Drugging the Undruggable

• Genentech

Introduction

When I first heard the phrase “Drugging the Undruggable” my mind raced between platitudes such as: “When the going gets tough the tough get going”,¹ “Work Smarter not Harder”² and the Goldilocks-Like metaphor of “not too big and not too small – just right”.³ My traditional drug discovery philosophy didn’t apply. After all, when designing small molecule pharmaceuticals, finding a protein binding target such as an enzyme or membrane receptor and building a selective molecule with affinity and selectivity isn’t new. Biologic therapies are newer, but finding an antigen target for MAB binding or dosing a therapeutic protein (hormone or enzyme) are tried and true paths. To paraphrase Dr. Suess⁴ regarding the evolution of my thoughts “As I stood puzzling and puzzling drugging the undruggable, how could it be so? I puzzled and puzzled ‘till my puzzler was sore. Then I thought of something I hadn’t before. What if drugging the undruggable, perhaps, means a little bit more.” After pushing past the platitudes and doing some research the key concepts of New Drug Modalities and Novel Biologic Targets emerged. The combination of New Drug Modalities to modulate Novel Biologic Targets equate to “Drugging the Undruggable”.

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New Drug Modalities to Modulate Novel Biologic Targets

Genomic and proteomic research is leading to an ever-increasing understanding of biological systems. The research is finding novel biological targets with potential for disease modification including protein–protein and protein–nucleic acid interactions. Since many of these novel targets are within the cell and also within specific organelles (e.g. ribosomes and the Endoplastic reticulum) surfacebinding biologics such as MABs are often too large to be effective in-vivo. Conventional small molecule pharmaceuticals can reach these targets; but, modulating the activity requires much broader interaction over extended binding sites (up to 3000 Å). Drugging these targets has led to the development of creative New Drug Modalities with some of the major Translational Medicine efforts being focused in the following areas:

Macrocycles

Macrocycles are not exactly new with many structures originating in nature including the therapeutic agents vancomycin, amphotericin B, romidepsin, and sirolimus.⁵ Macrocycles by definition contain one or more rings of at least 12 atoms. Macrocycles combine the benefits of large biomolecules, such as high potency and exquisite selectivity, with those of small molecules, including favorable pharmacokinetics, oral bioavailability and lack of immunogenicity. For targets with extended binding sites (e.g. G-protein-coupled receptors (GPCRs), and protein−protein interactions) Macrocycles are of increasing interest.⁶

Macrocycles can drug these large and spatially diverse targets with numerous interactions to increase both potency and selectivity. Cyclization provides a degree of structural organization not present in linear analogues without the high degree of rigidity found in small molecule heterocycles. The cyclization is a compromise between structural organization and flexibility to interact with these dynamic protein targets.

Peptides and Peptidomimetics Modulating protein-protein interactions (PPIs) with peptides is common in nature. A number of naturally occurring bioactive peptides have been found such as  eurotransmitters, anti-infective agents and growth factors. Several peptidomimetic protease inhibitors have already been approved by the FDA for treating HIV and hypertension.⁷

There are estimates of between 130,000 to 650,000 drugable human protein - protein interactions.⁸ A PPI is usually large in size (up to 3000 Å), and lacks the tertiary structure “pockets” for binding small molecules.

However; only a small number of crucial amino acid residues within the PPI are important to binding affinity and specificity. These regions have been termed ‘hot spots’. Hot spots are generally clustered at the center of small to medium size binding interfaces (250-900 Å)⁹ which make them attractive targets for bioactive peptides. The problem with most bioactive peptides is unfavorable DMPK properties due to: poor oral bioavailability, rapid metabolism, low cell-membrane permeability and rapid excretion.¹⁰ To overcome the DMPK limitations and target critical hot spots stabilized peptides and peptideomimetics are increasingly being investigated. Peptidomimetics can be defined as non-natural mimics of bioactive peptides. Strategies for developing peptides and peptidomimetics to target PPI Hot Spots with good DMPK properties generally include locking in secondary structures such as alpha helices or beta sheets with the right affinity while minimizing proteolysis. Techniques commonly used include: stapling, knotting, cyclization, side chain protecting, using non-natural amino acid residues, and including more sophisticated non-amino acid mimetic structures.

