, eRF1.eRF3.GTP)-dependent catalysis of translation termination. Here, using an in vitro reconstituted system (PURE-LITE) and both ensemble and single molecule assays, we demonstrate that PTC identity and the immediately adjacent mRNA sequence contexts modulate the catalytic activity of RFC in terminating peptide elongation. Such modulation largely determines the effectiveness of ataluren in stimulating readthrough, whether added alone or in combination with either the aminoglycoside G418 or an anticodon edited aa-tRNA, each of which stimulate readthrough by mechanisms orthogonal to that of ataluren. Our results provide an attractive rationale for the variability of ataluren effectiveness in stimulating readthrough in clinical trials. Patients harboring a PTC mutation with a sequence context promoting strong interaction with RFC are predicted to be resistant to ataluren, whereas ataluren treatment should be more effective for patient sequences conferring weaker interaction with RFC.

biochemistry↗

Single-Molecule Studies of Cognate and Near-Cognate Elongation in an in vitro Eukaryotic Translation System

The ribosome plays a central role in translation of the genetic code into amino acid sequences during synthesis of polypeptides. During each cycle of peptide elongation, the ribosome must discriminate between correct and incorrect aminoacyl-tRNAs according to the codon present in its A-site. Ribosomes rely on a complex sequence of proofreading mechanisms to minimize erroneous selection of incorrect aminoacyl-tRNAs that would lead to mistakes in translation. These mechanisms have been studied extensively in prokaryotic organisms, but eukaryotic elongation is less well understood. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) with an in vitro eukaryotic translation system to investigate tRNA selection and subsequent steps during peptide elongation. We compared accommodation of a tryptophan-aminoacyl-tRNA into the ribosomal A-site containing either a cognate or near-cognate codon and unexpectedly found that, following an initial slow sampling event, subsequent near-cognate sampling events proceeded more rapidly than the initial event. Further, we found a strong negative correlation between the concentration of near-cognate aminoacyl-tRNA and the efficiency of tRNA accommodation. These novel characteristics of near-cognate interaction with the eukaryotic ribosome suggest that rejection of a near-cognate tRNAs leads to formation of an altered ribosomal conformation that assists in rejecting subsequent incorrect tRNA interactions.

biophysics↗

A new in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery

Nonsense suppressors (NonSups) induce \"readthrough\", i.e., the selection of near cognate tRNAs at premature termination codons and insertion of the corresponding amino acid into nascent polypeptide. Prior readthrough measurements utilized contexts in which NonSups can promote readthrough directly, by binding to one or more of the components of the protein synthesis machinery, or indirectly, by several other mechanisms. Here we utilize a new, highly-purified in vitro assay to measure exclusively direct nonsense suppressor-induced readthrough. Of 16 NonSups tested, 12 display direct readthrough, with results suggesting that such NonSups act by at least two different mechanisms. In preliminary work we demonstrate the potential of single molecule fluorescence energy transfer measurements to elucidate mechanisms of NonSup-induced direct readthrough, which will aid efforts to identify NonSups having improved clinical efficacy.\n\nTable of Contents artwork\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC=\"FIGDIR/small/330506_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@a548dborg.highwire.dtl.DTLVardef@15237bcorg.highwire.dtl.DTLVardef@c93d3forg.highwire.dtl.DTLVardef@1d40e2c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