Miniproteins as a Powerful Modality in Drug Development.

Miniproteins are a diverse group of protein scaffolds characterized by small (1-10 kDa) size, stability, and versatility in drug-like roles. Coming largely from native sources, they have been widely adopted into drug development pipelines. While their structures and capabilities are diverse, the approaches to their utilization share more similarities with each other than with more widely used modalities (e.g., antibodies or small molecules). In this review, we highlight recent advances in miniprotein-based approaches to otherwise poorly addressed clinical needs, including structure-based and functional characterization. We also summarize their unique screening strategies and pharmacology considerations. Through a greater understanding of the unique properties that make them attractive for drug design, miniproteins can be effectively utilized against targets that are intractable by other approaches.

Dysregulation of dopamine receptor D2 as a sensitive measure for Huntington disease pathology in model mice.

The ability to quantitatively evaluate the impact of a potential therapeutic intervention for Huntington disease (HD) in animal models for the disease is a critical step in the pathway to development of an effective therapy for this devastating neurodegenerative disorder. We report here an approach that combines a cell-based assay's quantitative accuracy and direct relationship to molecular processes with the ability to directly monitor effects in HD model mouse neurons. To accomplish this goal, we have developed an accurate quantitative reporter assay for a transcript known to be down-regulated as an early consequence of mutant huntingtin expression. This system uses mouse strains carrying a GFP reporter for the expression of the dopamine receptor D2, expressed in the medium spiny neurons of the basal ganglion. This receptor consistently demonstrates reduced expression in patients and murine models, and the FACS-based assay gives a highly accurate and quantitative readout of this pathology in mouse neurons expressing mutant huntingtin. For four genetic models and one viral model, a highly reproducible time course of loss of reporter expression is observed. This quantitative measure of HD pathology can be used to measure the effects of HD therapeutics in small cohorts with high confidence. We further demonstrate that the introduction of an shRNA against the huntingtin transgene by virus can improve this pathological status in medium spiny neurons transduced with the construct. We believe this system can be of great utility in the validation of effective therapeutic interventions for HD.

CYpHER: Catalytic extracellular targeted protein degradation with high potency and durable effect.

Many disease-causing proteins have multiple pathogenic mechanisms, and conventional inhibitors struggle to reliably disrupt more than one. Targeted protein degradation (TPD) can eliminate the protein, and thus all its functions, by directing a cell's protein turnover machinery towards it. Two established strategies either engage catalytic E3 ligases or drive uptake towards the endolysosomal pathway. Here we describe CYpHER (CatalYtic pH-dependent Endolysosomal delivery with Recycling) technology with potency and durability from a novel catalytic mechanism that shares the specificity and straightforward modular design of endolysosomal uptake. By bestowing pH-dependent release on the target engager and using the rapid-cycling transferrin receptor as the uptake receptor, CYpHER induces endolysosomal target delivery while re-using drug, potentially yielding increased potency and reduced off-target tissue exposure risks. The TfR-based approach allows targeting to tumors that overexpress this receptor and offers the potential for transport to the CNS. CYpHER function was demonstrated in vitro with EGFR and PD-L1, and in vivo with EGFR in a model of EGFR-driven non-small cell lung cancer.

Ex silico engineering of cystine-dense peptides yielding a potent bispecific T cell engager.

Cystine-dense peptides (CDPs) are a miniprotein class that can drug difficult targets with high affinity and low immunogenicity. Tools for their design, however, are not as developed as those for small-molecule and antibody drugs. CDPs have diverse taxonomic origins, but structural characterization is lacking. Here, we adapted Iterative Threading ASSEmbly Refinement (I-TASSER) and Rosetta protein modeling software for structural prediction of 4298 CDP scaffolds and performed in silico prescreening for CDP binders to targets of interest. Mammalian display screening of a library of docking-enriched, methionine and tyrosine scanned (DEMYS) CDPs against PD-L1 yielded binders from four distinct CDP scaffolds. One was affinity-matured, and cocrystallography yielded a high-affinity (KD = 202 pM) PD-L1-binding CDP that competes with PD-1 for PD-L1 binding. Its subsequent incorporation into a CD3-binding bispecific T cell engager produced a molecule with pM-range in vitro T cell killing potency and which substantially extends survival in two different xenograft tumor-bearing mouse models. Both in vitro and in vivo, the CDP-incorporating bispecific molecule outperformed a comparator antibody-based molecule. This CDP modeling and DEMYS technique can accelerate CDP therapeutic development.

Mammalian Surface Display Screening of Diverse Cystine-Dense Peptide Libraries for Difficult-to-Drug Targets.

Many diseases are mediated by targets that are not amenable to conventional small-molecule drug approaches. While antibody-based drugs have undeniable utility, peptides of the 1-9 kDa size range (10-80 amino acids) have drawn interest as alternate drug scaffolds This is born of a desire to identify compounds with the advantages of antibody-based therapeutics (affinity, potency, specificity, and ability to disrupt protein:protein interactions) without all of their liabilities (large size, expensive manufacturing, and necessity of humanization). Of these alternate scaffolds, cystine-dense peptides (CDPs) have several specific benefits. Due to their stable intra-chain disulfide bridges, CDPs often demonstrate resistance to heat and proteolysis, along with low immunogenicity. These properties do not require chemical modifications, permitting CDP screening by conventional genetic means. The cystine topology of a typical CDP requires an oxidative environment, and we have found that the mammalian secretory pathway is most effective at allowing diverse CDPs to achieve a stable fold. As such, high-diversity screens to identify CDPs that interact with targets of interest can be efficiently conducted using mammalian surface display. In this protocol, we present the theory and tools to conduct a mammalian surface display screen for CDPs that bind with targets of interest, including the steps to validate binding and mature the affinity of preliminary candidates. With these methods, CDPs of all kinds can be brought to bear against targets that would benefit from a peptide-based intervention.