Oligonucleotides

Since the completion of the Human Genome Project finding oligonucleotide based targets with potential for modulating disease has increasing at a rapid rate. While proof of concept in vitro was demonstrated many years ago; drugging targets with oligonucleotides in vivo has been challenging. Oligonucleotides are inherently unstable, polyanionic, rapidly cleared by nuclease enzymes, and require a delivery vehicle for transport to the targeted cells. Modifications to stabilize oligonucleotides include changes in the sugar, base, or backbone to increase target affinity and specificity, decrease susceptibility to nuclease degradation, and improve PK¹¹ properties.

Even with all the challenges US regulators have recently approved the first therapy based on RNA interference (RNAi), a technique that can be used to silence specific genes linked to disease.¹² Events leading to translation of this first RNAi therapy involved: the discovery that encasing chemically modified RNAi molecules in fatty nanoparticles caused accumulation in the kidneys and liver; followed by targeting transthyretin, which is expressed mainly in the liver. In clinical trials 225 people with hereditary transthyretin amyloidosis showed signs of significant improvement after treatment leading to the FDA approval for this rare disease indication. With the approval renewed interest in oligonucleotide drug development is anticipated. In addition to RNAi, other novel oligonucleotide strategies include ASO (antisense oligonucleotides), siRNA (small interfering RNA), miRNA (micro RNA), ddRNAi (DNA-directed RNA interference), gRNA (guide RNA for CRISPER editing), and Aptamers (evolved random sequences of nucleic acids or proteins).

Novel Delivery Systems: Nanoparticles and Molecular Conjugates

Drugging novel biologic targets is often achievable in vitro with the key challenge being delivery in vivo. In addition to the complexities of novel biologic targets many therapies face factors such as systemic toxicity and multi-drug resistance as highlighted in the treatment of cancer.¹³ Bringing the drug to the target without general systemic exposure can overcome these complexities. Nanoparticles in the size range of 1–200 nm offer important and unique interactions with targets yet translation of these system to clinically-relevant therapies has proven to be difficult, with only six anticancer nanoparticle drug delivery systems FDA-approved. Nanoparticles are often engineered from biocompatible polymers and surfactants to avoid immunogenicity. The following are often investigated nanoparticle delivery systems; liposomes, solid–lipid nanoparticles, dendrimers, gold/silicon nanoparticles and polymeric nanoparticles. Molecular conjugates combine a pharmacologically active molecule with a “homing” molecule to direct the cargo (drug molecule) to the desired tissue and/or cell.¹⁴ ADC’s (Antibody Drug Conjugates) where a small molecule drug is coupled to a monoclonal antibody were the first molecular conjugates to be approved for oncology indications. An increasing number of molecular conjugates are now using synthetic ligands, and several of these approaches have recently entered the clinic. Molecular conjugates being investigated include: Peptides-Small Molecules Conjugates, Carbohydrate–Oligonucleotides Conjugates, Lipid–Oligonucleotides Conjugates, Small-Molecule-Oligonucleotides Conjugates, Peptide–Oligonucleotides Conjugates, and Aptamer Conjugates. Molecular conjugates open the possibilities from targeted delivery and synergistic binding of the same target to reaching more than one target. Proteolysis-Targeting Chimeras (PROTACs) for Protein Degradation are an interesting example where a bifunctional conjugate is used to bring a target protein and protein degradation systems such as ubiquitin ligase together to knock down activity of the target protein.

Conclusion

Drugging the undruggable is a combination of New Drug Modalities to modulate Novel Biologic Targets. Genomic and proteomic research is leading to an ever-increasing understanding of biological systems. The research is finding novel biological targets with potential for disease modification including protein–protein and protein–nucleic acid interactions. Some exciting New Drug Modalities with the potential to translate into effective therapies include: Macrocycles, Peptides and Peptidomimetics, Oligonucleotides, and Novel Delivery Systems such as Nanoparticles and Molecular Conjugates.

References

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