A TfR-Binding Cystine-Dense Peptide Promotes Blood-Brain Barrier Penetration of Bioactive Molecules.

The impenetrability of the blood-brain barrier (BBB) to most conventional drugs impedes the treatment of central nervous system (CNS) disorders. Interventions for diseases like brain cancer, neurodegeneration, or age-associated inflammatory processes require varied approaches to CNS drug delivery. Cystine-dense peptides (CDPs) have drawn recent interest as drugs or drug-delivery vehicles. Found throughout the phylogenetic tree, often in drug-like roles, their size, stability, and protein interaction capabilities make CDPs an attractive mid-size biologic scaffold to complement conventional antibody-based drugs. Here, we describe the identification, maturation, characterization, and utilization of a CDP that binds to the transferrin receptor (TfR), a native receptor and BBB transporter for the iron chaperone transferrin. We developed variants with varying binding affinities (KD as low as 216 pM), co-crystallized it with the receptor, and confirmed murine cross-reactivity. It accumulates in the mouse CNS at ~25% of blood levels (CNS blood content is only ~1%-6%) and delivers neurotensin, an otherwise non-BBB-penetrant neuropeptide, at levels capable of modulating CREB signaling in the mouse brain. Our work highlights the utility of CDPs as a diverse, easy-to-screen scaffold family worthy of inclusion in modern drug discovery strategies, demonstrated by the discovery of a candidate CNS drug delivery vehicle ready for further optimization and preclinical development.

Publisher Correction: Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets.

In the original version of this Article the colour key for the amino acid enrichment score was inadvertently omitted from the lower panel of Figure 5b during the production process. This has now been corrected in the PDF and HTML versions of the Article.

Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets.

Protein:protein interactions are among the most difficult to treat molecular mechanisms of disease pathology. Cystine-dense peptides have the potential to disrupt such interactions, and are used in drug-like roles by every clade of life, but their study has been hampered by a reputation for being difficult to produce, owing to their complex disulfide connectivity. Here we describe a platform for identifying target-binding cystine-dense peptides using mammalian surface display, capable of interrogating high quality and diverse scaffold libraries with verifiable folding and stability. We demonstrate the platform's capabilities by identifying a cystine-dense peptide capable of inhibiting the YAP:TEAD interaction at the heart of the oncogenic Hippo pathway, and possessing the potency and stability necessary for consideration as a drug development candidate. This platform provides the opportunity to screen cystine-dense peptides with drug-like qualities against targets that are implicated for the treatment of diseases, but are poorly suited for conventional approaches.

Surveying the landscape of Huntington's disease mechanisms, measurements, and medicines.

Though 20 years have now passed since the cloning of the huntingtin gene (HTT), there remains no treatment for Huntington's Disease (HD) that alters the course of disease or lifespan of patients. The reasons for this are manifold, and likely have to do with the diverse cellular pathways disrupted by mutant HTT (mHTT) protein expression. Furthermore, the evaluation of efficacy using a putative intervention is complex, largely due to the slow course of disease and variability in the classic techniques for evaluating patient symptoms and quality of life, which make the patient populations and duration of trials particularly imposing. However, there are signs for hope both in the clinic and at the bench. This review serves three purposes. It discusses the known cellular pathologies in HD, the current and upcoming methods for clinical evaluation of disease progress, and the tested and untested interventions proposed to counter the progression in animal models and patients. With the vast knowledge of pathology accumulated over two decades of modeling HD in animals and following it in patients, as well as the advances in intervention techniques both pharmaceutical and genetic, there is reason for optimism in the field. Such optimism can only be tempered by the lack of success in the clinic to this point, though patients, scientists, and clinicians all remain enthusiastic about each new trial, and progress can only continue until an effective treatment is found.

In vivo identification of therapeutic constructs from pooled candidates in HD model mice.

Rapidly identifying targets for Huntington's Disease (HD) therapeutics in relevant mouse models could hasten the development of patient interventions. We have recently described a method for rapidly and quantitatively measuring the progression of HD-like symptoms in mouse models. Because this method uses flow cytometry to measure GFP levels in affected neurons, it is amenable to pooled approaches. Here we describe a continuation of this work, using pools of shRNA-delivering AAV vectors and high throughput sequencing to determine which hairpins in a mixed population are most effective at preventing the transcriptional dysregulation phenotype of R6/2 mice.

Huntington's disease: can mice lead the way to treatment?

Mouse models for Huntington's Disease (HD) and HD patients demonstrate motor and behavioral dysfunctions, such as progressive loss of coordination and memory, and share similar transcriptional profiles and striatal neuron atrophy. Clear differences between the mouse and human diseases include almost complete striatal degeneration and rarity of intranuclear inclusions in HD, and the fact that mice expressing full-length mutant huntingtin do not demonstrate a shortened life span characteristic of HD. While no clinical interventions tested in mouse models to date have delayed disease progression, the mouse models provide an invaluable tool for both investigating the underlying pathogenic processes and developing new effective therapies. Inherent differences between humans and mice must be considered in the search for efficacious treatments for HD, but the striking similarities between human HD and mouse models support the view that these models are a biologically relevant system to support the identification and testing of potential clinical therapies.